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Sample records for 3-kinase p110beta gene

  1. Phosphoinositide 3-kinase δ gene mutation predisposes to respiratory infection and airway damage

    PubMed Central

    Angulo, Ivan; Vadas, Oscar; Garçon, Fabien; Banham-Hall, Edward; Plagnol, Vincent; Leahy, Timothy R.; Baxendale, Helen; Coulter, Tanya; Curtis, James; Wu, Changxin; Blake-Palmer, Katherine; Perisic, Olga; Smyth, Deborah; Maes, Mailis; Fiddler, Christine; Juss, Jatinder; Cilliers, Deirdre; Markelj, Gašper; Chandra, Anita; Farmer, George; Kielkowska, Anna; Clark, Jonathan; Kracker, Sven; Debré, Marianne; Picard, Capucine; Pellier, Isabelle; Jabado, Nada; Morris, James A.; Barcenas-Morales, Gabriela; Fischer, Alain; Stephens, Len; Hawkins, Phillip; Barrett, Jeffrey C.; Abinun, Mario; Clatworthy, Menna; Durandy, Anne; Doffinger, Rainer; Chilvers, Edwin; Cant, Andrew J.; Kumararatne, Dinakantha; Okkenhaug, Klaus; Williams, Roger L.; Condliffe, Alison; Nejentsev, Sergey

    2014-01-01

    Genetic mutations cause primary immunodeficiencies (PIDs), which predispose to infections. Here we describe Activated PI3K-δ Syndrome (APDS), a PID associated with a dominant gain-of-function mutation E1021K in the p110δ protein, the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3,346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased IgM and reduced IgG2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110δ. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110δ inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, suggesting a therapeutic approach for patients with APDS. PMID:24136356

  2. 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

  3. A genomewide overexpression screen identifies genes involved in the phosphatidylinositol 3-kinase pathway in the human protozoan parasite Entamoeba histolytica.

    PubMed

    Koushik, Amrita B; Welter, Brenda H; Rock, Michelle L; Temesvari, Lesly A

    2014-03-01

    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.

  4. Epidermal growth factor-dependent association of phosphatidylinositol 3-kinase with the erbB3 gene product.

    PubMed

    Kim, H H; Sierke, S L; Koland, J G

    1994-10-07

    The ErbB3 protein is a member of the ErbB subfamily of receptor protein tyrosine kinases. In the present study, the mechanism by which the ErbB3 protein is phosphorylated and the signal-transducing functions of this phosphorylated protein were investigated. When phosphorylated by the epidermal growth factor receptor in vitro, the ErbB3 protein strongly associated with the regulatory p85 subunit and the catalytic activity of phosphatidylinositol (PI) 3-kinase. The association of PI 3-kinase with ErbB3 in human breast cancer cells was found to be correlated with the constitutive phosphorylation of ErbB3 on tyrosine residues. In MDA-MB-468 breast cancer cells in which the ErbB3 protein is not constitutively phosphorylated, stimulation with epidermal growth factor led to the phosphorylation of ErbB3 on tyrosine residues and the formation of a functional signal transduction complex involving the ErbB3 protein and PI 3-kinase. These results suggest that the ErbB3 protein can be phosphorylated on tyrosine residues by a cross-phosphorylation mechanism and that the phosphorylated ErbB3 protein can couple other growth factor receptor protein tyrosine kinases to the PI 3-kinase pathway in a manner similar to the insulin receptor substrate 1 protein.

  5. MGMT-independent temozolomide resistance in pediatric glioblastoma cells associated with a PI3-kinase-mediated HOX/stem cell gene signature.

    PubMed

    Gaspar, Nathalie; Marshall, Lynley; Perryman, Lara; Bax, Dorine A; Little, Suzanne E; Viana-Pereira, Marta; Sharp, Swee Y; Vassal, Gilles; Pearson, Andrew D J; Reis, Rui M; Hargrave, Darren; Workman, Paul; Jones, Chris

    2010-11-15

    Sensitivity to temozolomide is restricted to a subset of glioblastoma patients, with the major determinant of resistance being a lack of promoter methylation of the gene encoding the repair protein DNA methyltransferase MGMT, although other mechanisms are thought to be active. There are, however, limited preclinical data in model systems derived from pediatric glioma patients. We screened a series of cell lines for temozolomide efficacy in vitro, and investigated the differential mechanisms of resistance involved. In the majority of cell lines, a lack of MGMT promoter methylation and subsequent protein overexpression were linked to temozolomide resistance. An exception was the pediatric glioblastoma line KNS42. Expression profiling data revealed a coordinated upregulation of HOX gene expression in resistant lines, especially KNS42, which was reversed by phosphoinositide 3-kinase pathway inhibition. High levels of HOXA9/HOXA10 gene expression were associated with a shorter survival in pediatric high-grade glioma patient samples. Combination treatment in vitro of pathway inhibition and temozolomide resulted in a highly synergistic interaction in KNS42 cells. The resistance gene signature further included contiguous genes within the 12q13-q14 amplicon, including the Akt enhancer PIKE, significantly overexpressed in the KNS42 line. These cells were also highly enriched for CD133 and other stem cell markers. We have thus shown an in vitro link between phosphoinositide 3-kinase-mediated HOXA9/HOXA10 expression, and a drug-resistant, progenitor cell phenotype in MGMT-independent pediatric glioblastoma.

  6. Down-regulation of the tumor suppressor gene retinoic acid receptor beta2 through the phosphoinositide 3-kinase/Akt signaling pathway.

    PubMed

    Lefebvre, Bruno; Brand, Céline; Flajollet, Sébastien; Lefebvre, Philippe

    2006-09-01

    The retinoic acid receptor beta2 (RARbeta2) is a potent, retinoid-inducible tumor suppressor gene, which is a critical molecular relay for retinoid actions in cells. Its down-regulation, or loss of expression, leads to resistance of cancer cells to retinoid treatment. Up to now, no primary mechanism underlying the repression of the RARbeta2 gene expression, hence affecting cellular retinoid sensitivity, has been identified. Here, we demonstrate that the phosphoinositide 3-kinase/Akt signaling pathway affects cellular retinoid sensitivity, by regulating corepressor recruitment to the RARbeta2 promoter. Through direct phosphorylation of the corepressor silencing mediator for retinoic and thyroid hormone receptors (SMRT), Akt stabilized RAR/SMRT interaction, leading to an increased tethering of SMRT to the RARbeta2 promoter, decreased histone acetylation, down-regulation of the RARbeta2 expression, and impaired cellular differentiation in response to retinoid. The phosphoinositide 3-kinase/Akt signaling pathway, an important modulator of cellular survival, has thus a direct impact on cellular retinoid sensitivity, and its deregulation may be the triggering event in retinoid resistance of cancer cells.

  7. The phosphoinositide 3-kinase pathway.

    PubMed

    Cantley, Lewis C

    2002-05-31

    Phosphorylated lipids are produced at cellular membranes during signaling events and contribute to the recruitment and activation of various signaling components. The role of phosphoinositide 3-kinase (PI3K), which catalyzes the production of phosphatidylinositol-3,4,5-trisphosphate, in cell survival pathways; the regulation of gene expression and cell metabolism; and cytoskeletal rearrangements are highlighted. The PI3K pathway is implicated in human diseases including diabetes and cancer, and understanding the intricacies of this pathway may provide new avenues for therapuetic intervention.

  8. ARIA/HRG regulates AChR epsilon subunit gene expression at the neuromuscular synapse via activation of phosphatidylinositol 3-kinase and Ras/MAPK pathway

    PubMed Central

    1996-01-01

    AChR-inducing activity (ARIA)/heregulin, a ligand for erbB receptor tyrosine kinases (RTKs), is likely to be one nerve-supplied signal that induces expression of acetylcholine receptor (AChR) genes at the developing neuromuscular junction. Since some RTKs act through Ras and phosphatidylinositol 3-kinase (PI3K), we investigated the role of these pathways in ARIA signaling. Expression of activated Ras or Raf mimicked ARIA-induction of AChR epsilon subunit genes in muscle cells; whereas dominant negative Ras or Raf blocked the effect of ARIA. ARIA rapidly activated erk1 and erk2 and inhibition of both erks also abolished the effect of ARIA. ARIA stimulated association of PI3K with erbB3, expression of an activated PI3K led to ARIA-independent AChR epsilon subunit expression, and inhibition of PI3K abolished the action of ARIA. Thus, synaptic induction of AChR genes requires activation of both Ras/MAPK and PI3K signal transduction pathways. PMID:8707830

  9. Phosphoinositide 3-kinase targeting by the β galactoside binding protein cytokine negates akt gene expression and leads aggressive breast cancer cells to apoptotic death

    PubMed Central

    Wells, Valerie; Mallucci, Livio

    2009-01-01

    Introduction Phosphoinositide 3-kinase (PI3K)-activated signalling has a critical role in the evolution of aggressive tumourigenesis and is therefore a prime target for anticancer therapy. Previously we have shown that the β galactoside binding protein (βGBP) cytokine, an antiproliferative molecule, induces functional inhibition of class 1A and class 1B PI3K. Here, we have investigated whether, by targeting PI3K, βGBP has therapeutic efficacy in aggressive breast cancer cells where strong mitogenic input is fuelled by overexpression of the ErbB2 (also known as HER/neu, for human epidermal growth factor receptor 2) oncoprotein receptor and have used immortalised ductal cells and non-aggressive mammary cancer cells, which express ErbB2 at low levels, as controls. Methods Aggressive BT474 and SKBR3 cancer cells where ErbB2 is overexpressed, MCF10A immortalised ductal cells and non-invasive MCF-7 cancer cells which express low levels of ErbB2, both in their naive state and when forced to mimic aggressive behaviour, were used. Class IA PI3K was immunoprecipitated and the conversion of phosphatidylinositol (4,5)-biphosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3) assessed by ELISA. The consequences of PI3K inhibition by βGBP were analysed at proliferation level, by extracellular signal-regulated kinase (ERK) activation, by akt gene expression and by apoptosis. Apoptosis was documented by changes in mitochondrial membrane potential, alteration of the plasma membrane, caspase 3 activation and DNA fragmentation. Phosphorylated and total ERK were measured by Western blot analysis and akt mRNA levels by Northern blot analysis. The results obtained with the BT474 and SKBR3 cells were validated in the MCF10A ductal cells and in non-invasive MCF-7 breast cancer cells forced into mimicking the in vitro behaviour of the BT474 and SKBR3 cells. Results In aggressive breast cancer cells, where mitogenic signalling is enforced by the ErbB2 oncoprotein receptor

  10. The wavy Mutation Maps to the Inositol 1,4,5-Trisphosphate 3-Kinase 2 (IP3K2) Gene of Drosophila and Interacts with IP3R to Affect Wing Development.

    PubMed

    Dean, Derek M; Maroja, Luana S; Cottrill, Sarah; Bomkamp, Brent E; Westervelt, Kathleen A; Deitcher, David L

    2015-11-27

    Inositol 1,4,5-trisphosphate (IP3) regulates a host of biological processes from egg activation to cell death. When IP3-specific receptors (IP3Rs) bind to IP3, they release calcium from the ER into the cytoplasm, triggering a variety of cell type- and developmental stage-specific responses. Alternatively, inositol polyphosphate kinases can phosphorylate IP3; this limits IP3R activation by reducing IP3 levels, and also generates new signaling molecules altogether. These divergent pathways draw from the same IP3 pool yet cause very different cellular responses. Therefore, controlling the relative rates of IP3R activation vs. phosphorylation of IP3 is essential for proper cell functioning. Establishing a model system that sensitively reports the net output of IP3 signaling is crucial for identifying the controlling genes. Here we report that mutant alleles of wavy (wy), a classic locus of the fruit fly Drosophila melanogaster, map to IP3 3-kinase 2 (IP3K2), a member of the inositol polyphosphate kinase gene family. Mutations in wy disrupt wing structure in a highly specific pattern. RNAi experiments using GAL4 and GAL80(ts) indicated that IP3K2 function is required in the wing discs of early pupae for normal wing development. Gradations in the severity of the wy phenotype provide high-resolution readouts of IP3K2 function and of overall IP3 signaling, giving this system strong potential as a model for further study of the IP3 signaling network. In proof of concept, a dominant modifier screen revealed that mutations in IP3R strongly suppress the wy phenotype, suggesting that the wy phenotype results from reduced IP4 levels, and/or excessive IP3R signaling.

  11. Isoform-selective phosphoinositide 3'-kinase inhibitors inhibit CXCR4 signaling and overcome stromal cell-mediated drug resistance in chronic lymphocytic leukemia: a novel therapeutic approach.

    PubMed

    Niedermeier, Matthias; Hennessy, Bryan T; Knight, Zachary A; Henneberg, Marina; Hu, Jianhua; Kurtova, Antonina V; Wierda, William G; Keating, Michael J; Shokat, Kevan M; Burger, Jan A

    2009-05-28

    Phosphoinositide 3-kinases (PI3Ks) are among the most frequently activated signaling pathways in cancer. In chronic lymphocytic leukemia (CLL), signals from the microenvironment are critical for expansion of the malignant B cells, and cause constitutive activation of PI3Ks. CXCR4 is a key receptor for CLL cell migration and adhesion to marrow stromal cells (MSCs). Because of the importance of CXCR4 and PI3Ks for CLL-microenvironment cross-talk, we investigated the activity of novel, isoform-selective PI3K inhibitors that target different isoforms of the p110-kDa subunit. Inhibition with p110alpha inhibitors (PIK-90 and PI-103) resulted in a significant reduction of chemotaxis and actin polymerization to CXCL12 and reduced migration beneath MSC (pseudoemperipolesis). Western blot and reverse phase protein array analyses consistently demonstrated that PIK-90 and PI-103 inhibited phosphorylation of Akt and S6, whereas p110delta or p110beta/p110delta inhibitors were less effective. In suspension and MSC cocultures, PI-103 and PIK-90 were potent inducers of CLL cell apoptosis. Moreover, these p110alpha inhibitors enhanced the cytotoxicity of fludarabine and reversed the protective effect of MSC on fludarabine-induced apoptosis. Collectively, our data demonstrate that p110alpha inhibitors antagonize stromal cell-derived migration, survival, and drug-resistance signals and therefore provide a rational to explore the therapeutic activity of these promising agents in CLL.

  12. 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.

  13. Concerted transcriptional activation of the low density lipoprotein receptor gene by insulin and luteinizing hormone in cultured porcine granulosa-luteal cells: possible convergence of protein kinase a, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase signaling pathways.

    PubMed

    Sekar, N; Veldhuis, J D

    2001-07-01

    -repressive region in this gene. Non-LH receptor-dependent agonists of protein kinase A (PKA), 8-bromo-cAMP (1 mM), and forskolin (10 microM) with or without insulin/IGF-I costimulation likewise augmented LDL receptor promoter expression with similar strong dependency on the -255 to -139 bp 5'-upstream region. To assess more specific PKA-dependent mediation of LH's contribution to combined hormonal drive, the LDL receptor (-1076 to +11 bp) reporter plasmid was cotransfected with a full-sequence rabbit muscle protein kinase inhibitor (PKI) minigene driven constitutively by a Rous sarcoma virus promoter. Expression of the latter PKA antagonist blocked transcriptional stimulation by LH alone as well as that by LH combined with insulin (or IGF-I) by 70-85% without reducing basal transcriptional activity. Transfection of a mutant inactive (Arg to Gly) Rous sarcoma virus/PKI gene confirmed the specificity of the PKI effect. To investigate the convergent role of the insulin/IGF-I effector pathway mediating bihormonal stimulation of LDL receptor promoter expression, transfected granulosa-luteal cells were pretreated for 30 min with two specific inhibitors of phophatidylinositol 3-kinase, wortmannin (100 nM) and LY 294002 (10 microM), or of mitogen-activated protein kinase kinase, PD 98059 (50 microM), U0126 (10 microM), or the latter's inactive derivative, U0124 (10 microM). Both classes of antagonists impeded the ability of insulin or IGF-I to enhance LH-stimulated LDL receptor promoter expression by 60-80%. In conclusion, the present analyses indicate that LH and insulin (or IGF-I) can up-regulate LDL receptor transcriptional activity supraadditively in porcine granulosa-luteal cells 1) via one or more agonistic cis-acting DNA regions located between -255 and -139 bp 5'- upstream of the transcriptional start site, 2) without abrogating sterol-sensitive repressive of this promoter, and 3) by way of intracellular mechanisms that include the PKA, phophatidylinositol 3-kinase, and mitogen

  14. 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

  15. Natural variation in Drosophila melanogaster diapause due to the insulin-regulated PI3-kinase

    PubMed Central

    Williams, Karen D.; Busto, Macarena; Suster, Maximiliano L.; So, Anthony K.-C.; Ben-Shahar, Yehuda; Leevers, Sally J.; Sokolowski, Marla B.

    2006-01-01

    This study links natural variation in a Drosophila melanogaster overwintering strategy, diapause, to the insulin-regulated phosphatidylinositol 3-kinase (PI3-kinase) gene, Dp110. Variation in diapause, a reproductive arrest, was associated with Dp110 by using Dp110 deletions and genomic rescue fragments in transgenic flies. Deletions of Dp110 increased the proportion of individuals in diapause, whereas expression of Dp110 in the nervous system, but not including the visual system, decreased it. The roles of phosphatidylinositol 3-kinase for both diapause in D. melanogaster and dauer formation in Caenorhabditis elegans suggest a conserved role for this kinase in both reproductive and developmental arrests in response to environmental stresses. PMID:17043223

  16. 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

  17. Two distinct functions for PI3-kinases in macropinocytosis

    PubMed Central

    Hoeller, Oliver; Bolourani, Parvin; Clark, Jonathan; Stephens, Len R.; Hawkins, Phillip T.; Weiner, Orion D.; Weeks, Gerald; Kay, Robert R.

    2013-01-01

    Summary Class-1 PI3-kinases are major regulators of the actin cytoskeleton, whose precise contributions to chemotaxis, phagocytosis and macropinocytosis remain unresolved. We used systematic genetic ablation to examine this question in growing Dictyostelium cells. Mass spectroscopy shows that a quintuple mutant lacking the entire genomic complement of class-1 PI3-kinases retains only 10% of wild-type PtdIns(3,4,5)P3 levels. Chemotaxis to folate and phagocytosis of bacteria proceed normally in the quintuple mutant but macropinocytosis is abolished. In this context PI3-kinases show specialized functions, only one of which is directly linked to gross PtdIns(3,4,5)P3 levels: macropinosomes originate in patches of PtdIns(3,4,5)P3, with associated F-actin-rich ruffles, both of which depend on PI3-kinase 1/2 (PI3K1/2) but not PI3K4, whereas conversion of ruffles into vesicles requires PI3K4. A biosensor derived from the Ras-binding domain of PI3K1 suggests that Ras is activated throughout vesicle formation. Binding assays show that RasG and RasS interact most strongly with PI3K1/2 and PI3K4, and single mutants of either Ras have severe macropinocytosis defects. Thus, the fundamental function of PI3-kinases in growing Dictyostelium cells is in macropinocytosis where they have two distinct functions, supported by at least two separate Ras proteins. PMID:23843627

  18. 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.

  19. Neuroprotective Role of the PI3 Kinase/Akt Signaling Pathway in Zebrafish

    PubMed Central

    Chen, Shuang; Liu, Yunzhang; Rong, Xiaozhi; Li, Yun; Zhou, Jianfeng; Lu, Ling

    2017-01-01

    Neuronal survival and growth in the embryo is controlled partly by trophic factors. For most trophic factors (such as Insulin-like growth factor-1), the ability to regulate cell survival has been attributed to the phosphoinositide 3-kinase (PI3K)/Akt kinase cascade. This study presents data illustrating the role of PI3K/Akt in attainment of normal brain size during zebrafish embryogenesis. Blocking PI3K with inhibitor LY294002 caused a significant reduction in brain size (in addition to global growth retardation) during zebrafish embryogenesis. This PI3 Kinase inhibition-induced brain size decrease was recovered by the overexpression of myristoylated Akt (myr-Akt), a constitutive form of Akt. Further analysis reveals that expressing exogenous myr-Akt significantly augmented brain size. Whole mount in situ hybridization analysis of several marker genes showed that myr-Akt overexpression did not alter brain patterning. Furthermore, the expression of myr-Akt was found to protect neuronal cells from apoptosis induced by heat shock and UV light, suggesting that inhibition of neuronal cell death may be part of the underlying cause of the increased brain size. These data provide a foundation for addressing the role of PI3K/Akt in brain growth during zebrafish embryogenesis. PMID:28228749

  20. Phosphatidylinositol 3-kinase (PI3K) inhibitors as cancer therapeutics

    PubMed Central

    2013-01-01

    Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that regulate diverse cellular processes including proliferation, adhesion, survival, and motility. Dysregulated PI3K pathway signaling occurs in one-third of human tumors. Aberrantly activated PI3K signaling also confers sensitivity and resistance to conventional therapies. PI3K has been recognized as an attractive molecular target for novel anti-cancer molecules. In the last few years, several classes of potent and selective small molecule PI3K inhibitors have been developed, and at least fifteen compounds have progressed into clinical trials as new anticancer drugs. Among these, idelalisib has advanced to phase III trials in patients with advanced indolent non-Hodgkin’s lymphoma and mantle cell lymphoma. In this review, we summarized the major molecules of PI3K signaling pathway, and discussed the preclinical models and clinical trials of potent small-molecule PI3K inhibitors. PMID:24261963

  1. Phosphoinositide 3-kinase signaling in the vertebrate retina

    PubMed Central

    Rajala, Raju V. S.

    2010-01-01

    The phosphoinositide (PI) cycle, discovered over 50 years ago by Mabel and Lowell Hokin, describes a series of biochemical reactions that occur on the inner leaflet of the plasma membrane of cells in response to receptor activation by extracellular stimuli. Studies from our laboratory have shown that the retina and rod outer segments (ROSs) have active PI metabolism. Biochemical studies revealed that the ROSs contain the enzymes necessary for phosphorylation of phosphoinositides. We showed that light stimulates various components of the PI cycle in the vertebrate ROS, including diacylglycerol kinase, PI synthetase, phosphatidylinositol phosphate kinase, phospholipase C, and phosphoinositide 3-kinase (PI3K). This article describes recent studies on the PI3K-generated PI lipid second messengers in the control and regulation of PI-binding proteins in the vertebrate retina. PMID:19638643

  2. Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex

    PubMed Central

    Baskaran, Sulochanadevi; Carlson, Lars-Anders; Stjepanovic, Goran; Young, Lindsey N; Kim, Do Jin; Grob, Patricia; Stanley, Robin E; Nogales, Eva; Hurley, James H

    2014-01-01

    The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen–deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity. DOI: http://dx.doi.org/10.7554/eLife.05115.001 PMID:25490155

  3. Phosphoinositide 3-kinase inhibitors induce DNA damage through nucleoside depletion

    PubMed Central

    Juvekar, Ashish; Hu, Hai; Yadegarynia, Sina; Lyssiotis, Costas A.; Ullas, Soumya; Lien, Evan C.; Bellinger, Gary; Son, Jaekyoung; Hok, Rosanna C.; Seth, Pankaj; Daly, Michele B.; Kim, Baek; Scully, Ralph; Asara, John M.; Cantley, Lewis C.; Wulf, Gerburg M.

    2016-01-01

    We previously reported that combining a phosphoinositide 3-kinase (PI3K) inhibitor with a poly-ADP Rib polymerase (PARP)-inhibitor enhanced DNA damage and cell death in breast cancers that have genetic aberrations in BRCA1 and TP53. Here, we show that enhanced DNA damage induced by PI3K inhibitors in this mutational background is a consequence of impaired production of nucleotides needed for DNA synthesis and DNA repair. Inhibition of PI3K causes a reduction in all four nucleotide triphosphates, whereas inhibition of the protein kinase AKT is less effective than inhibition of PI3K in suppressing nucleotide synthesis and inducing DNA damage. Carbon flux studies reveal that PI3K inhibition disproportionately affects the nonoxidative pentose phosphate pathway that delivers Rib-5-phosphate required for base ribosylation. In vivo in a mouse model of BRCA1-linked triple-negative breast cancer (K14-Cre BRCA1f/fp53f/f), the PI3K inhibitor BKM120 led to a precipitous drop in DNA synthesis within 8 h of drug treatment, whereas DNA synthesis in normal tissues was less affected. In this mouse model, combined PI3K and PARP inhibition was superior to either agent alone to induce durable remissions of established tumors. PMID:27402769

  4. Endosomal Phosphatidylinositol 3-Kinase Is Essential for Canonical GPCR Signaling.

    PubMed

    Uchida, Yasunori; Rutaganira, Florentine U; Jullié, Damien; Shokat, Kevan M; von Zastrow, Mark

    2017-01-01

    G protein-coupled receptors (GPCRs), the largest family of signaling receptors, are critically regulated by endosomal trafficking, suggesting that endosomes might provide new strategies for manipulating GPCR signaling. Here we test this hypothesis by focusing on class III phosphatidylinositol 3-kinase (Vps34), which is an essential regulator of endosomal trafficking. We verify that Vps34 is required for recycling of the β2-adrenoceptor (β2AR), a prototypical GPCR, and then investigate the effects of Vps34 inhibition on the canonical cAMP response elicited by β2AR activation. Vps34 inhibition impairs the ability of cells to recover this response after prolonged activation, which is in accord with the established role of recycling in GPCR resensitization. In addition, Vps34 inhibition also attenuates the short-term cAMP response, and its effect begins several minutes after initial agonist application. These results establish Vps34 as an essential determinant of both short-term and long-term canonical GPCR signaling, and support the potential utility of the endosomal system as a druggable target for signaling.

  5. 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

  6. PI3 kinase enzymology on fluid lipid bilayers.

    PubMed

    Dutta, Debjit; Pulsipher, Abigail; Luo, Wei; Yousaf, Muhammad N

    2014-10-21

    We report the use of fluid lipid bilayer membrane as a model platform to study the influence of the bilayer microenvironment and composition on the enzymology in membrane. As a model system we determined the enzyme kinetics on membranes for the transformation of bilayers containing phosphoinositol(4,5)-bisphosphate (PI(4,5)P2) to phosphoinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) by the enzyme phosphoinositol-3-kinase (PI3K) using radiolabeled ATP. The activity of the enzyme was monitored as a function of the radioactivity incorporated within the bilayer. The transformation of PI(4,5)P2 to PI(3,4,5)P3 was determined using a mass strip assay. The fluidity of the bilayer was confirmed by Fluorescence Recovery After Photobleaching (FRAP) experiments. Kinetic simulations were performed based on Langmuir adsorption and Michaelis-Menton kinetics equations to generate the rate constants for the enzymatic reaction. The effect of cholesterol on the enzyme kinetics was studied by doping the bilayer with 1% cholesterol. This leads to significant reduction in reaction rate due to change in membrane microenvironment. This strategy provides a method to study the enzymology of various kinases and phosphatases occurring at the membrane and also how these reactions are affected by the membrane composition and surface microenvironment.

  7. Clinical development of phosphatidylinositol 3-kinase inhibitors for cancer treatment

    PubMed Central

    2012-01-01

    The phosphatidylinositol 3-kinase (PI3K) pathway is commonly deregulated in cancer. In recent years, the results of the first phase I clinical trials with PI3K inhibitors have become available. In comparison to other targeted agents such v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors in melanoma or crizotinib in anaplastic lymphoma receptor tyrosine kinase (ALK) translocated tumors, the number of objective responses to PI3K inhibitors is less dramatic. In this review we propose possible strategies to optimize the clinical development of PI3K inhibitors: by exploring the potential role of PI3K isoform-specific inhibitors in improving the therapeutic index, molecular characterization as a basis for patient selection, and the relevance of performing serial tumor biopsies to understand the associated mechanisms of drug resistance. The main focus of this review will be on PI3K isoform-specific inhibitors by describing the functions of different PI3K isoforms, the preclinical activity of selective PI3K isoform-specific inhibitors and the early clinical data of these compounds. PMID:23232172

  8. Targeting phosphoinositide 3-kinase δ for allergic asthma.

    PubMed

    Rowan, Wendy C; Smith, Janet L; Affleck, Karen; Amour, Augustin

    2012-02-01

    Chronic inflammation in the lung has long been linked to the pathogenesis of asthma. Central to this airway inflammation is a T-cell response to allergens, with Th2 cytokines driving the differentiation, survival and function of the major inflammatory cells involved in the allergic cascade. PI3Kδ (phosphoinositide 3-kinase δ) is a lipid kinase, expressed predominantly in leucocytes, where it plays a critical role in immune receptor signalling. A selective PI3Kδ inhibitor is predicted to block T-cell activation in the lung, reducing the production of pro-inflammatory Th2 cytokines. PI3Kδ is also involved in B-cell and mast cell activation. Therefore the inhibition of PI3Kδ should dampen down the inflammatory cascade involved in the asthmatic response through a wide breadth of pharmacology. Current anti-inflammatory therapies, which are based on corticosteroids, are effective in controlling inflammation in mild asthmatics, but moderate/severe asthmatic patients remain poorly controlled, experiencing recurrent exacerbations. Corticosteroids have no effect on mast cell degranulation and do not act directly on B-cells, so, overall, a PI3Kδ inhibitor has the potential to deliver improvements in onset of action, efficacy and reduced exacerbations in moderate/severe asthmatics. Additionally, PI3Kδ inhibition is expected to block effects of Th17 cells, which are increasingly implicated in steroid-insensitive asthma.

  9. Dual regulation of glucocorticoid-induced leucine zipper (GILZ) by the glucocorticoid receptor and the PI3-kinase/AKT pathways in multiple myeloma.

    PubMed

    Grugan, Katharine D; Ma, Chunguang; Singhal, Seema; Krett, Nancy L; Rosen, Steven T

    2008-06-01

    Glucocorticoids (GCs) are effective therapeutics commonly used in multiple myeloma (MM) treatment. Clarifying the pathway of GC-induced apoptosis is crucial to understanding the process of drug resistance and to the development of new targets for MM treatment. We have previously published results of a micro-array identifying glucocorticoid-induced leucine zipper (GILZ) as GC-regulated gene in MM.1S cells. Consistent with those results, GCs increased GILZ in MM cell lines and patient samples. Reducing the levels of GILZ with siRNA decreased GC-induced cell death suggesting GILZ may mediate GC-killing. We conducted a screen to identify other pathways that affect GILZ regulation and report that inhibitors of PI3-kinase/AKT enhanced GILZ expression in MM cell lines and clinical samples. The combination of dexamethasone (Dex) and LY294002, wortmannin, triciribine, or AKT inhibitor VIII dramatically up regulated GILZ levels and enhanced apoptosis. Addition of interleukin-6 (IL-6) or insulin-like growth factor (IGF1), both which activate the PI3-kinase/AKT pathway and inhibit GC killing, blocked up regulation of GILZ by GC and PI3-kinase/AKT inhibitors. In summary, these results identify GILZ as a mediator of GC killing, indicate a role of PI3-kinase/AKT in controlling GILZ regulation and suggest that the combination of PI3-kinase/AKT inhibitors and GCs may be a beneficial MM treatment.

  10. Class (I) Phosphoinositide 3-Kinases in the Tumor Microenvironment

    PubMed Central

    Gyori, David; Chessa, Tamara; Hawkins, Phillip T.; Stephens, Len R.

    2017-01-01

    Phosphoinositide 3-kinases (PI3Ks) are a diverse family of enzymes which regulate various critical biological processes, such as cell proliferation and survival. Class (I) PI3Ks (PI3Kα, PI3Kβ, PI3Kγ and PI3Kδ) mediate the phosphorylation of the inositol ring at position D3 leading to the generation of PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 can be dephosphorylated by several phosphatases, of which the best known is the 3-phosphatase PTEN (phosphatase and tensin homolog). The Class (I) PI3K pathway is frequently disrupted in human cancers where mutations are associated with increased PI3K-activity or loss of PTEN functionality within the tumor cells. However, the role of PI3Ks in the tumor stroma is less well understood. Recent evidence suggests that the white blood cell-selective PI3Kγ and PI3Kδ isoforms have an important role in regulating the immune-suppressive, tumor-associated myeloid cell and regulatory T cell subsets, respectively, and as a consequence are also critical for solid tumor growth. Moreover, PI3Kα is implicated in the direct regulation of tumor angiogenesis, and dysregulation of the PI3K pathway in stromal fibroblasts can also contribute to cancer progression. Therefore, pharmacological inhibition of the Class (I) PI3K family in the tumor microenvironment can be a highly attractive anti-cancer strategy and isoform-selective PI3K inhibitors may act as potent cancer immunotherapeutic and anti-angiogenic agents. PMID:28273837

  11. 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.

  12. Class IA phosphatidylinositol 3-kinase in pancreatic β cells controls insulin secretion by multiple mechanisms.

    PubMed

    Kaneko, Kazuma; Ueki, Kohjiro; Takahashi, Noriko; Hashimoto, Shinji; Okamoto, Masayuki; Awazawa, Motoharu; Okazaki, Yukiko; Ohsugi, Mitsuru; Inabe, Kazunori; Umehara, Toshihiro; Yoshida, Masashi; Kakei, Masafumi; Kitamura, Tadahiro; Luo, Ji; Kulkarni, Rohit N; Kahn, C Ronald; Kasai, Haruo; Cantley, Lewis C; Kadowaki, Takashi

    2010-12-01

    Type 2 diabetes is characterized by insulin resistance and pancreatic β cell dysfunction, the latter possibly caused by a defect in insulin signaling in β cells. Inhibition of class IA phosphatidylinositol 3-kinase (PI3K), using a mouse model lacking the pik3r1 gene specifically in β cells and the pik3r2 gene systemically (βDKO mouse), results in glucose intolerance and reduced insulin secretion in response to glucose. β cells of βDKO mice had defective exocytosis machinery due to decreased expression of soluble N-ethylmaleimide attachment protein receptor (SNARE) complex proteins and loss of cell-cell synchronization in terms of Ca(2+) influx. These defects were normalized by expression of a constitutively active form of Akt in the islets of βDKO mice, preserving insulin secretion in response to glucose. The class IA PI3K pathway in β cells in vivo is important in the regulation of insulin secretion and may be a therapeutic target for type 2 diabetes.

  13. Involvement of phosphoinositide 3-kinase in insulin- or IGF-1-induced membrane ruffling.

    PubMed Central

    Kotani, K; Yonezawa, K; Hara, K; Ueda, H; Kitamura, Y; Sakaue, H; Ando, A; Chavanieu, A; Calas, B; Grigorescu, F

    1994-01-01

    Insulin, IGF-1 or EGF induce membrane ruffling through their respective tyrosine kinase receptors. To elucidate the molecular link between receptor activation and membrane ruffling, we microinjected phosphorylated peptides containing YMXM motifs or a mutant 85 kDa subunit of phosphoinositide (PI) 3-kinase (delta p85) which lacks a binding site for the catalytic 110 kDa subunit of PI 3-kinase into the cytoplasm of human epidermoid carcinoma KB cells. Both inhibited the association of insulin receptor substrate-1 (IRS-1) with PI 3-kinase in a cell-free system and also inhibited insulin- or IGF-1-induced, but not EGF-induced, membrane ruffling in KB cells. Microinjection of nonphosphorylated analogues, phosphorylated peptides containing the EYYE motif or wild-type 85 kDa subunit (Wp85), all of which did not inhibit the association of IRS-1 with PI 3-kinase in a cell-free system, did not inhibit membrane ruffling in KB cells. In addition, wortmannin, an inhibitor of PI 3-kinase activity, inhibited insulin- or IGF-1-induced membrane ruffling. These results suggest that the association of IRS-1 with PI 3-kinase followed by the activation of PI 3-kinase are required for insulin- or IGF-1-induced, but not for EGF-induced, membrane ruffling. Images PMID:8194523

  14. Class IA phosphoinositide 3-kinase regulates heart size and physiological cardiac hypertrophy.

    PubMed

    Luo, Ji; McMullen, Julie R; Sobkiw, Cassandra L; Zhang, Li; Dorfman, Adam L; Sherwood, Megan C; Logsdon, M Nicole; Horner, James W; DePinho, Ronald A; Izumo, Seigo; Cantley, Lewis C

    2005-11-01

    Class I(A) phosphoinositide 3-kinases (PI3Ks) are activated by growth factor receptors, and they regulate, among other processes, cell growth and organ size. Studies using transgenic mice overexpressing constitutively active and dominant negative forms of the p110alpha catalytic subunit of class I(A) PI3K have implicated the role of this enzyme in regulating heart size and physiological cardiac hypertrophy. To further understand the role of class I(A) PI3K in controlling heart growth and to circumvent potential complications from the overexpression of dominant negative and constitutively active proteins, we generated mice with muscle-specific deletion of the p85alpha regulatory subunit and germ line deletion of the p85beta regulatory subunit of class I(A) PI3K. Here we show that mice with cardiac deletion of both p85 subunits exhibit attenuated Akt signaling in the heart, reduced heart size, and altered cardiac gene expression. Furthermore, exercise-induced cardiac hypertrophy is also attenuated in the p85 knockout hearts. Despite such defects in postnatal developmental growth and physiological hypertrophy, the p85 knockout hearts exhibit normal contractility and myocardial histology. Our results therefore provide strong genetic evidence that class I(A) PI3Ks are critical regulators for the developmental growth and physiological hypertrophy of the heart.

  15. Polycystin-1 Induces Resistance to Apoptosis through the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway

    PubMed Central

    Boca, Manila; Distefano, Gianfranco; Boletta, Alessandra; Qian, Feng; Bhunia, Anil K.; Germino, Gregory G.

    2006-01-01

    Polycystin-1 (PC-1), the PKD1 gene product, is a large receptor whose expression in renal epithelial cells results in resistance to apoptosis and tubulogenesis, a model consistent with the phenotype observed in patients. This study links PC-1 expression to a signaling pathway that is known to be both antiapoptotic and important for normal tubulogenesis. This study found that PC-1 expression results in phosphorylation of Akt and downstream effectors and that phosphatidylinositol 3-kinase (PI3-K) inhibitors prevent this process. In addition, it is shown that dominant negative Akt can revert PC-1-induced protection from apoptosis. Furthermore, it was observed that increased PI3-K β activity in PC-1- expressing MDCK cells seems to be dependent on both tyrosine-kinase activity and heterotrimeric G proteins. It also was found that PC-1-induced tubulogenesis is inhibited by PI3-K inhibitors. Taken together, these data suggest that the PI3-K/Akt cascade may be a central modulator of PC-1 function and that its deregulation might be important in autosomal dominant polycystic kidney disease. PMID:16452497

  16. Role of class III phosphatidylinositol 3-kinase during programmed nuclear death of Tetrahymena thermophila.

    PubMed

    Akematsu, Takahiko; Fukuda, Yasuhiro; Attiq, Rizwan; Pearlman, Ronald E

    2014-02-01

    Programmed nuclear death (PND) in the ciliate protozoan Tetrahymena thermophila is a novel type of autophagy that occurs during conjugation, in which only the parental somatic macronucleus is destined to die and is then eliminated from the progeny cytoplasm. Other coexisting nuclei, however, such as new micro- and macronuclei are unaffected. PND starts with condensation in the nucleus followed by apoptotic DNA fragmentation, lysosomal acidification, and final resorption. Because of the peculiarity in the process and the absence of some ATG genes in this organism, the mechanism of PND has remained unclear. In this study, we focus on the role of class III phosphatidylinositol 3-kinase (PtdIns3K, corresponding to yeast Vps34) in order to identify central regulators of PND. We identified the sole Tetrahymena thermophila ortholog (TtVPS34) to yeast Vps34 and human PIK3C3 (the catalytic subunit of PtdIns3K), through phylogenetic analysis, and generated the gene knockdown mutant for functional analysis. Loss of TtVPS34 activity prevents autophagosome formation on the parental macronucleus, and this nucleus escapes from the lysosomal pathway. In turn, DNA fragmentation and final resorption of the nucleus are drastically impaired. These phenotypes are similar to the situation in the ATG8Δ mutants of Tetrahymena, implying an inextricable link between TtVPS34 and TtATG8s in controlling PND as well as general macroautophagy. On the other hand, TtVPS34 does not appear responsible for the nuclear condensation and does not affect the progeny nuclear development. These results demonstrate that TtVPS34 is critically involved in the nuclear degradation events of PND in autophagosome formation rather than with an involvement in commitment to the death program.

  17. Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits.

    PubMed Central

    Holt, K H; Olson, L; Moye-Rowley, W S; Pessin, J E

    1994-01-01

    Domains of interaction between the p85 and p110 subunits of phosphatidylinositol 3-kinase (PI 3-kinase) were studied with the yeast two-hybrid expression system. A gene fusion between the GAL4 transactivation domain and p85 activated transcription from a GAL1-lacZ reporter gene when complemented with a gene fusion between the GAL4 DNA binding domain and p110. To define subdomains responsible for this interaction, a series of p85 deletion mutants were analyzed. A 192-amino-acid inter-SH2 (IS) fragment (residues 429 to 621) was the smallest determinant identified that specifically associated with p110. In analogous experiments, the subdomain within p110 responsible for interaction with p85 was localized to an EcoRI fragment encoding the amino-terminal 127 residues. Expression of these two subdomains [p85(IS) with p110RI] resulted in 100-fold greater reporter activity than that obtained with full-length p85 and p110. Although the p85(IS) domain conferred a strong interaction with the p110 catalytic subunit, this region was not sufficient to impart phosphotyrosine peptide stimulation of PI 3-kinase activity. In contrast, coexpression of the p110 subunit with full-length p85 or with constructs containing the IS sequences flanked by both SH2 domains of p85 [p85(n/cSH2)] or either of the individual SH2 domains [p85(nSH2+IS) or p85(IS+cSH2)] resulted in PI 3-kinase activity that was activated by a phosphotyrosine peptide. These data suggest that phosphotyrosine peptide binding to either SH2 domain generates an intramolecular signal propagated through the IS region to allosterically activate p110. Images PMID:8264609

  18. Phosphoinositide 3-kinase p110δ mediates estrogen- and FSH-stimulated ovarian follicle growth.

    PubMed

    Li, Qian; He, Hui; Zhang, Yin-Li; Li, Xiao-Meng; Guo, Xuejiang; Huo, Ran; Bi, Ye; Li, Jing; Fan, Heng-Yu; Sha, Jiahao

    2013-09-01

    In the mammalian ovary, primordial follicles are generated early in life and remain dormant for prolonged periods. Their growth resumes via primordial follicle activation, and they continue to grow until the preovulatory stage under the regulation of hormones and growth factors, such as estrogen, FSH, and IGF-1. Both FSH and IGF-1 activate the phosphatidylinositol-3 kinase (PI3K)/Akt (acute transforming retrovirus thymoma protein kinase) signaling pathway in granulosa cells (GCs), yet it remains inconclusive whether the PI3K pathway is crucial for follicle growth. In this study, we investigated the p110δ isoform (encoded by the Pik3cd gene) of PI3K catalytic subunit expression in the mouse ovary and its function in fertility. Pik3cd-null females were subfertile, exhibited fewer growing follicles and more atretic antral follicles in the ovary, and responded poorly to exogenous gonadotropins compared with controls. Ovary transplantation showed that Pik3cd-null ovaries responded poorly to FSH stimulation in vitro; this confirmed that the follicle growth defect was intrinsically ovarian. In addition, estradiol (E2)-stimulated follicle growth and GC proliferation in preantral follicles was impaired in Pik3cd-null ovaries. FSH and E2 substantially activated the PI3K/Akt pathway in GCs of control mice but not in those of Pik3cd-null mice. However, primordial follicle activation and oocyte meiotic maturation were not affected by Pik3cd knockout. Taken together, our findings indicate that the p110δ isoform of the PI3K catalytic subunit is a key component of the PI3K pathway for both FSH and E2-stimulated follicle growth in ovarian GCs; however, it is not required for primordial follicle activation and oocyte development.

  19. The role of phosphoinositide 3-kinases in neutrophil migration in 3D collagen gels.

    PubMed

    Martin, Kayleigh J S; Muessel, Michelle J; Pullar, Christine E; Willars, Gary B; Wardlaw, Andrew J

    2015-01-01

    The entry of neutrophils into tissue has been well characterised; however the fate of these cells once inside the tissue microenvironment is not fully understood. A variety of signal transduction pathways including those involving class I PI3 Kinases have been suggested to be involved in neutrophil migration. This study aims to determine the involvement of PI3 Kinases in chemokinetic and chemotactic neutrophil migration in response to CXCL8 and GM-CSF in a three-dimensional collagen gel, as a model of tissue. Using a three-dimensional collagen assay chemokinetic and chemotactic migration induced by CXCL8 was inhibited with the pan PI3 Kinase inhibitor wortmannin. Analysis of the specific Class I PI3 Kinase catalytic isoforms alpha, delta and gamma using the inhibitors PIK-75, PIK-294 and AS-605240 respectively indicated differential roles in CXCL8-induced neutrophil migration. PIK-294 inhibited both chemokinetic and chemotactic CXCL8-induced migration. AS-605240 markedly reduced CXCL8 induced chemokinetic migration but had no effect on CXCL8 induced chemotactic migration. In contrast PIK-75 inhibited chemotactic migration but not chemokinetic migration. At optimal concentrations of GM-CSF the inhibitors had no effect on the percentage of neutrophil migration in comparison to the control however at suboptimal concentrations wortmannin, AS-605240 and PIK-294 inhibited chemokinesis. This study suggests that PI3 Kinase is necessary for CXCL8 induced migration in a 3D tissue environment but that chemokinetic and chemotactic migration may be controlled by different isoforms with gamma shown to be important in chemokinesis and alpha important in chemotaxis. Neutrophil migration in response to suboptimal concentrations of GM-CSF is dependent on PI3 Kinase, particularly the gamma and delta catalytic isoforms.

  20. Hexamethylenebisacetamide modulation of thyroglobulin and protein levels in thyroid cells is not mediated by phosphatidylinositol-3-kinase: a study with wortmannin.

    PubMed

    Aouani, A; Samih, N; Amphoux-Fazekas, T; Hovsépian, S; Fayet, G

    1999-04-01

    Hexamethylenebisacetamide (HMBA) induces in murine erythroleukemia cells (MELC) the commitment to terminal differentiation leading to globin gene expression. In the thyroid, HMBA acts as a growth factor and also as a differentiating agent. In the present paper, we studied the effect of HMBA on the very specific thyroid marker thyroglobulin (Tg) in two different thyroid cell systems, i.e., porcine cells in primary culture and ovine cells in long term culture. Using wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, we investigated whether this enzyme is involved in HMBA mode of action. We found that HMBA is a positive modulator of Tg production in porcine cells, but a negative effector in the OVNIS cell line. As all HMBA effects studied in the present paper, i.e., Tg production and total protein levels, are not inhibited by wortmannin, we suggest the non-involvement of phosphatidylinositol-3-kinase in HMBA mode of action.

  1. Involvement of phosphatidylinositol 3-kinase in stromal cell-derived factor-1 alpha-induced lymphocyte polarization and chemotaxis.

    PubMed

    Vicente-Manzanares, M; Rey, M; Jones, D R; Sancho, D; Mellado, M; Rodriguez-Frade, J M; del Pozo, M A; Yáñez-Mó, M; de Ana, A M; Martínez-A, C; Mérida, I; Sánchez-Madrid, F

    1999-10-01

    The role of phosphatidylinositol 3-kinase (PI3-kinase), an important enzyme involved in signal transduction events, has been studied in the polarization and chemotaxis of lymphocytes induced by the chemokine stromal cell-derived factor-1 alpha (SDF-1 alpha). This chemokine was able to directly activate p85/p110 PI3-kinase in whole human PBL and to induce the association of PI3-kinase to the SDF-1 alpha receptor, CXCR4, in a pertussis toxin-sensitive manner. Two unrelated chemical inhibitors of PI3-kinase, wortmannin and Ly294002, prevented ICAM-3 and ERM protein moesin polarization as well as the chemotaxis of PBL in response to SDF-1 alpha. However, they did not interfere with the reorganization of either tubulin or the actin cytoskeleton. Moreover, the transient expression of a dominant negative form of the PI3-kinase 85-kDa regulatory subunit in the constitutively polarized Peer T cell line inhibited ICAM-3 polarization and markedly reduced SDF-1 alpha-induced chemotaxis. Conversely, overexpression of a constitutively activated mutant of the PI3-kinase 110-kDa catalytic subunit in the round-shaped PM-1 T cell line induced ICAM-3 polarization. These results underline the role of PI3-kinase in the regulation of lymphocyte polarization and motility and indicate that PI3-kinase plays a selective role in the regulation of adhesion and ERM proteins redistribution in the plasma membrane of lymphocytes.

  2. 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

  3. 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

  4. Effects of eicosapentaenoic acid on synaptic plasticity, fatty acid profile and phosphoinositide 3-kinase signaling in rat hippocampus and differentiated PC12 cells.

    PubMed

    Kawashima, Akiko; Harada, Tsuyoshi; Kami, Hideaki; Yano, Takashi; Imada, Kazunori; Mizuguchi, Kiyoshi

    2010-04-01

    Placebo-controlled clinical studies suggest that intake of n-3 polyunsaturated fatty acids improves neurological disorders such as Alzheimer's disease, Huntington's disease and schizophrenia. To evaluate the impact of eicosapentaenoic acid (EPA), we orally administered highly purified ethyl EPA (EPA-E) to rats at a dose of 1.0 mg/g per day and measured long-term potentiation of the CA1 hippocampal region, a physiological correlate of synaptic plasticity that is thought to underlie learning and memory. The mean field excitatory postsynaptic potential slope of the EPA-E group was significantly greater than that of the control group in the CA1 region. Gene expression of hippocampal p85alpha, one of the regulatory subunits of phosphatidylinositol 3-kinase (PI3-kinase), was increased with EPA-E administration. Investigation of fatty acid profiles of neuronal and glia-enriched fractions demonstrated that a single administration of EPA-E significantly increased neuronal and glial EPA content and glial docosahexaenoic acid content, clearly suggesting that EPA was indeed taken up by both neurons and glial cells. In addition, we investigated the direct effects of EPA on the PI3-kinase/Akt pathway in differentiated PC12 cells. Phosphorylated-Akt expression was significantly increased in EPA-treated cells, and nerve growth factor withdrawal-induced increases in cell death and caspase-3 activity were suppressed by EPA treatment. These findings suggest that EPA protects against neurodegeneration by modulating synaptic plasticity and activating the PI3-kinase/Akt pathway, possibly by its own functional effects in neurons and glial cells and by its capacity to increase brain docosahexaenoic acid.

  5. Phosphatidylinositol 3'-kinase associates with an insulin receptor substrate-1 serine kinase distinct from its intrinsic serine kinase.

    PubMed Central

    Cengel, K A; Kason, R E; Freund, G G

    1998-01-01

    Serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been proposed as a counter-regulatory mechanism in insulin and cytokine signalling. Here we report that IRS-1 is phosphorylated by a wortmannin insensitive phosphatidylinositol 3'-kinase (PI 3-kinase)-associated serine kinase (PAS kinase) distinct from PI 3-kinase serine kinase. We found that PI 3-kinase immune complexes contain 5-fold more wortmannin-insensitive serine kinase activity than SH2-containing protein tyrosine phosphatase-2 (SHP2) and IRS-1 immune complexes. Affinity chromatography of cell lysates with a glutathione S-transferase fusion protein for the p85 subunit of PI 3-kinase showed that PAS kinase associated with the p85 subunit of PI 3-kinase. This interaction required unoccupied SH2 domain(s) but did not require the PI 3-kinase p110 subunit binding domain. In terms of function, PAS kinase phosphorylated IRS-1 and, after insulin stimulation, PAS kinase phosphorylated IRS-1 in PI 3-kinase-IRS-1 complexes. Phosphopeptide mapping showed that insulin-dependent in vivo sites of IRS-1 serine phosphorylation were comparable to those of PAS kinase phosphorylated IRS-1. More importantly, PAS kinase-dependent phosphorylation of IRS-1 reduced by 4-fold the ability of IRS-1 to act as an insulin receptor substrate. Taken together, these findings indicate that: (a) PAS kinase is distinct from the intrinsic serine kinase activity of PI 3-kinase, (b) PAS kinase associates with the p85 subunit of PI 3-kinase through SH2 domain interactions, and (c) PAS kinase is an IRS-1 serine kinase that can reduce the ability of IRS-1 to serve as an insulin receptor substrate. PMID:9761740

  6. Phosphoinositide lipid phosphatases: natural regulators of phosphoinositide 3-kinase signaling in T lymphocytes.

    PubMed

    Harris, Stephanie J; Parry, Richard V; Westwick, John; Ward, Stephen G

    2008-02-01

    The phosphoinositide 3-kinase signaling pathway has been implicated in a range of T lymphocyte cellular functions, particularly growth, proliferation, cytokine secretion, and survival. Dysregulation of phosphoinositide 3-kinase-dependent signaling and function in leukocytes, including B and T lymphocytes, has been implicated in many inflammatory and autoimmune diseases. As befits a pivotal signaling cascade, several mechanisms exist to ensure that the pathway is tightly regulated. This minireview focuses on two lipid phosphatases, viz. the 3'-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP (Src homology 2 domain-containing inositol-5-phosphatase). We discuss their role in regulating T lymphocyte signaling as well their potential as future therapeutic targets.

  7. The association of phosphoinositide 3-kinase enhancer A with hepatic insulin receptor enhances its kinase activity.

    PubMed

    Chan, Chi Bun; Liu, Xia; He, Kunyan; Qi, Qi; Jung, Dae Y; Kim, Jason K; Ye, Keqiang

    2011-07-01

    Dysfunction of hepatic insulin receptor tyrosine kinase (IRTK) causes the development of type 2 diabetes. However, the molecular mechanism regulating IRTK activity in the liver remains poorly understood. Here, we show that phosphoinositide 3-kinase enhancer A (PIKE-A) is a new insulin-dependent enhancer of hepatic IRTK. Liver-specific Pike-knockout (LPKO) mice display glucose intolerance with impaired hepatic insulin sensitivity. Specifically, insulin-provoked phosphoinositide 3-kinase/Akt signalling is diminished in the liver of LPKO mice, leading to the failure of insulin-suppressed gluconeogenesis and hyperglycaemia. Thus, hepatic PIKE-A has a key role in mediating insulin signal transduction and regulating glucose homeostasis in the liver.

  8. Recent development of ATP-competitive small molecule phosphatidylinostitol-3-kinase inhibitors as anticancer agents

    PubMed Central

    Liu, Yu; Wan, Wen-zhu; Li, Yan; Zhou, Guan-lian; Liu, Xin-guang

    2017-01-01

    Phosphatidylinostitol-3-kinase (PI3K) is the potential anticancer target in the PI3K/Akt/ mTOR pathway. Here we reviewed the ATP-competitive small molecule PI3K inhibitors in the past few years, including the pan Class I PI3K inhibitors, the isoform-specific PI3K inhibitors and/or the PI3K/mTOR dual inhibitors. PMID:27769061

  9. Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

    PubMed Central

    Cafferkey, R; Young, P R; McLaughlin, M M; Bergsma, D J; Koltin, Y; Sathe, G M; Faucette, L; Eng, W K; Johnson, R K; Livi, G P

    1993-01-01

    Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the

  10. 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

  11. Activation of phosphoinositide 3-kinase by D2 receptor prevents apoptosis in dopaminergic cell lines.

    PubMed

    Nair, Venugopalan D; Olanow, C Warren; Sealfon, Stuart C

    2003-07-01

    Whereas dopamine agonists are known to provide symptomatic benefits for Parkinson's disease, recent clinical trials suggest that they might also be neuroprotective. Laboratory studies demonstrate that dopamine agonists can provide neuroprotective effects in a number of model systems, but the role of receptor-mediated signalling in these effects is controversial. We find that dopamine agonists have robust, concentration-dependent anti-apoptotic activity in PC12 cells that stably express human D(2L) receptors from cell death due to H(2)O(2) or trophic withdrawal and that the protective effects are abolished in the presence of D(2)-receptor antagonists. D(2) agonists are also neuroprotective in the nigral dopamine cell line SN4741, which express endogenous D(2) receptors, whereas no anti-apoptotic activity is observed in native PC12 cells, which do not express detectable D(2) receptors. Notably, the agonists studied differ in their relative efficacy to mediate anti-apoptotic effects and in their capacity to stimulate [(35)S]guanosine 5'-[gamma-thio]triphosphate ([(35)S]GTP[S]) binding, an indicator of G-protein activation. Studies with inhibitors of phosphoinositide 3-kinase (PI 3-kinase), extracellular-signal-regulated kinase or p38 mitogen-activated protein kinase indicate that the PI 3-kinase pathway is required for D(2) receptor-mediated cell survival. These studies indicate that certain dopamine agonists can complex with D(2) receptors to preferentially transactivate neuroprotective signalling pathways and to mediate increased cell survival.

  12. Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L.

    PubMed

    Matsunaga, Kohichi; Morita, Eiji; Saitoh, Tatsuya; Akira, Shizuo; Ktistakis, Nicholas T; Izumi, Tetsuro; Noda, Takeshi; Yoshimori, Tamotsu

    2010-08-23

    Autophagy is a catabolic process that allows cells to digest their cytoplasmic constituents via autophagosome formation and lysosomal degradation. Recently, an autophagy-specific phosphatidylinositol 3-kinase (PI3-kinase) complex, consisting of hVps34, hVps15, Beclin-1, and Atg14L, has been identified in mammalian cells. Atg14L is specific to this autophagy complex and localizes to the endoplasmic reticulum (ER). Knockdown of Atg14L leads to the disappearance of the DFCP1-positive omegasome, which is a membranous structure closely associated with both the autophagosome and the ER. A point mutation in Atg14L resulting in defective ER localization was also defective in the induction of autophagy. The addition of the ER-targeting motif of DFCP1 to this mutant fully complemented the autophagic defect in Atg14L knockout embryonic stem cells. Thus, Atg14L recruits a subset of class III PI3-kinase to the ER, where otherwise phosphatidylinositol 3-phosphate (PI3P) is essentially absent. The Atg14L-dependent appearance of PI3P in the ER makes this organelle the platform for autophagosome formation.

  13. DHEA improves glucose uptake via activations of protein kinase C and phosphatidylinositol 3-kinase.

    PubMed

    Ishizuka, T; Kajita, K; Miura, A; Ishizawa, M; Kanoh, Y; Itaya, S; Kimura, M; Muto, N; Mune, T; Morita, H; Yasuda, K

    1999-01-01

    We have examined the effect of adrenal androgen, dehydroepiandrosterone (DHEA), on glucose uptake, phosphatidylinositol (PI) 3-kinase, and protein kinase C (PKC) activity in rat adipocytes. DHEA (1 microM) provoked a twofold increase in 2-[3H]deoxyglucose (DG) uptake for 30 min. Pretreatment with DHEA increased insulin-induced 2-[3H]DG uptake without alterations of insulin specific binding and autophosphorylation of insulin receptor. DHEA also stimulated PI 3-kinase activity. [3H]DHEA bound to purified PKC containing PKC-alpha, -beta, and -gamma. DHEA provoked the translocation of PKC-beta and -zeta from the cytosol to the membrane in rat adipocytes. These results suggest that DHEA stimulates both PI 3-kinase and PKCs and subsequently stimulates glucose uptake. Moreover, to clarify the in vivo effect of DHEA on Goto-Kakizaki (GK) and Otsuka Long-Evans fatty (OLETF) rats, animal models of non-insulin-dependent diabetes mellitus (NIDDM) were treated with 0.4% DHEA for 2 wk. Insulin- and 12-O-tetradecanoyl phorbol-13-acetate-induced 2-[3H]DG uptakes of adipocytes were significantly increased, but there was no significant increase in the soleus muscles in DHEA-treated GK/Wistar or OLETF/Long-Evans Tokushima (LETO) rats when compared with untreated GK/Wistar or OLETF/LETO rats. These results indicate that in vivo DHEA treatment can result in increased insulin-induced glucose uptake in two different NIDDM rat models.

  14. Andrographolide inhibits hypoxia-inducible factor-1 through phosphatidylinositol 3-kinase/AKT pathway and suppresses breast cancer growth

    PubMed Central

    Li, Jie; Zhang, Chao; Jiang, Hongchuan; Cheng, Jiao

    2015-01-01

    Hypoxia-inducible factor-1 (HIF-1) is a master regulator of the transcriptional response to hypoxia. HIF-1α is one of the most compelling anticancer targets. Andrographolide (Andro) was newly identified to inhibit HIF-1 in T47D cells (a half maximal effective concentration [EC50] of 1.03×10−7 mol/L), by a dual-luciferase reporter assay. It suppressed HIF-1α protein and gene accumulation, which was dependent on the inhibition of upstream phosphatidylinositol 3-kinase (PI3K)/AKT pathway. It also abrogated the expression of HIF-1 target vascular endothelial growth factor (VEGF) gene and protein. Further, Andro inhibited T47D and MDA-MB-231 cell proliferation and colony formation. In addition, it exhibited significant in vivo efficacy and antitumor potential against the MDA-MB-231 xenograft in nude mice. In conclusion, these results highlighted the potential effects of Andro, which inhibits HIF-1, and hence may be developed as an antitumor agent for breast cancer therapy in future. PMID:25709476

  15. Receptor-mediated endocytosis of albumin by kidney proximal tubule cells is regulated by phosphatidylinositide 3-kinase.

    PubMed Central

    Brunskill, N J; Stuart, J; Tobin, A B; Walls, J; Nahorski, S

    1998-01-01

    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

  16. Structural basis for isoform selectivity in a class of benzothiazole inhibitors of phosphoinositide 3-kinase γ.

    PubMed

    Collier, Philip N; Martinez-Botella, Gabriel; Cornebise, Mark; Cottrell, Kevin M; Doran, John D; Griffith, James P; Mahajan, Sudipta; Maltais, François; Moody, Cameron S; Huck, Emilie Porter; Wang, Tiansheng; Aronov, Alex M

    2015-01-08

    Phosphoinositide 3-kinase γ (PI3Kγ) is an attractive target to potentially treat a range of disease states. Herein, we describe the evolution of a reported phenylthiazole pan-PI3K inhibitor into a family of potent and selective benzothiazole inhibitors. Using X-ray crystallography, we discovered that compound 22 occupies a previously unreported hydrophobic binding cleft adjacent to the ATP binding site of PI3Kγ, and achieves its selectivity by exploiting natural sequence differences among PI3K isoforms in this region.

  17. 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

  18. Src-homology 3 domain of protein kinase p59fyn mediates binding to phosphatidylinositol 3-kinase in T cells.

    PubMed Central

    Prasad, K V; Janssen, O; Kapeller, R; Raab, M; Cantley, L C; Rudd, C E

    1993-01-01

    The Src-related tyrosine kinase p59fyn(T) plays an important role in the generation of intracellular signals from the T-cell antigen receptor TCR zeta/CD3 complex. A key question concerns the nature and the binding sites of downstream components that interact with this Src-related kinase. p59fyn(T) contains Src-homology 2 and 3 domains (SH2 and SH3) with a capacity to bind to intracellular proteins. One potential downstream target is phosphatidylinositol 3-kinase (PI 3-kinase). In this study, we demonstrate that anti-CD3 and anti-Fyn immunoprecipitates possess PI 3-kinase activity as assessed by TLC and HPLC. Both free and receptor-bound p59fyn(T) were found to bind to the lipid kinase. Further, our results indicate that Src-related kinases have developed a novel mechanism to interact with PI 3-kinase. Precipitation using GST fusion proteins containing Fyn SH2, SH3, and SH2/SH3 domains revealed that PI 3-kinase bound principally to the SH3 domain of Fyn. Fyn SH3 bound directly to the p85 subunit of PI 3-kinase as expressed in a baculoviral system. Anti-CD3 crosslinking induced an increase in the detection of Fyn SH3-associated PI 3-kinase activity. Thus PI 3-kinase is a target of SH3 domains and is likely to play a major role in the signals derived from the TCR zeta/CD3-p59fyn complex. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8394019

  19. Expression of beta-catenin is regulated by PI-3 kinase and sodium butyrate in colorectal cancer cells.

    PubMed

    Turecková, Jolana; Kucerová, Dana; Vojtechová, Martina; Sloncová, Eva; Tuhácková, Zdena

    2006-01-01

    beta-catenin has a dual function; it is implicated in intercellular junctions and transcriptional co-activation. Here we examined the regulation of the expression and localization of beta-catenin in HT29 colorectal adenocarcinoma cells. Our results showed that inhibition of PI-3 kinase with wortmannin was accompanied by a considerably reduced expression of beta-catenin. This effect was overcome by butyrate and occurred at the protein level, not at the level of mRNA. Moreover, NaBT significantly increased the phosphorylation of the ribosomal protein, S6, known to participate in the translational control of gene expression. This was accompanied by the increased phosphorylation of p70 S6K and MAPKs, the effector proteins that are upstream of protein S6 in the distinct signaling pathways. These facts indicate that different signaling pathways may be involved in the regulation of beta-catenin synthesis. Modulation of beta-catenin expression induced by NaBT appeared to occur at the level of protein translation, suggesting that NaBT may act as a translational regulator.

  20. Phosphatidylinositol 3-Kinase (PI3K) Signaling via Glycogen Synthase Kinase-3 (Gsk-3) Regulates DNA Methylation of Imprinted Loci*

    PubMed Central

    Popkie, Anthony P.; Zeidner, Leigh C.; Albrecht, Ashley M.; D'Ippolito, Anthony; Eckardt, Sigrid; Newsom, David E.; Groden, Joanna; Doble, Bradley W.; Aronow, Bruce; McLaughlin, K. John; White, Peter; Phiel, Christopher J.

    2010-01-01

    Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3β, are constitutively active, largely inhibitory kinases involved in signal transduction. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, Alzheimer disease, schizophrenia, and bipolar disorder. Here, we demonstrate that deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions. Treatment of wild-type embryonic stem cells and neural stem cells with the Gsk-3 inhibitor, lithium, phenocopies the DNA hypomethylation at these imprinted loci. We show that inhibition of Gsk-3 by phosphatidylinositol 3-kinase (PI3K)-mediated activation of Akt also results in reduced DNA methylation at these imprinted loci. Finally, we find that N-Myc is a potent Gsk-3-dependent regulator of Dnmt3a2 expression. In summary, we have identified a signal transduction pathway that is capable of altering the DNA methylation of imprinted loci. PMID:21047779

  1. Constitutively activated phosphatidylinositol 3-kinase primes platelets from patients with chronic myelogenous leukemia for thrombopoietin-induced aggregation.

    PubMed

    Kubota, Y; Tanaka, T; Ohnishi, H; Kitanaka, A; Okutani, Y; Taminato, T; Ishida, T; Kamano, H

    2004-06-01

    In this study, we examined the effect of thrombopoietin (TPO) on the aggregation of platelets from 40 patients with myeloproliferative disorders (MPDs), including 17 patients with chronic myelogenous leukemia in the chronic phase (CML-CP), 10 with polycythemia vera, 10 with essential thrombocythemia, and three with myelofibrosis. TPO by itself dose-dependently induced the aggregation of platelets from patients with CML-CP but not from those with other MPDs or with CML-CP in cytogenetical complete remission. The expression of CD63 in CML-CP platelets was induced by TPO treatment. Phosphatidylinositol 3-kinase (PI3-kinase) was constitutively activated in CML-CP platelets. Pretreatment with PI3-kinase inhibitors (wortmannin and LY294002) dose-dependently inhibited TPO-induced aggregation of CML-CP platelets. The Abl kinase inhibitor imatinib mesylate and the Jak inhibitor AG490 suppressed TPO-induced aggregation of CML-CP platelets. Pretreatment with imatinib mesylate, but not with AG490, inhibited the activity of PI3-kinase in CML-CP platelets. In addition, tyrosine phosphorylation of Jak2 was undetected in CML-CP platelets before TPO treatment. These findings indicate that the constitutive activation of PI3-kinase primes CML-CP platelets for the aggregation induced by TPO, and that Bcr-Abl, but not Jak family protein tyrosine kinases, are involved in the constitutive activation of PI3-kinase in CML-CP platelets.

  2. Cross-Talk between NFkB and the PI3-Kinase/AKT Pathway Can Be Targeted in Primary Effusion Lymphoma (PEL) Cell Lines for Efficient Apoptosis

    PubMed Central

    Hussain, Azhar R.; Ahmed, Saeeda O.; Ahmed, Maqbool; Khan, Omar S.; Al AbdulMohsen, Sally; Platanias, Leonidas C.; Al-Kuraya, Khawla S.; Uddin, Shahab

    2012-01-01

    Background A number of constitutively activated signaling pathways play critical roles in the survival and growth of primary effusion lymphoma cells (PELs) including NFkB and PI3/AKT kinase cascades. NFkBis constitutively activated in a number of malignancies, including multiple myeloma, Burkitt’s lymphoma and diffuse large cell B-cell lymphoma. However, its role in primary effusion lymphoma has not been fully explored. Methodology/Principal Findings We used pharmacological inhibition and gene silencing to define the role of NFkB in growth and survival of PEL cells. Inhibition of NFkB activity by Bay11-7085 resulted in decreased expression of p65 in the nuclear compartment as detected by EMSA assays. In addition, Bay11-7085 treatment caused de-phosphorylation of AKT and its downstream targets suggesting a cross-talk between NFkB and the PI3-kinase/AKT pathway. Importantly, treatment of PEL cells with Bay11-7085 led to inhibition of cell viability and induced apoptosis in a dose dependent manner. Similar apoptotic effects were found when p65 was knocked down using specific small interference RNA. Finally, co-treatment of PEL cells with suboptimal doses of Bay11-7085 and LY294002 led to synergistic apoptotic responses in PEL cells. Conclusion/Significance These data support a strong biological-link between NFkB and the PI3-kinase/AKT pathway in the modulation of anti-apoptotic effects in PEL cells. Synergistic targeting of these pathways using NFKB- and PI3-kinase/AKT- inhibitors may have a therapeutic potential for the treatment of PEL and possibly other malignancies with constitutive activation of these pathways. PMID:22768179

  3. 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.

  4. The involvement of Gab1 and PI 3-kinase in {beta}1 integrin signaling in keratinocytes

    SciTech Connect

    Kuwano, Yoshihiro; Fujimoto, Manabu . E-mail: fujimoto-m@umin.ac.jp; Watanabe, Rei; Ishiura, Nobuko; Nakashima, Hiroko; Komine, Mayumi; Hamazaki, Tatsuo S.; Tamaki, Kunihiko; Okochi, Hitoshi

    2007-09-14

    The control of the stem cell compartment in epidermis is closely linked to the regulation of keratinocyte proliferation and differentiation. {beta}1 integrins are expressed 2-fold higher by stem cells than transit-amplifying cells. Signaling from these {beta}1 integrins is critical for the regulation of the epidermal stem cell compartment. To clarify the functional relevance of this differential expression of {beta}1 integrins, we established HaCaT cells with high {beta}1integrin expression by repeated flow cytometric sorting of this population from the parental cell line. In these obtained cells expressing {beta}1 integrins by 5-fold, MAPK activation was markedly increased. Regarding the upstream of MAPK, Gab1 phosphorylation was also higher with high {beta}1 integrin expression, while Shc phosphorylation was not altered. In addition, enhanced phosphatidylinositol 3-kinase activation was also observed. These observations suggest that Gab1 and phosphatidylinositol 3-kinase play pivotal roles in the {beta}1 integrin-mediated regulation of the epidermal stem cell compartment.

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

    PubMed

    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.

  6. 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.

  7. Pathophysiological roles of Pim-3 kinase in pancreatic cancer development and progression.

    PubMed

    Li, Ying-Yi; Mukaida, Naofumi

    2014-07-28

    Pim-3 is a member of the provirus integration site for Moloney murine leukemia virus (Pim) family proteins that exhibit serine/threonine kinase activity. Similar to the other Pim kinases (Pim-1 and Pim-2), Pim-3 is involved in many cellular processes, including cell proliferation, survival, and protein synthesis. Although Pim-3 is expressed in normal vital organs, it is overexpressed particularly in tumor tissues of endoderm-derived organs, including the liver, pancreas, and colon. Silencing of Pim-3 expression can retard in vitro cell proliferation of hepatocellular, pancreatic, and colon carcinoma cell lines by promoting cell apoptosis. Pim-3 lacks the regulatory domains similarly as Pim-1 and Pim-2 lack, and therefore, Pim-3 can exhibit its kinase activity once it is expressed. Pim-3 expression is regulated at transcriptional and post-transcriptional levels by transcription factors (e.g., Ets-1) and post-translational modifiers (e.g., translationally-controlled tumor protein), respectively. Pim-3 could promote growth and angiogenesis of human pancreatic cancer cells in vivo in an orthotopic nude mouse model. Furthermore, a Pim-3 kinase inhibitor inhibited cell proliferation when human pancreatic cancer cells were injected into nude mice, without inducing any major adverse effects. Thus, Pim-3 kinase may serve as a novel molecular target for developing targeting drugs against pancreatic and other types of cancer.

  8. Identification of small molecule inhibitors of phosphatidylinositol 3-kinase and autophagy.

    PubMed

    Farkas, Thomas; Daugaard, Mads; Jäättelä, Marja

    2011-11-11

    Macroautophagy (hereafter autophagy) is a lysosomal catabolic pathway that controls cellular homeostasis and survival. It has recently emerged as an attractive target for the treatment of a variety of degenerative diseases and cancer. The targeting of autophagy has, however, been hampered by the lack of specific small molecule inhibitors. Thus, we screened two small molecule kinase inhibitor libraries for inhibitors of rapamycin-induced autophagic flux. The three most potent inhibitors identified conferred profound inhibition of autophagic flux by inhibiting the formation of autophagosomes. Notably, the autophagy inhibitory effects of all three compounds were independent of their established kinase targets, i.e. ataxia telangiectasia mutated for KU55933, protein kinase C for Gö6976, and Janus kinase 3 for Jak3 inhibitor VI. Instead, we identified phosphatidylinositol 3-kinase (PtdIns3K) as a direct target of KU55933 and Gö6976. Importantly, and in contrast to the currently available inhibitors of autophagosome formation (e.g. 3-methyladenine), none of the three compounds inhibited the cell survival promoting class I phosphoinositide 3-kinase-Akt signaling at the concentrations required for effective autophagy inhibition. Accordingly, they proved to be valuable tools for investigations of autophagy-associated cell death and survival. Employing KU55399, we demonstrated that autophagy protects amino acid-starved cells against both apoptosis and necroptosis. Taken together, our data introduce new possibilities for the experimental study of autophagy and can form a basis for the development of clinically relevant autophagy inhibitors.

  9. Inhibition of neointimal formation by trans-resveratrol: role of phosphatidyl inositol 3-kinase-dependent Nrf2 activation in heme oxygenase-1 induction.

    PubMed

    Kim, Jung Woo; Lim, Sung Chul; Lee, Moo Yeol; Lee, Jeong Woon; Oh, Won Keun; Kim, Sang Kyum; Kang, Keon Wook

    2010-10-01

    Neointima, defined as abnormal growth of the intimal layer of blood vessels, is believed to be a critical event in the development of vascular occlusive disease. Although resveratrol's inhibitory effects on proliferation and migration of vascular smooth muscle cells has been reported, its activity on neointimal formation is still unclear. Oral administration of trans-resveratrol significantly suppressed intimal hyperplasia in a wire-injured femoral artery mouse model. In cultured vascular smooth muscle cells, trans-resveratrol inhibited platelet-derived growth factor-stimulated DNA synthesis and cell proliferation with down-regulation of cyclin D and pRB. Moreover, platelet-derived growth factor-induced production of reactive oxygen species was inhibited by trans-resveratrol and the compound induced heme oxygenase-1 (HO-1). The anti-proliferative activity of trans-resveratrol was reversed by an HO-1 inhibitor, ZnPPIX. Subcellular fractionation and reporter gene analyses revealed that trans-resveratrol increased the level of nuclear Nrf2 and antioxidant response element reporter activity, and that these were essential for the induction of HO-1. Trans-resveratrol also enhanced the activities of phosphatidyl inositol 3-kinase and extracellular signal regulated kinase, and phosphatidyl inositol 3-kinase was required for Nrf2/antioxidant response element-dependent HO-1 induction. These data have significant implications for the elucidation of the pharmacological mechanism by which trans-resveratrol prevents vascular occlusive diseases.

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

    PubMed Central

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

    1997-01-01

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

  11. Inhibition of PI-3 kinase for treating respiratory disease: good idea or bad idea?

    PubMed

    Thomas, Matt; Owen, Charles

    2008-06-01

    Inhibition of one or more members of the phosphoinositide 3-kinase (PI3K) family for the treatment of respiratory diseases remains the goal of many pharmaceutical companies over the past 20 years. Here we briefly review the PI3K family, then focus on the assessment of each isoform as a drug discovery target. The rationale for PI3Kalpha inhibition in the treatment of lung cancer, and PI3Kbeta inhibitors in pulmonary thrombotic processes, are balanced with a potential side effect profile affecting metabolism and/or foetal development. Roles for PI3Kdelta in inflammatory lung diseases and PI3Kgamma in asthma are weighed against the consequences of manipulating key immune cell populations. We also discuss the current status and future potential of PI3K inhibitors in respiratory disease.

  12. Targeting the Phosphatidylinositol-3-kinase Pathway in Gastric Cancer: Can Omics Improve Outcomes?

    PubMed Central

    Tran, Phu; Nguyen, Cham

    2016-01-01

    Phosphatidylinositol-3-kinase (PI3K) pathway signaling is an established oncogenic signal transduction pathway implicated in multiple malignancies. Therapeutic targeting of PI3K pathway components has improved outcomes in chronic lymphocytic leukemia, kidney cancer, breast cancer, and neuroendocrine tumors. Gastric cancers harbor some of the highest rates of oncogenic alterations in PI3K but attempts to translate this genomic observation have met with limited clinical success and novel approaches are needed. In the following review we discuss PI3K signaling, previous preclinical and clinical investigations in gastric cancer, and discuss future strategies aimed at overcoming resistance and improving efficacy. Identification and refinement of molecular tumor subtypes, development of predictive biomarkers along, and rational drug combination strategies are key to capitalizing on the therapeutic potential of PI3K pathway directed therapies in gastric cancers. PMID:27915478

  13. Measurement of phosphoinositide 3-kinase products in cultured Mammalian cells by HPLC.

    PubMed

    Cooke, Frank T

    2010-01-01

    The phosphoinositide 3-kinase (PI3K) family catalyses the addition of a phosphate group to the D-3 position of polyphosphoinositides (PPIn). Since the discovery in the late 80s that phosphatidylinositol is phosphorylated in the D-3 position in eukaryotic cells, there has been an explosion of interest in these PPIn. Although the four D-3 PPIn (phosphatidylinositol 3-phophate (PtdIns3P), PtdIns(3,4)P(2), PtdIns(3,5)P(2), and PtdIns(3,4,5)P(3)) represent only a small proportion of PPIn, production of D-3 PPIn is required for an ever-increasing number of processes. Measurement of the PPIn levels in intact cells cultured cells has been vital to our understanding of the metabolism and function of these important signalling molecules; methods are described herein that allow measurement of PPIn levels in cultured cells, with emphasis on the 3-OH PPIn.

  14. 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.

  15. LINGO-1 receptor promotes neuronal apoptosis by inhibiting WNK3 kinase activity.

    PubMed

    Zhang, Zhaohuan; Xu, Xiaohui; Xiang, Zhenghua; Yu, Zhongwang; Feng, Jifeng; He, Cheng

    2013-04-26

    LINGO-1 is a functional component of the Nogo receptor 1 · p75(NTR) · LINGO-1 and Nogo receptor 1 · TAJ (TNFRSF19/TROY)·LINGO-1 signaling complexes. It has recently been shown that LINGO-1 antagonists significantly improve neuronal survival after neural injury. However, the mechanism by which LINGO-1 signaling influences susceptibility to apoptosis remains unknown. In an effort to better understand how LINGO-1 regulates these signaling pathways, we used an established model of serum deprivation (SD) to induce neuronal apoptosis. We demonstrate that treatment either with a construct containing the intracellular domain of LINGO-1 or with Nogo66, a LINGO-1 receptor complex agonist, resulted in an enhanced rate of apoptosis in primary cultured cortical neurons under SD. Reducing the expression levels of the serine/threonine kinase WNK3 using shRNA or inhibiting its kinase activity had similar effects on the survival of serum-deprived neurons. Consistent with these observations, we found that LINGO-1 and WNK3 co-localized and co-precipitated in cultured cortical neurons and brain tissue. Significantly, this co-association was enhanced by Nogo66 treatment. Binding of WNK3 to the intracellular domain of LINGO-1 led to a reduction in WNK3 kinase activity, as did Nogo66 stimulation. Moreover, in vitro and in vivo evidence indicates that endogenous WNK3 suppresses SD-induced neuronal apoptosis in a kinase-dependent manner, as the expression of either a WNK3 RNAi construct or a kinase-dead N-terminal fragment of WNK3 led to increased apoptosis. Taken together, our results show that LINGO-1 potentiates neuronal apoptosis, likely by inhibiting WNK3 kinase activity.

  16. FgSsn3 kinase, a component of the mediator complex, is important for sexual reproduction and pathogenesis in Fusarium graminearum

    PubMed Central

    Cao, Shulin; Zhang, Shijie; Hao, Chaofeng; Liu, Huiquan; Xu, Jin-Rong; Jin, Qiaojun

    2016-01-01

    Fusarium graminearum is an important pathogen of wheat and barley. In addition to severe yield losses, infested grains are often contaminated with harmful mycotoxins. In this study, we characterized the functions of FgSSN3 kinase gene in different developmental and infection processes and gene regulation in F. graminearum. The FgSSN3 deletion mutant had a nutrient-dependent growth defects and abnormal conidium morphology. It was significantly reduced in DON production, TRI gene expression, and virulence. Deletion of FgSSN3 also resulted in up-regulation of HTF1 and PCS1 expression in juvenile cultures, and repression of TRI genes in DON-producing cultures. In addition, Fgssn3 was female sterile and defective in hypopodium formation and infectious growth. RNA-seq analysis showed that FgSsn3 is involved in the transcriptional regulation of a wide variety genes acting as either a repressor or activator. FgSsn3 physically interacted with C-type cyclin Cid1 and the cid1 mutant had similar phenotypes with Fgssn3, indicating that FgSsn3 and Cid1 form the CDK-cyclin pair as a component of the mediator complex in F. graminearum. Taken together, our results indicate that FgSSN3 is important for secondary metabolism, sexual reproduction, and plant infection, as a subunit of mediator complex contributing to transcriptional regulation of diverse genes. PMID:26931632

  17. Frequent PTEN genomic alterations and activated phosphatidylinositol 3-kinase pathway in basal-like breast cancer cells

    PubMed Central

    Marty, Bérengère; Maire, Virginie; Gravier, Eléonore; Rigaill, Guillem; Vincent-Salomon, Anne; Kappler, Marion; Lebigot, Ingrid; Djelti, Fathia; Tourdès, Audrey; Gestraud, Pierre; Hupé, Philippe; Barillot, Emmanuel; Cruzalegui, Francisco; Tucker, Gordon C; Stern, Marc-Henri; Thiery, Jean-Paul; Hickman, John A; Dubois, Thierry

    2008-01-01

    Introduction Basal-like carcinomas (BLCs) and human epidermal growth factor receptor 2 overexpressing (HER2+) carcinomas are the subgroups of breast cancers that have the most aggressive clinical behaviour. In contrast to HER2+ carcinomas, no targeted therapy is currently available for the treatment of patients with BLCs. In order to discover potential therapeutic targets, we aimed to discover deregulated signalling pathways in human BLCs. Methods In this study, we focused on the oncogenic phosphatidylinositol 3-kinase (PI3K) pathway in 13 BLCs, and compared it with a control series of 11 hormonal receptor negative- and grade III-matched HER2+ carcinomas. The two tumour populations were first characterised by immunohistochemistry and gene expression. The PI3K pathway was then investigated by gene copy-number analysis, gene expression profiling and at a proteomic level using reverse-phase protein array technology and tissue microarray. The effects of the PI3K inhibition pathway on proliferation and apoptosis was further analysed in three human basal-like cell lines. Results The PI3K pathway was found to be activated in BLCs and up-regulated compared with HER2+ tumours as shown by a significantly increased activation of the downstream targets Akt and mTOR (mammalian target of rapamycin). BLCs expressed significantly lower levels of the tumour suppressor PTEN and PTEN levels were significantly negatively correlated with Akt activity within that population. PTEN protein expression correlated significantly with PTEN DNA copy number and more importantly, reduced PTEN DNA copy numbers were observed specifically in BLCs. Similar to human samples, basal-like cell lines exhibited an activation of PI3K/Akt pathway and low/lack PTEN expression. Both PI3K and mTOR inhibitors led to basal-like cell growth arrest. However, apoptosis was specifically observed after PI3K inhibition. Conclusions These data provide insight into the molecular pathogenesis of BLCs and implicate the

  18. Sphingosine-1-phosphate stimulates human glioma cell proliferation through Gi-coupled receptors: role of ERK MAP kinase and phosphatidylinositol 3-kinase beta.

    PubMed

    Van Brocklyn, James; Letterle, Catherine; Snyder, Pamela; Prior, Thomas

    2002-07-26

    The regulation of glioma cell proliferation by sphingosine-1-phosphate (S1P) was studied using the human glioblastoma cell line U-373 MG. U-373 MG cells responded mitogenically to nanomolar concentrations of S1P, and express mRNA encoding the S1P receptors S1P1/endothelial differentiation gene (EDG)-1, S1P3/EDG-3 and S1P2/EDG-5. S1P-induced proliferation required extracellular signal-regulated kinase activation and was partially sensitive to pertussis toxin and wortmannin, indicating involvement of a Gi-coupled receptor and phosphatidylinositol 3-kinase. Moreover, S1P1, S1P3 and S1P2 receptors are expressed in the majority of human glioblastomas as determined by reverse transcriptase-polymerase chain reaction analysis. Thus, S1P signaling through EDG receptors may contribute to glioblastoma growth in vivo.

  19. Chemokine-cytokine cross-talk. The ELR+ CXC chemokine LIX (CXCL5) amplifies a proinflammatory cytokine response via a phosphatidylinositol 3-kinase-NF-kappa B pathway.

    PubMed

    Chandrasekar, Bysani; Melby, Peter C; Sarau, Henry M; Raveendran, Muthuswamy; Perla, Rao P; Marelli-Berg, Federica M; Dulin, Nickolai O; Singh, Ishwar S

    2003-02-14

    It is well established that cytokines can induce the production of chemokines, but the role of chemokines in the regulation of cytokine expression has not been fully investigated. Exposure of rat cardiac-derived endothelial cells (CDEC) to lipopolysaccharide-induced CXC chemokine (LIX), and to a lesser extent to KC and MIP-2, activated NF-kappaB and induced kappaB-driven promoter activity. LIX did not activate Oct-1. LIX-induced interleukin-1beta and tumor necrosis factor-alpha promoter activity, and up-regulated mRNA expression. Increased transcription and mRNA stability both contributed to cytokine expression. LIX-mediated cytokine gene transcription was inhibited by interleukin-10. Transient overexpression of kinase-deficient NF-kappaB-inducing kinase (NIK) and IkappaB kinase (IKK), and dominant negative IkappaB significantly inhibited LIX-mediated NF-kappaB activation in rat CDEC. Inhibition of G(i) protein-coupled signal transduction, poly(ADP-ribose) polymerase, phosphatidylinositol 3-kinase, and the 26 S proteasome significantly inhibited LIX-mediated NF-kappaB activation and cytokine gene transcription. Blocking CXCR2 attenuated LIX-mediated kappaB activation and kappaB-driven promoter activity in rat CDEC that express both CXCR1 and -2, and abrogated its activation in mouse CDEC that express only CXCR2. These results indicate that LIX activates NF-kappaB and induces kappaB-responsive proinflammatory cytokines via either CXCR1 or CXCR2, and involved phosphatidylinositol 3-kinase, NIK, IKK, and IkappaB. Thus, in addition to attracting and activating neutrophils, the ELR(+) CXC chemokines amplify the inflammatory cascade, stimulating local production of cytokines that have negative inotropic and proapoptotic effects.

  20. Regulation of phosphatidylinositol 3-kinase activity in liver and muscle of animal models of insulin-resistant and insulin-deficient diabetes mellitus.

    PubMed Central

    Folli, F; Saad, M J; Backer, J M; Kahn, C R

    1993-01-01

    Insulin stimulates tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), which in turn binds to and activates phosphatidylinositol 3-kinase (PI 3-kinase). In the present study, we have examined these processes in animal models of insulin-resistant and insulin-deficient diabetes mellitus. After in vivo insulin stimulation, there was a 60-80% decrease in IRS-1 phosphorylation in liver and muscle of the ob/ob mouse. There was no insulin stimulation of PI 3-kinase (85 kD subunit) association with IRS-1, and IRS-1-associated PI 3-kinase activity was reduced 90%. Insulin-stimulated total PI 3-kinase activity was also absent in both tissues of the ob/ob mouse. By contrast, in the streptozotocin diabetic rat, IRS-1 phosphorylation increased 50% in muscle, IRS-1-associated PI 3-kinase activity was increased two- to threefold in liver and muscle, and there was a 50% increase in the p85 associated with IRS-1 after insulin stimulation in muscle. In conclusion, (a) IRS-1-associated PI 3-kinase activity is differentially regulated in hyperinsulinemic and hypoinsulinemic diabetic states; (b) PI 3-kinase activation closely correlates with IRS-1 phosphorylation; and (c) reduced PI 3-kinase activity may play a role in the pathophysiology of insulin resistant diabetic states, such as that seen in the ob/ob mouse. Images PMID:7691886

  1. Class A scavenger receptor-mediated cell adhesion requires the sequential activation of Lyn and PI3-kinase.

    PubMed

    Nikolic, Dejan M; Cholewa, Jill; Gass, Cecelia; Gong, Ming C; Post, Steven R

    2007-04-01

    Class A scavenger receptors (SR-A) participate in multiple macrophage functions including macrophage adhesion to modified proteins. SR-A-mediated adhesion may therefore contribute to chronic inflammation by promoting macrophage accumulation at sites of protein modification. The mechanisms that couple SR-A binding to modified proteins with increased cell adhesion have not been defined. In this study, SR-A expressing HEK cells and SR-A+/+ or SR-A-/- macrophages were used to delineate the signaling pathways required for SR-A-mediated adhesion to modified protein. Inhibiting G(i/o) activation, which decreases initial SR-A-mediated cell attachment, did not prevent the subsequent spreading of attached cells. In contrast, inhibition of Src kinases or PI3-kinase abolished SR-A-dependent cell spreading without affecting SR-A-mediated cell attachment. Consistent with these results, the Src kinase Lyn and PI3-kinase were sequentially activated during SR-A-mediated cell spreading. Furthermore, activation of both Lyn and PI3-kinase was required for enhancing paxillin phosphorylation. Activation of a Src kinase-PI3-kinase-Akt pathway was also observed in cells expressing a truncated SR-A protein that does not internalize indicating that SR-A-mediated activation of intracellular signaling cascades following adhesion to MDA-BSA is independent of receptor internalization. Thus SR-A binding to modified protein activates signaling cascades that have distinct roles in regulating initial cell attachment and subsequent cell spreading.

  2. Kinetic analysis of platelet-derived growth factor receptor/phosphoinositide 3-kinase/Akt signaling in fibroblasts.

    PubMed

    Park, Chang Shin; Schneider, Ian C; Haugh, Jason M

    2003-09-26

    Isoforms of the serine-threonine kinase Akt coordinate multiple cell survival pathways in response to stimuli such as platelet-derived growth factor (PDGF). Activation of Akt is a multistep process, which relies on the production of 3'-phosphorylated phosphoinositide (PI) lipids by PI 3-kinases. To quantitatively assess the kinetics of PDGF receptor/PI 3-kinase/Akt signaling in fibroblasts, a systematic study of this pathway was performed, and a mechanistic mathematical model that describes its operation was formulated. We find that PDGF receptor phosphorylation exhibits positive cooperativity with respect to PDGF concentration, and its kinetics are quantitatively consistent with a mechanism in which receptor dimerization is initially mediated by the association of two 1:1 PDGF/PDGF receptor complexes. Receptor phosphorylation is transient at high concentrations of PDGF, consistent with the loss of activated receptors upon endocytosis. By comparison, Akt activation responds to lower PDGF concentrations and exhibits more sustained kinetics. Further analysis and modeling suggest that the pathway is saturated at the level of PI 3-kinase activation, and that the p110alpha catalytic subunit of PI 3-kinase contributes most to PDGF-stimulated 3'-PI production. Thus, at high concentrations of PDGF the kinetics of 3'-PI production are limited by the turnover rate of these lipids, while the Akt response is additionally influenced by the rate of Akt deactivation.

  3. Phosphatidylinositol 3-kinase mediates the ability of retinol to decrease colorectal cancer cell invasion.

    PubMed

    Lengyel, Jennifer N Griffin; Park, Eun Young; Brunson, Anna R; Pinali, Daniel; Lane, Michelle A

    2014-01-01

    Previously, we showed that retinol (vitamin A) decreased both colorectal cancer cell invasion and phosphatidylinositol 3-kinase (PI3K) activity through a retinoic acid receptor-independent mechanism. Here, we determined if these phenomena were related by using parental HCT-116 cells that harbor 1 allele of wild-type PI3K and 1 allele of constitutively active (ca) PI3K and 2 mutant HCT-116 cell lines homozygous for caPI3K. In vitro, treatment of parental HCT-116 cells with 10 μM retinol reduced cell invasion whereas treatment of mutant HCT-116 cell lines with retinol did not. Treatment with 10 μM retinol also decreased the activity of matrixmetalloproteinase-9 and increased tissue inhibitor of matrixmetalloproteinase-I levels in parental, but not mutant, HCT-116 cells. Finally, parental or mutant cells were intrasplenically injected into athymic mice consuming diets with or without supplemental vitamin A. As expected, vitamin A supplementation tended (P = 0.18) to reduce the incidence of metastases in mice injected with the parental cell line and consuming the supplemented diet. In contrast, metastatic incidence was not affected (P = 1.00) by vitamin A supplementation in mice injected with mutant cells. These data indicate that the capacity of retinol to inhibit PI3K activity confers its ability to decrease colorectal cancer metastasis.

  4. ERK kinases modulate the activation of PI3 kinase related kinases (PIKKs) in DNA damage response.

    PubMed

    Lin, Xiaozeng; Yan, Judy; Tang, Damu

    2013-12-01

    DNA damage response (DDR) is the critical surveillance mechanism in maintaining genome integrity. The mechanism activates checkpoints to prevent cell cycle progression in the presence of DNA lesions, and mediates lesion repair. DDR is coordinated by three apical PI3 kinase related kinases (PIKKs), including ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), and DNA-PKcs (the catalytic subunit of the DNA dependent protein kinase). These kinases are activated in response to specific DNA damage or lesions, resulting in checkpoint activation and DNA lesion repair. While it is clear that the pathways of ATM, ATR, and DNA-PK are the core components of DDR, there is accumulating evidence revealing the involvement of other cellular pathways in regulating DDR; this is in line with the concept that in addition to being a nuclear event DDR is also a cellular process. One of these pathways is the extracellular signal-regulated kinase (ERK) MAPK (mitogen-activated protein kinase) pathway. ERK is a converging point of multiple signal transduction pathways involved in cell proliferation, differentiation, and apoptosis. Adding to this list of pathways is the recent development of ERK in DDR. The ERK kinases (ERK1 and ERK2) contribute to the proper execution of DDR in terms of checkpoint activation and the repair of DNA lesions. This review summarizes the contributions of ERK to DDR with emphasis on the relationship of ERK kinases with the activation of ATM, ATR, and DNA-PKcs.

  5. 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

  6. Diosgenin inhibits melanogenesis through the activation of phosphatidylinositol-3-kinase pathway (PI3K) signaling.

    PubMed

    Lee, Jongsung; Jung, Kwangseon; Kim, Yeong Shik; Park, Deokhoon

    2007-06-27

    An increased level of melanin is characteristic of a large number of skin diseases, including acquired hyperpigmentation conditions such as melasma, post inflammatory melanoderma, and solar lentigo. Thus, there is an increasing need for the development of depigmenting agents. In order to evaluate the depigmenting capacity of diosgenin and elucidate its mechanism of action, several experiments were performed in B16 melanoma cells. Melanin content and Western blots for proteins that are involved in melanogenesis were assessed in this study. The melanin content was significantly inhibited by diosgenin. To clarify the mechanism of the depigmenting property of diosgenin, we examined the involvement of diosgenin in the phosphatidylinositol-3-kinase (PI3K) pathway. In this study, diosgenin inhibited the reduction of Akt and GSK 3beta phosphorylation induced by LY294,002, a PI3K inhibitor. In accordance with this result, production levels of MITF (microphthalmia-associated transcription factor) and tyrosinase were increased by diosgenin. These data suggest that diosgenin inhibits melanogenesis through the activation of the PI3K pathway. This suggestion was further confirmed by the fact that the increased production level of melanin by LY294,002 was reduced by diosgenin in B16 melanoma cells. Our study shows that diosgenin inhibits melanogenesis by activating the PI3K pathway, and also suggests that diosgenin may be an effective inhibitor of hyperpigmentation.

  7. Vav3 modulates B cell receptor responses by regulating phosphoinositide 3-kinase activation.

    PubMed

    Inabe, Kazunori; Ishiai, Masamichi; Scharenberg, Andrew M; Freshney, Norman; Downward, Julian; Kurosaki, Tomohiro

    2002-01-21

    To elucidate the mechanism(s) by which Vav3, a new member of the Vav family proteins, participates in B cell antigen receptor (BCR) signaling, we have generated a B cell line deficient in Vav3. Here we report that Vav3 influences phosphoinositide 3-kinase (PI3K) function through Rac1 in that phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation was attenuated by loss of Vav3 or by expression of a dominant negative form of Rac1. The functional interaction between PI3K and Rac1 was also demonstrated by increased PI3K activity in the presence of GTP-bound Rac1. In addition, we show that defects of calcium mobilization and c-Jun NH2-terminal kinase (JNK) activation in Vav3-deficient cells are relieved by deletion of a PIP3 hydrolyzing enzyme, SH2 domain-containing inositol polyphosphate 5'-phosphatase (SHIP). Hence, our results suggest a role for Vav3 in regulating the B cell responses by promoting the sustained production of PIP3 and thereby calcium flux.

  8. Vav3 Modulates B Cell Receptor Responses by Regulating Phosphoinositide 3-Kinase Activation

    PubMed Central

    Inabe, Kazunori; Ishiai, Masamichi; Scharenberg, Andrew M.; Freshney, Norman; Downward, Julian; Kurosaki, Tomohiro

    2002-01-01

    To elucidate the mechanism(s) by which Vav3, a new member of the Vav family proteins, participates in B cell antigen receptor (BCR) signaling, we have generated a B cell line deficient in Vav3. Here we report that Vav3 influences phosphoinositide 3-kinase (PI3K) function through Rac1 in that phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation was attenuated by loss of Vav3 or by expression of a dominant negative form of Rac1. The functional interaction between PI3K and Rac1 was also demonstrated by increased PI3K activity in the presence of GTP-bound Rac1. In addition, we show that defects of calcium mobilization and c-Jun NH2-terminal kinase (JNK) activation in Vav3-deficient cells are relieved by deletion of a PIP3 hydrolyzing enzyme, SH2 domain-containing inositol polyphosphate 5′-phosphatase (SHIP). Hence, our results suggest a role for Vav3 in regulating the B cell responses by promoting the sustained production of PIP3 and thereby calcium flux. PMID:11805146

  9. IPD-196, a novel phosphatidylinositol 3-kinase inhibitor with potent anticancer activity against hepatocellular carcinoma.

    PubMed

    Lee, Ju-Hee; Lee, Hyunseung; Yun, Sun-Mi; Jung, Kyung Hee; Jeong, Yujeong; Yan, Hong Hua; Hong, Sungwoo; Hong, Soon-Sun

    2013-02-01

    As the activation of phosphatidylinositol 3-kinase (PI3K) is associated with a wide variety of human malignancies, it is emerging as an attractive target for cancer treatment. In this study we synthesized a novel PI3Kα inhibitor, IPD-196 [ethyl 6-(5-(2,4-difluorophenylsulfonamido)pyridin-3-yl)imidazo[1,2-a]pyridine-3-carboxylate], and evaluated its anticancer effects on human hepatocellular carcinoma (HCC) cells. IPD-196 effectively inhibited the phosphorylation of downstream PI3K effectors such as Akt, mTOR, p70S6K, and 4E-BP1, and its antiproliferative effect was more potent than that of sorafenib or LY294002. It also induced cell cycle arrest at the G0/G1 phase as well as apoptosis by increasing the proportion of sub-G1 apoptotic cells, and the levels of cleaved PARP, caspase-3, and caspase-9. Furthermore, it decreased the expression of HIF-1α and VEGF in Huh-7 cells, and inhibited tube formation and migration of human umbilical vein endothelial cells, which was confirmed by a Matrigel plug assay in mice. Taken together, IPD-196 exhibited its anticancer activity through disruption of the PI3K/Akt pathway that caused cell cycle arrest, apoptosis induction, and inhibition of angiogenesis in human HCC cells. We therefore suggest that IPD-196 may be a potential candidate drug for targeted HCC therapy.

  10. Phosphoinositide-3-kinases as the novel therapeutic targets for the inflammatory diseases: Current and future perspectives.

    PubMed

    Vyas, Preeti; Vohora, Divya

    2016-10-13

    Recent findings have publicized phosphoinositide-3-kinases (PI3Ks) as novel therapeutic targets, which are also purported to be involved in the complex pathophysiology of inflammatory and various other diseases. They are recognized to participate in the inflammatory cellular responses by modulating the growth, development and proliferation of various immune cells and hence, affect the release of various cytokines and other inflammatory mediators involved in these manifestations. The review presents a brief synopsis of the PI3K/AKT/mTOR signalling pathway along with the current and future prospects of targeting PI3Ks for various diseases, like malignant, autoimmune, inflammatory, cardiovascular, neurological disorders etc., laying special emphasis on the inflammatory diseases and associated cellular responses. The recent literature relating this pathway with these diseases is highlighted, with a hope, which remains for the progression of PI3K inhibitors in the market as a treatment option. With Idelalisib entering the market for cancer, PI3K/AKT signalling has also gained significance as an investigational target for other diseases, particularly for inflammation. Further exploration of this pathway may also uncover its involvement in these disorders, which may further contribute to developing the new treatments and can turn out to be an innovative brainwave in the field of experimental and clinical pharmacology in future.

  11. Nuclear but Not Cytosolic Phosphoinositide 3-Kinase Beta Has an Essential Function in Cell Survival ▿

    PubMed Central

    Kumar, Amit; Redondo-Muñoz, Javier; Perez-García, Vicente; Cortes, Isabel; Chagoyen, Monica; Carrera, Ana C.

    2011-01-01

    Class IA phosphoinositide 3-kinases (PI3Ks) are heterodimeric enzymes composed of a p85 regulatory and a p110 catalytic subunit that induce the formation of 3-polyphosphoinositides, which mediate cell survival, division, and migration. There are two ubiquitous PI3K isoforms p110α and p110β that have nonredundant functions in embryonic development and cell division. However, whereas p110α concentrates in the cytoplasm, p110β localizes to the nucleus and modulates nuclear processes such as DNA replication and repair. At present, the structural features that determine p110β nuclear localization remain unknown. We describe here that association with the p85β regulatory subunit controls p110β nuclear localization. We identified a nuclear localization signal (NLS) in p110β C2 domain that mediates its nuclear entry, as well as a nuclear export sequence (NES) in p85β. Deletion of p110β induced apoptosis, and complementation with the cytoplasmic C2-NLS p110β mutant was unable to restore cell survival. These studies show that p110β NLS and p85β NES regulate p85β/p110β nuclear localization, supporting the idea that nuclear, but not cytoplasmic, p110β controls cell survival. PMID:21383062

  12. Phosphoinositide 3-kinase beta controls replication factor C assembly and function

    PubMed Central

    Redondo-Muñoz, Javier; Josefa Rodríguez, María; Silió, Virginia; Pérez-García, Vicente; María Valpuesta, José; Carrera, Ana C.

    2013-01-01

    Genomic integrity is preserved by the action of protein complexes that control DNA homeostasis. These include the sliding clamps, trimeric protein rings that are arranged around DNA by clamp loaders. Replication factor C (RFC) is the clamp loader for proliferating cell nuclear antigen, which acts on DNA replication. Other processes that require mobile contact of proteins with DNA use alternative RFC complexes that exchange RFC1 for CTF18 or RAD17. Phosphoinositide 3-kinases (PI3K) are lipid kinases that generate 3-poly-phosphorylated-phosphoinositides at the plasma membrane following receptor stimulation. The two ubiquitous isoforms, PI3Kalpha and PI3Kbeta, have been extensively studied due to their involvement in cancer and nuclear PI3Kbeta has been found to regulate DNA replication and repair, processes controlled by molecular clamps. We studied here whether PI3Kbeta directly controls the process of molecular clamps loading. We show that PI3Kbeta associated with RFC1 and RFC1-like subunits. Only when in complex with PI3Kbeta, RFC1 bound to Ran GTPase and localized to the nucleus, suggesting that PI3Kbeta regulates RFC1 nuclear import. PI3Kbeta controlled not only RFC1– and RFC–RAD17 complexes, but also RFC–CTF18, in turn affecting CTF18-mediated chromatid cohesion. PI3Kbeta thus has a general function in genomic stability by controlling the localization and function of RFC complexes. PMID:23175608

  13. Cell Activation-Induced Phosphoinositide 3-Kinase Alpha/Beta Dimerization Regulates PTEN Activity

    PubMed Central

    Pérez-García, Vicente; Redondo-Muñoz, Javier; Kumar, Amit

    2014-01-01

    The phosphoinositide 3-kinase (PI3K)/PTEN (phosphatase and tensin homolog) pathway is one of the central routes that enhances cell survival, division, and migration, and it is frequently deregulated in cancer. PI3K catalyzes formation of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] after cell activation; PTEN subsequently reduces these lipids to basal levels. Activation of the ubiquitous p110α isoform precedes that of p110β at several points during the cell cycle. We studied the potential connections between p110α and p110β activation, and we show that cell stimulation promotes p110α and p110β association, demonstrating oligomerization of PI3K catalytic subunits within cells. Cell stimulation also promoted PTEN incorporation into this complex, which was necessary for PTEN activation. Our results show that PI3Ks dimerize in vivo and that PI3K and PTEN activities modulate each other in a complex that controls cell PI(3,4,5)P3 levels. PMID:24958106

  14. Phosphoinositide 3-Kinase Beta Protects Nuclear Envelope Integrity by Controlling RCC1 Localization and Ran Activity

    PubMed Central

    Redondo-Muñoz, Javier; Pérez-García, Vicente; Rodríguez, María J.; Valpuesta, José M.

    2014-01-01

    The nuclear envelope (NE) forms a barrier between the nucleus and the cytosol that preserves genomic integrity. The nuclear lamina and nuclear pore complexes (NPCs) are NE components that regulate nuclear events through interaction with other proteins and DNA. Defects in the nuclear lamina are associated with the development of laminopathies. As cells depleted of phosphoinositide 3-kinase beta (PI3Kβ) showed an aberrant nuclear morphology, we studied the contribution of PI3Kβ to maintenance of NE integrity. pik3cb depletion reduced the nuclear membrane tension, triggered formation of areas of lipid bilayer/lamina discontinuity, and impaired NPC assembly. We show that one mechanism for PI3Kβ regulation of NE/NPC integrity is its association with RCC1 (regulator of chromosome condensation 1), the activator of nuclear Ran GTPase. PI3Kβ controls RCC1 binding to chromatin and, in turn, Ran activation. These findings suggest that PI3Kβ regulates the nuclear envelope through upstream regulation of RCC1 and Ran. PMID:25348717

  15. 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

  16. Creatine inhibits adipogenesis by downregulating insulin-induced activation of the phosphatidylinositol 3-kinase signaling pathway.

    PubMed

    Lee, Nayeon; Kim, Inhee; Park, Soojeong; Han, Dasol; Ha, Soobong; Kwon, Mookwang; Kim, Juwan; Byun, Sung-Hyun; Oh, Wonil; Jeon, Hong Bae; Kweon, Dae-Hyuk; Cho, Jae Youl; Yoon, Keejung

    2015-04-15

    Creatine is a nitrogenous organic acid known to function in adenosine triphosphate (ATP) metabolism. Recent evidence indicates that creatine regulates the differentiation of mesenchymal stem cells (MSCs) in processes such as osteogenesis and myogenesis. In this study, we show that creatine also has a negative regulatory effect on fat cell formation. Creatine inhibits the accumulation of cytoplasmic triglycerides in a dose-dependent manner irrespective of the adipogenic cell models used, including a C3H10T1/2 MSC line, 3T3-L1 preadipocytes, and primary human MSCs. Consistently, a dramatic reduction in mRNA expression of adipogenic transcription factors, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), glucose transporters, 1 and 4 (Glut1, Glut4), and adipocyte markers, aP2 and adipsin, was observed in the presence of creatine. Creatine appears to exert its inhibitory effects on adipogenesis during early differentiation, but not late differentiation, or proliferation stages through inhibition of the PI3K-Akt-PPARγ signaling pathway. In an in vivo model, administration of creatine into mice resulted in body mass increase without fat accumulation. In summary, our results indicate that creatine downregulates adipogenesis through inhibition of phosphatidylinositol 3-kinase (PI3K) activation and imply the potent therapeutic value of creatine in treating obesity and obesity-related metabolic disorders.

  17. Dynamics and architecture of the NRBF2-containing phosphatidylinositol 3-kinase complex I of autophagy

    PubMed Central

    Young, Lindsey N.; Cho, Kelvin; Lawrence, Rosalie; Zoncu, Roberto; Hurley, James H.

    2016-01-01

    The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is central to autophagy initiation. We previously reported the V-shaped architecture of the four-subunit version of PI3KC3-C1 consisting of VPS (vacuolar protein sorting) 34, VPS15, BECN1 (Beclin 1), and ATG (autophagy-related) 14. Here we show that a putative fifth subunit, nuclear receptor binding factor 2 (NRBF2), is a tightly bound component of the complex that profoundly affects its activity and architecture. NRBF2 enhances the lipid kinase activity of the catalytic subunit, VPS34, by roughly 10-fold. We used hydrogen–deuterium exchange coupled to mass spectrometry and negative-stain electron microscopy to map NRBF2 to the base of the V-shaped complex. NRBF2 interacts primarily with the N termini of ATG14 and BECN1. We show that NRBF2 is a homodimer and drives the dimerization of the larger PI3KC3-C1 complex, with implications for the higher-order organization of the preautophagosomal structure. PMID:27385829

  18. Biliverdin Reductase Mediates Hypoxia-Induced EMT via PI3-Kinase and Akt

    PubMed Central

    Zeng, Rui; Yao, Ying; Han, Min; Zhao, Xiaoqin; Liu, Xiao-Cheng; Wei, Juncheng; Luo, Yun; Zhang, Juan; Zhou, Jianfeng; Wang, Shixuan; Ma, Ding; Xu, Gang

    2008-01-01

    Chronic hypoxia in the renal parenchyma is thought to induce epithelial-to-mesenchymal transition (EMT), leading to fibrogenesis and ultimately end-stage renal failure. Biliverdin reductase, recently identified as a serine/threonine/tyrosine kinase that may activate phosphatidylinositol 3-kinase (PI3K) and Akt, is upregulated in response to reactive oxygen species that may accompany hypoxia. We investigated this potential role of biliverdin reductase in hypoxia-induced renal tubular EMT. Expression of biliverdin reductase was upregulated in a human proximal tubule cell line (HK-2) cultured in hypoxic conditions (1% O2), and this was accompanied by reduced expression of E-cadherin and increased expression of the mesenchymal marker vimentin. Inhibiting PI3K reversed these changes, consistent with EMT. In normoxic conditions, overexpression of biliverdin reductase promoted similar characteristics of EMT, which were also reversed by inhibiting PI3K. Furthermore, using small interfering RNA (siRNA) to knockdown biliverdin reductase, we demonstrated that the enzyme associates with phosphorylated Akt and mediates the hypoxia-induced EMT phenotype. In vivo, expression of biliverdin reductase increased in the tubular epithelia of 5/6-nephrectomized rats, and immunohistochemistry of serial sections demonstrated similar localization of phosphorylated Akt and biliverdin reductase. In conclusion, biliverdin reductase mediates hypoxia-induced EMT through a PI3K/Akt-dependent pathway. PMID:18184861

  19. Tyrosol Suppresses Allergic Inflammation by Inhibiting the Activation of Phosphoinositide 3-Kinase in Mast Cells.

    PubMed

    Je, In-Gyu; Kim, Duk-Sil; Kim, Sung-Wan; Lee, Soyoung; Lee, Hyun-Shik; Park, Eui Kyun; Khang, Dongwoo; Kim, Sang-Hyun

    2015-01-01

    Allergic diseases such as atopic dermatitis, rhinitis, asthma, and anaphylaxis are attractive research areas. Tyrosol (2-(4-hydroxyphenyl)ethanol) is a polyphenolic compound with diverse biological activities. In this study, we investigated whether tyrosol has anti-allergic inflammatory effects. Ovalbumin-induced active systemic anaphylaxis and immunoglobulin E-mediated passive cutaneous anaphylaxis models were used for the immediate-type allergic responses. Oral administration of tyrosol reduced the allergic symptoms of hypothermia and pigmentation in both animal models. Mast cells that secrete allergic mediators are key regulators on allergic inflammation. Tyrosol dose-dependently decreased mast cell degranulation and expression of inflammatory cytokines. Intracellular calcium levels and activation of inhibitor of κB kinase (IKK) regulate cytokine expression and degranulation. Tyrosol blocked calcium influx and phosphorylation of the IKK complex. To define the molecular target for tyrosol, various signaling proteins involved in mast cell activation such as Lyn, Syk, phosphoinositide 3-kinase (PI3K), and Akt were examined. Our results showed that PI3K could be a molecular target for tyrosol in mast cells. Taken together, these findings indicated that tyrosol has anti-allergic inflammatory effects by inhibiting the degranulation of mast cells and expression of inflammatory cytokines; these effects are mediated via PI3K. Therefore, we expect tyrosol become a potential therapeutic candidate for allergic inflammatory disorders.

  20. Targeting phosphoinositide 3-kinase δ for the treatment of respiratory diseases.

    PubMed

    Sriskantharajah, Srividya; Hamblin, Nicole; Worsley, Sally; Calver, Andrew R; Hessel, Edith M; Amour, Augustin

    2013-03-01

    Asthma and chronic obstructive pulmonary disease (COPD) are characterized in their pathogenesis by chronic inflammation in the airways. Phosphoinositide 3-kinase δ (PI3Kδ), a lipid kinase expressed predominantly in leukocytes, is thought to hold much promise as a therapeutic target for such inflammatory conditions. Of particular interest for the treatment of severe respiratory disease is the observation that inhibition of PI3Kδ may restore steroid effectiveness under conditions of oxidative stress. PI3Kδ inhibition may also prevent recruitment of inflammatory cells, including T lymphocytes and neutrophils, as well as the release of proinflammatory mediators, such as cytokines, chemokines, reactive oxygen species, and proteolytic enzymes. In addition, targeting the PI3Kδ pathway could reduce the incidence of pathogen-induced exacerbations by improving macrophage-mediated bacterial clearance. In this review, we discuss the potential and highlight the unknowns of targeting PI3Kδ for the treatment of respiratory disease, focusing on recent developments in the role of the PI3Kδ pathway in inflammatory cell types believed to be critical to the pathogenesis of COPD.

  1. Phosphatidylinositol 3-Kinase γ is required for the development of experimental cerebral malaria.

    PubMed

    Lacerda-Queiroz, Norinne; Brant, Fatima; Rodrigues, David Henrique; Vago, Juliana Priscila; Rachid, Milene Alvarenga; Sousa, Lirlândia Pires; Teixeira, Mauro Martins; Teixeira, Antonio Lucio

    2015-01-01

    Experimental cerebral malaria (ECM) is characterized by a strong immune response, with leukocyte recruitment, blood-brain barrier breakdown and hemorrhage in the central nervous system. Phosphatidylinositol 3-kinase γ (PI3Kγ) is central in signaling diverse cellular functions. Using PI3Kγ-deficient mice (PI3Kγ-/-) and a specific PI3Kγ inhibitor, we investigated the relevance of PI3Kγ for the outcome and the neuroinflammatory process triggered by Plasmodium berghei ANKA (PbA) infection. Infected PI3Kγ-/- mice had greater survival despite similar parasitemia levels in comparison with infected wild type mice. Histopathological analysis demonstrated reduced hemorrhage, leukocyte accumulation and vascular obstruction in the brain of infected PI3Kγ-/- mice. PI3Kγ deficiency also presented lower microglial activation (Iba-1+ reactive microglia) and T cell cytotoxicity (Granzyme B expression) in the brain. Additionally, on day 6 post-infection, CD3+CD8+ T cells were significantly reduced in the brain of infected PI3Kγ-/- mice when compared to infected wild type mice. Furthermore, expression of CD44 in CD8+ T cell population in the brain tissue and levels of phospho-IkB-α in the whole brain were also markedly lower in infected PI3Kγ-/- mice when compared with infected wild type mice. Finally, AS605240, a specific PI3Kγ inhibitor, significantly delayed lethality in infected wild type mice. In brief, our results indicate a pivotal role for PI3Kγ in the pathogenesis of ECM.

  2. Phosphoinositide 3-kinase: a new kid on the block in vascular anomalies.

    PubMed

    Castillo, Sandra D; Vanhaesebroeck, Bart; Sebire, Neil J

    2016-12-01

    Vascular anomalies are broadly divided into vascular tumours and malformations. These lesions are composed of abnormal vascular elements of various types, and mainly affect infants, children, and young adults. Vascular anomalies may be painful, may be complicated by bleeding, infection, or organ dysfunction, and can have secondary effects on other tissues. Current treatment strategies include surgical excision, pulsed laser, and sclerotherapy, which are invasive, with risks of recurrence. There are growing pharmacological options for these vascular anomalies, but, to date, no specific targeted therapies have been developed. Phosphoinositide 3-kinases (PI3Ks) constitute a family of lipid kinases that are involved in signal transduction and vesicular traffic, and that modulate important cellular processes such as proliferation, growth, and migration. Recent findings have indicated that the PI3K signalling pathway is important in the pathogenesis of vascular anomalies. This provides an opportunity to use PI3K inhibitors, which are in clinical trials for cancer treatment, for such lesions. Here, we provide an update on the classification of vascular anomalies, with their major features, and discuss the role of the PI3K signalling pathway in the pathogenesis of vascular anomalies, and their clinical implications and therapeutic opportunities. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  3. 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

  4. ZSTK474, a novel phosphatidylinositol 3-kinase inhibitor identified using the JFCR39 drug discovery system.

    PubMed

    Kong, De-xin; Yamori, Takao

    2010-09-01

    JFCR39 is an informatic anticancer drug discovery system that utilizes a panel of 39 human cancer cells coupled with a drug-activity database. This system not only provides disease-oriented information but can also predict the mechanism of action of a given antitumor agent. Development of a phosphatidylinositol 3-kinase (PI3K) inhibitor as an anticancer drug candidate has attracted a great deal of attention from both academia and industry because PI3K is known to be closely involved in carcinogenesis. ZSTK474 was identified as a PI3K inhibitor using JFCR39 system in combination with COMPARE analysis program. These findings were based on the similar fingerprint (growth inhibition profiles for JFCR39 human cancer cell line panel) with that of a classical PI3K inhibitor LY294002. Biochemical experiments confirmed ZSTK474 to be a potent pan-class I PI3K inhibitor, with high selectivity over other classes of PI3K and protein kinases. We previously reported the in vitro and in vivo antitumor efficacy of ZSTK474, together with the G(0)/G(1) arrest and antiangiogenic activity. Here, we review the JFCR39 system and summarize recent studies on PI3K biology and the development of PI3K inhibitors before discussing ZSTK474 in some detail.

  5. RhoG regulates anoikis through a phosphatidylinositol 3-kinase-dependent mechanism

    SciTech Connect

    Yamaki, Nao; Negishi, Manabu; Katoh, Hironori . E-mail: hirokato@pharm.kyoto-u.ac.jp

    2007-08-01

    In normal epithelial cells, cell-matrix interaction is required for cell survival and proliferation, whereas disruption of this interaction causes epithelial cells to undergo apoptosis called anoikis. Here we show that the small GTPase RhoG plays an important role in the regulation of anoikis. HeLa cells are capable of anchorage-independent cell growth and acquire resistance to anoikis. We found that RNA interference-mediated knockdown of RhoG promoted anoikis in HeLa cells. Previous studies have shown that RhoG activates Rac1 and induces several cellular functions including promotion of cell migration through its effector ELMO and the ELMO-binding protein Dock180 that function as a Rac-specific guanine nucleotide exchange factor. However, RhoG-induced suppression of anoikis was independent of the ELMO- and Dock180-mediated activation of Rac1. On the other hand, the regulation of anoikis by RhoG required phosphatidylinositol 3-kinase (PI3K) activity, and constitutively active RhoG bound to the PI3K regulatory subunit p85{alpha} and induced the PI3K-dependent phosphorylation of Akt. Taken together, these results suggest that RhoG protects cells from apoptosis caused by the loss of anchorage through a PI3K-dependent mechanism, independent of its activation of Rac1.

  6. Molecular cloning and biochemical characterization of a Drosophila phosphatidylinositol-specific phosphoinositide 3-kinase.

    PubMed

    Linassier, C; MacDougall, L K; Domin, J; Waterfield, M D

    1997-02-01

    Molecular, biochemical and genetic characterization of phosphoinositide 3-kinases (PI3Ks) have identified distinct classes of enzymes involved in processes mediated by activation of cell-surface receptors and in constitutive intracellular protein trafficking events. The latter process appears to involve a PtdIns-specific PI3K first described in yeast as a mutant, vps34, defective in the sorting of newly synthesized proteins from the Golgi to the vacuole. We have identified a representative member of each class of PI3Ks in Drosophila using a PCR-based approach. In the present paper we describe the molecular cloning of a PI3K from Drosophila, P13K_59F, that shows sequence similarity to Vps34. PI3K_59F encodes a protein of 108 kDa co-linear with Vps34 homologues, and with three regions of sequence similarity to other PI3Ks. Biochemical characterization of the enzyme, by expression of the complete coding sequence as a glutathione S-transferase fusion protein in Sf9 cells, demonstrates that PI3K_59F is a PtdIns-specific PI3K that can utilize either Mg2+ or Mn2+. This activity is sensitive to inhibition both by non-ionic detergent (Nonidet P40) and by wortmannin (IC50 10 nM). PI3K_59F, therefore, conserves both the structural and biochemical properties of the Vps34 class of enzymes.

  7. Involvement of phosphoinositide 3-kinases in neutrophil activation and the development of acute lung injury.

    PubMed

    Yum, H K; Arcaroli, J; Kupfner, J; Shenkar, R; Penninger, J M; Sasaki, T; Yang, K Y; Park, J S; Abraham, E

    2001-12-01

    Activated neutrophils contribute to the development and severity of acute lung injury (ALI). Phosphoinositide 3-kinases (PI3-K) and the downstream serine/threonine kinase Akt/protein kinase B have a central role in modulating neutrophil function, including respiratory burst, chemotaxis, and apoptosis. In the present study, we found that exposure of neutrophils to endotoxin resulted in phosphorylation of Akt, activation of NF-kappaB, and expression of the proinflammatory cytokines IL-1beta and TNF-alpha through PI3-K-dependent pathways. In vivo, endotoxin administration to mice resulted in activation of PI3-K and Akt in neutrophils that accumulated in the lungs. The severity of endotoxemia-induced ALI was significantly diminished in mice lacking the p110gamma catalytic subunit of PI3-K. In PI3-Kgamma(-/-) mice, lung edema, neutrophil recruitment, nuclear translocation of NF-kappaB, and pulmonary levels of IL-1beta and TNF-alpha were significantly lower after endotoxemia as compared with PI3-Kgamma(+/+) controls. Among neutrophils that did accumulate in the lungs of the PI3-Kgamma(-/-) mice after endotoxin administration, activation of NF-kappaB and expression of proinflammatory cytokines was diminished compared with levels present in lung neutrophils from PI3-Kgamma(+/+) mice. These results show that PI3-K, and particularly PI3-Kgamma, occupies a central position in regulating endotoxin-induced neutrophil activation, including that involved in ALI.

  8. Class IA phosphoinositide 3-kinases are obligate p85-p110 heterodimers

    PubMed Central

    Geering, Barbara; Cutillas, Pedro R.; Nock, Gemma; Gharbi, Severine I.; Vanhaesebroeck, Bart

    2007-01-01

    Class IA phosphoinositide 3-kinases (PI3Ks) signal downstream of tyrosine kinases and Ras and control a wide variety of biological responses. In mammals, these heterodimeric PI3Ks consist of a p110 catalytic subunit (p110α, p110β, or p110δ) bound to any of five distinct regulatory subunits (p85α, p85β, p55γ, p55α, and p50α, collectively referred to as “p85s”). The relative expression levels of p85 and p110 have been invoked to explain key features of PI3K signaling. For example, free (i.e., non-p110-bound) p85α has been proposed to negatively regulate PI3K signaling by competition with p85/p110 for recruitment to phosphotyrosine docking sites. Using affinity and ion exchange chromatography and quantitative mass spectrometry, we demonstrate that the p85 and p110 subunits are present in equimolar amounts in mammalian cell lines and tissues. No evidence for free p85 or p110 subunits could be obtained. Cell lines contain 10,000–15,000 p85/p110 complexes per cell, with p110β and p110δ being the most prevalent catalytic subunits in nonleukocytes and leukocytes, respectively. These results argue against a role of free p85 in PI3K signaling and provide insights into the nonredundant functions of the different class IA PI3K isoforms. PMID:17470792

  9. 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.

  10. PKC-ι promotes glioblastoma cell survival by phosphorylating and inhibiting BAD through a phosphatidylinositol 3-kinase pathway.

    PubMed

    Desai, S; Pillai, P; Win-Piazza, H; Acevedo-Duncan, M

    2011-06-01

    The focus of this research was to investigate the role of protein kinase C-iota (PKC-ι) in regulation of Bad, a pro-apoptotic BH3-only molecule of the Bcl-2 family in glioblastoma. Robust expression of PKC-ι is a hallmark of human glioma and benign and malignant meningiomas. The results were obtained from the two human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-ι co-localized and directly associated with Bad, as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-ι directly phosphorylated Bad at phospho specific residues, Ser-112, Ser-136 and Ser-155 which in turn induced inactivation of Bad and disruption of Bad/Bcl-XL dimer. Knockdown of PKC-ι by siRNA exhibited a corresponding reduction in Bad phosphorylation suggesting that PKC-ι may be a Bad kinase. PKC-ι knockdown also induced apoptosis in both the cell lines. Since, PKC-ι is an essential downstream mediator of the PI (3)-kinase, we hypothesize that glioma cell survival is mediated via a PI (3)-kinase/PDK1/PKC-ι/Bad pathway. Treatment with PI (3)-kinase inhibitors Wortmannin and LY294002, as well as PDK1 siRNA, inhibited PKC-ι activity and subsequent phosphorylation of Bad suggesting that PKC-ι regulates the activity of Bad in a PI (3)-kinase dependent manner. Thus, our data suggest that glioma cell survival occurs through a novel PI (3)-kinase/PDK1/PKC-ι/BAD mediated pathway.

  11. 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

  12. Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: Essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase

    SciTech Connect

    Hien, Tran Thi; Kim, Nak Doo; Pokharel, Yuba Raj; Oh, Seok Jeong; Lee, Moo Yeol; Kang, Keon Wook

    2010-08-01

    We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10 {mu}g/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.

  13. Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase.

    PubMed

    Hien, Tran Thi; Kim, Nak Doo; Pokharel, Yuba Raj; Oh, Seok Jeong; Lee, Moo Yeol; Kang, Keon Wook

    2010-08-01

    We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10μg/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.

  14. The inability of phosphatidylinositol 3-kinase activation to stimulate GLUT4 translocation indicates additional signaling pathways are required for insulin-stimulated glucose uptake.

    PubMed

    Isakoff, S J; Taha, C; Rose, E; Marcusohn, J; Klip, A; Skolnik, E Y

    1995-10-24

    Recent experimental evidence has focused attention to the role of two molecules, insulin receptor substrate 1 (IRS-1) and phosphatidylinositol 3-kinase (PI3-kinase), in linking the insulin receptor to glucose uptake; IRS-1 knockout mice are insulin resistant, and pharmacological inhibitors of PI3-kinase block insulin-stimulated glucose uptake. To investigate the role of PI3-kinase and IRS-1 in insulin-stimulated glucose uptake we examined whether stimulation of insulin-sensitive cells with platelet-derived growth factor (PDGF) or with interleukin 4 (IL-4) stimulates glucose uptake; the activated PDGF receptor (PDGFR) directly binds and activates PI3-kinase, whereas the IL-4 receptor (IL-4R) activates PI3-kinase via IRS-1 or the IRS-1-related molecule 4PS. We found that stimulation of 3T3-L1 adipocytes with PDGF resulted in tyrosine phosphorylation of the PDGFR and activation of PI3-kinase in these cells. To examine whether IL-4 stimulates glucose uptake, L6 myoblasts were engineered to overexpress GLUT4 as well as both chains of the IL-4R (L6/IL-4R/GLUT4); when these L6/IL-4R/GLUT4 myoblasts were stimulated with IL-4, IRS-1 became tyrosine phosphorylated and associated with PI3-kinase. Although PDGF and IL-4 can activate PI3-kinase in the respective cell lines, they do not possess insulin's ability to stimulate glucose uptake and GLUT4 translocation to the plasma membrane. These findings indicate that activation of PI3-kinase is not sufficient to stimulate GLUT4 translocation to the plasma membrane. We postulate that activation of a second signaling pathway by insulin, distinct from PI3-kinase, is necessary for the stimulation of glucose uptake in insulin-sensitive cells.

  15. 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

  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. A new calmodulin-binding motif for inositol 1,4,5-trisphosphate 3-kinase regulation.

    PubMed

    Franco-Echevarría, Elsa; Baños-Sanz, Jose I; Monterroso, Begoña; Round, Adam; Sanz-Aparicio, Julia; González, Beatriz

    2014-11-01

    IP3-3K [Ins(1,4,5)P3 3-kinase] is a key enzyme that catalyses the synthesis of Ins(1,3,4,5)P4, using Ins(1,4,5)P3 and ATP as substrates. Both inositides, substrate and product, present crucial roles in the cell. Ins(1,4,5)P3 is a key point in Ca2+ metabolism that promotes Ca2+ release from intracellular stores and together with Ins(1,3,4,5)P4 regulates Ca2+ homoeostasis. In addition, Ins(1,3,4,5)P4 is involved in immune cell development. It has been proved that Ca2+/CaM (calmodulin) regulates the activity of IP3-3K, via direct interaction between both enzymes. Although we have extensive structural knowledge of the kinase domains of the three IP3-3K isoforms, no structural information is available about the interaction between IP3-3K and Ca2+/CaM. In the present paper we describe the crystal structure of the complex between human Ca2+/CaM and the CaM-binding region of human IP3-3K isoform A (residues 158-183) and propose a model for a complex including the kinase domain. The structure obtained allowed us to identify all of the key residues involved in the interaction, which have been evaluated by site-directed mutagenesis, pull-down and fluorescence anisotropy experiments. The results allowed the identification of a new CaM-binding motif, expanding our knowledge about how CaM interacts with its partners.

  18. Rapamycin regulates connective tissue growth factor expression of lung epithelial cells via phosphoinositide 3-kinase.

    PubMed

    Xu, Xuefeng; Wan, Xuan; Geng, Jing; Li, Fei; Yang, Ting; Dai, Huaping

    2013-09-01

    The pathogenesis of idiopathic pulmonary fibrosis (IPF) remains largely unknown. It is believed that IPF is mainly driven by activated alveolar epithelial cells that have a compromised migration capacity, and that also produce substances (such as connective tissue growth factor, CTGF) that contribute to fibroblast activation and matrix protein accumulation. Because the mechanisms regulating these processes are unclear, the aim of this study was to determine the role of rapamycin in regulating epithelial cell migration and CTGF expression. Transformed epithelial cell line A549 and normal human pulmonary alveolar or bronchial epithelial cells were cultured in regular medium or medium containing rapamycin. Real time reverse transcriptase polymerase chain reaction was employed to determine CTGF mRNA expression. Western blotting and an enzyme-linked immunosorbent assay were used for detecting CTGF protein. Wound healing and migration assays were used to determine the cell migration potential. Transforming growth factor (TGF)-β type I receptor (TβRI) inhibitor, SB431542 and phosphoinositide 3-kinase (PI3K) inhibitor, LY294002 were used to determine rapamycin's mechanism of action. It was found that treatment of A549 and normal human alveolar or bronchial epithelial cells with rapamycin significantly promoted basal or TGF-β1 induced CTGF expression. LY294002, not SB431542 attenuated the promotional effect of rapamycin on CTGF expression. Cell mobility was not affected by rapamycin in wound healing and migration assays. These data suggest rapamycin has a profibrotic effect in vitro and underscore the potential of combined therapeutic approach with PI3K and mammalian target of rapamycin inhibitors for the treatment of animal or human lung fibrosis.

  19. Supramolecular nanoparticles that target phosphatidylinositol-3-kinase overcome insulin resistance and exert pronounced antitumor efficacy

    PubMed Central

    Kulkarni, Ashish A.; Roy, Bhaskar; Rao, Poornima S.; Wyant, Gregory A.; Mahmoud, Ayaat; Ramachandran, Madhumitha; Sengupta, Poulomi; Goldman, Aaron; Kotamraju, Venkata Ramana; Basu, Sudipta; Mashelkar, Raghunath A; Ruoslahti, Erkki; Dinulescu, Daniela M.; Sengupta, Shiladitya

    2013-01-01

    The centrality of phosphatidylinositol-3-kinase (PI3K) in cancer etiology is well established, but clinical translation of PI3K inhibitors has been limited by feedback signaling, suboptimal intra-tumoral concentration and an insulin resistance ‘class effect’. The current study was designed to explore the use of supramolecular nanochemistry for targeting PI3K to enhance antitumor efficacy and potentially overcome these limitations. PI3K inhibitor structures were rationally modified using a cholesterol-based derivative, facilitating supramolecular nanoassembly with L-α-phosphatidylcholine and DSPE-PEG. The supramolecular nanoparticles that were assembled were physicochemically characterized and functionally evaluated in vitro. Antitumor efficacy was quantified in vivo using 4T1 breast cancer and K-RasLSL/+/Ptenfl/fl ovarian cancer models, with effects on glucose homeostasis evaluated using an insulin sensitivity test. The use of PI103 and PI828 as surrogate molecules to engineer the supramolecular nanoparticles highlighted the need to keep design principles in perspective; specifically, potency of the active molecule and the linker chemistry were critical principles for efficacy, similar to antibody-drug conjugates. We found that the supramolecular nanoparticles exerted a temporally-sustained inhibition of phosphorylation of Akt, mTOR, S6K and 4EBP in vivo. These effects were associated with increased antitumor efficacy and survival as compared with PI103 and PI828. Efficacy was further increased by decorating the nanoparticle surface with tumor-homing peptides. Notably, the use of supramolecular nanoparticles abrogated the insulin resistance that has been associated widely with other PI3K inhibitors. This study provides a preclinical foundation for the use of supramolecular nanochemistry to overcome current challenges associated with PI3K inhibitors, offering a paradigm for extension to other molecularly targeted therapeutics being explored for cancer treatment

  20. Carbamazepine enhances the activity of glutamate transporter type 3 via phosphatidylinositol 3-kinase.

    PubMed

    Lee, Gwanwoo; Huang, Yueming; Washington, Jacqueline M; Briggs, Nicole W; Zuo, Zhiyi

    2005-01-01

    Glutamate transporters (also called excitatory amino acid transporters, EAAT) participate in maintaining extracellular homeostasis of glutamate, a major excitatory neurotransmitter, and regulating glutamate neurotransmission. EAAT3, the major neuronal EAAT, may also regulate gamma-aminobutyric acid-mediated inhibitory neurotransmission. Dysfunction of EAAT3 has been shown to induce seizure in rats. We hypothesize that carbamazepine, a commonly used antiepileptic agent, enhances EAAT3 activity. We tested this hypothesis using oocytes artificially expressing EAAT3 and C6 rat glioma cells expressing endogenous EAAT3. In oocytes, carbamazepine dose-dependently enhanced EAAT3 activity. The EC50 of this carbamazepine effect was 12.2muM. The concentrations of carbamazepine to significantly enhance EAAT3 activity were within the therapeutic serum levels (17-51muM) of carbamazepine for the antiepileptic effect. Carbamazepine decreased the Km but did not change the maximal response of EAAT3 to glutamate. Carbamazepine-increased EAAT3 activity was inhibited by wortmannin or LY-294002, phosphatidylinositol 3-kinase (PI3K) inhibitors, but was not affected by staurosporine, chelerythrine or calphostin C, protein kinase C inhibitors. In C6 cells, carbamazepine also enhanced the endogenous EAAT3 activity. However, carbamazepine did not affect the activity of EAAT4 expressed in Cos7 cells. These results suggest that carbamazepine at clinically relevant concentrations specifically enhances the affinity of EAAT3 for glutamate to increase EAAT3 activity via a PI3K-dependent pathway. EAAT3 may be a therapeutic target for carbamazepine in the central nervous system.

  1. Supramolecular nanoparticles that target phosphoinositide-3-kinase overcome insulin resistance and exert pronounced antitumor efficacy.

    PubMed

    Kulkarni, Ashish A; Roy, Bhaskar; Rao, Poornima S; Wyant, Gregory A; Mahmoud, Ayaat; Ramachandran, Madhumitha; Sengupta, Poulomi; Goldman, Aaron; Kotamraju, Venkata Ramana; Basu, Sudipta; Mashelkar, Raghunath A; Ruoslahti, Erkki; Dinulescu, Daniela M; Sengupta, Shiladitya

    2013-12-01

    The centrality of phosphoinositide-3-kinase (PI3K) in cancer etiology is well established, but clinical translation of PI3K inhibitors has been limited by feedback signaling, suboptimal intratumoral concentration, and an insulin resistance "class effect." This study was designed to explore the use of supramolecular nanochemistry for targeting PI3K to enhance antitumor efficacy and potentially overcome these limitations. PI3K inhibitor structures were rationally modified using a cholesterol-based derivative, facilitating supramolecular nanoassembly with L-α-phosphatidylcholine and DSPE-PEG [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polythylene glycol)]. The supramolecular nanoparticles (SNP) that were assembled were physicochemically characterized and functionally evaluated in vitro. Antitumor efficacy was quantified in vivo using 4T1 breast cancer and K-Ras(LSL/+)/Pten(fl/fl) ovarian cancer models, with effects on glucose homeostasis evaluated using an insulin sensitivity test. The use of PI103 and PI828 as surrogate molecules to engineer the SNPs highlighted the need to keep design principles in perspective; specifically, potency of the active molecule and the linker chemistry were critical principles for efficacy, similar to antibody-drug conjugates. We found that the SNPs exerted a temporally sustained inhibition of phosphorylation of Akt, mTOR, S6K, and 4EBP in vivo. These effects were associated with increased antitumor efficacy and survival as compared with PI103 and PI828. Efficacy was further increased by decorating the nanoparticle surface with tumor-homing peptides. Notably, the use of SNPs abrogated the insulin resistance that has been associated widely with other PI3K inhibitors. This study provides a preclinical foundation for the use of supramolecular nanochemistry to overcome current challenges associated with PI3K inhibitors, offering a paradigm for extension to other molecularly targeted therapeutics being explored for cancer

  2. 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.

  3. PHOSPHOINOSITIDE 3-KINASE REGULATES THE ROLE OF RETROMER IN TRANSCYTOSIS OF THE POLYMERIC IMMUNOGLOBULIN RECEPTOR

    PubMed Central

    Vergés, Marcel; Sebastián, Isabel; Mostov, Keith E.

    2007-01-01

    Retromer is a multimeric protein complex that mediates intracellular receptor sorting. One of the roles of retromer is to promote transcytosis of the polymeric immunoglobulin receptor (pIgR) and its ligand polymeric immunoglobulin A (pIgA) in polarized epithelial cells. In Madin-Darby Canine Kidney (MDCK) cells, overexpression of Vps35, the retromer subunit key for cargo recognition, restores transcytosis to a pIgR mutant that is normally degraded. Here we show that pIgA transcytosis was not restored in these cells when treated with the specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Likewise, the decrease in pIgA transcytosis by wild-type pIgR seen upon PI3K inhibition was not reverted by Vps35 overexpression. PI3K inhibition reduced membrane association of sorting-nexins (SNX) 1 and 2, which constitute the retromer subcomplex involved in membrane deformation, while association of the Vps35-Vps26-Vps29 subcomplex, involved in cargo recognition, remained virtually unaffected. Colocalization between the two retromer subcomplexes was reduced upon the treatment. Whereas the interaction among the subunits of the Vps35-Vps26-Vps29 subcomplex remained unchanged, less Vps35 was found associated with pIgR upon PI3K inhibition. In addition, colocalization of internalized pIgA with subunits of both retromer subcomplexes throughout the transcytotic pathway was substantially reduced by LY294002 treatment. These data implicate PI3K in controlling retromer’s role in pIgR-pIgA transcytosis. PMID:17184770

  4. Dual roles of hemidesmosomal proteins in the pancreatic epithelium: the phosphoinositide 3-kinase decides.

    PubMed

    Laval, S; Laklai, H; Fanjul, M; Pucelle, M; Laurell, H; Billon-Galés, A; Le Guellec, S; Delisle, M-B; Sonnenberg, A; Susini, C; Pyronnet, S; Bousquet, C

    2014-04-10

    Given the failure of chemo- and biotherapies to fight advanced pancreatic cancer, one major challenge is to identify critical events that initiate invasion. One priming step in epithelia carcinogenesis is the disruption of epithelial cell anchorage to the basement membrane which can be provided by hemidesmosomes (HDs). However, the existence of HDs in pancreatic ductal epithelium and their role in carcinogenesis remain unexplored. HDs have been explored in normal and cancer pancreatic cells, and patient samples. Unique cancer cell models where HD assembly can be pharmacologically manipulated by somatostatin/sst2 signaling have been then used to investigate the role and molecular mechanisms of dynamic HD during pancreatic carcinogenesis. We surprisingly report the presence of mature type-1 HDs comprising the integrin α6β4 and bullous pemphigoid antigen BP180 in the human pancreatic ductal epithelium. Importantly, HDs are shown to disassemble during pancreatic carcinogenesis. HD breakdown requires phosphoinositide 3-kinase (PI3K)-dependent induction of the matrix-metalloprotease MMP-9, which cleaves BP180. Consequently, integrin α6β4 delocalizes to the cell-leading edges where it paradoxically promotes cell migration and invasion through S100A4 activation. As S100A4 in turn stimulates MMP-9 expression, a vicious cycle maintains BP180 cleavage. Inactivation of this PI3K-MMP-9-S100A4 signaling loop conversely blocks BP180 cleavage, induces HD reassembly and inhibits cell invasion. We conclude that mature type-1 HDs are critical anchoring structures for the pancreatic ductal epithelium whose disruption, upon PI3K activation during carcinogenesis, provokes pancreatic cancer cell migration and invasion.

  5. Role of phosphoinositide 3-kinase in the pathogenesis of acute pancreatitis

    PubMed Central

    Lupia, Enrico; Pigozzi, Luca; Goffi, Alberto; Hirsch, Emilio; Montrucchio, Giuseppe

    2014-01-01

    A large body of experimental and clinical data supports the notion that inflammation in acute pancreatitis has a crucial role in the pathogenesis of local and systemic damage and is a major determinant of clinical severity. Thus, research has recently focused on molecules that can regulate the inflammatory processes, such as phosphoinositide 3-kinases (PI3Ks), a family of lipid and protein kinases involved in intracellular signal transduction. Studies using genetic ablation or pharmacologic inhibitors of different PI3K isoforms, in particular the class I PI3Kδ and PI3Kγ, have contributed to a greater understanding of the roles of these kinases in the modulation of inflammatory and immune responses. Recent data suggest that PI3Ks are also involved in the pathogenesis of acute pancreatitis. Activation of the PI3K signaling pathway, and in particular of the class IB PI3Kγ isoform, has a significant role in those events which are necessary for the initiation of acute pancreatic injury, namely calcium signaling alteration, trypsinogen activation, and nuclear factor-κB transcription. Moreover, PI3Kγ is instrumental in modulating acinar cell apoptosis, and regulating local neutrophil infiltration and systemic inflammatory responses during the course of experimental acute pancreatitis. The availability of PI3K inhibitors selective for specific isoforms may provide new valuable therapeutic strategies to improve the clinical course of this disease. This article presents a brief summary of PI3K structure and function, and highlights recent advances that implicate PI3Ks in the pathogenesis of acute pancreatitis. PMID:25386068

  6. Structural basis for decreased induction of class IB PI3-kinases expression by MIF inhibitors.

    PubMed

    Singh, Abhay Kumar; Pantouris, Georgios; Borosch, Sebastian; Rojanasthien, Siripong; Cho, Thomas Yoonsang

    2017-01-01

    Macrophage migration inhibitory factor (MIF) is a master regulator of proinflammatory cytokines and plays pathological roles when not properly regulated in rheumatoid arthritis, lupus, atherosclerosis, asthma and cancer. Unlike canonical cytokines, MIF has vestigial keto-enol tautomerase activity. Most of the current MIF inhibitors were screened for the inhibition of this enzymatic activity. However, only some of the enzymatic inhibitors inhibit receptor-mediated biological functions of MIF, such as cell recruitment, through an unknown molecular mechanism. The goal of this study was to understand the molecular basis underlying the pharmacological inhibition of biological functions of MIF. Here, we demonstrate how the structural changes caused upon inhibitor binding translate into the alteration of MIF-induced downstream signalling. Macrophage migration inhibitory factor activates phosphoinositide 3-kinases (PI3Ks) that play a pivotal role in immune cell recruitment in health and disease. There are several different PI3K isoforms, but little is known about how they respond to MIF. We demonstrate that MIF up-regulates the expression of Class IB PI3Ks in leucocytes. We also demonstrate that MIF tautomerase active site inhibitors down-regulate the expression of Class IB PI3Ks as well as leucocyte recruitment in vitro and in vivo. Finally, based on our MIF:inhibitor complex crystal structures, we hypothesize that the reduction in Class IB PI3K expression occurs because of the displacement of Pro1 towards the second loop of MIF upon inhibitor binding, which results in increased flexibility of the loop 2 and sub-optimal MIF binding to its receptors. These results will provide molecular insights for fine-tuning the biological functions of MIF.

  7. Selective Inhibition of Phosphoinositide 3-Kinase p110α Preserves Lymphocyte Function*

    PubMed Central

    So, Lomon; Yea, Sung Su; Oak, Jean S.; Lu, Mengrou; Manmadhan, Arun; Ke, Qiao Han; Janes, Matthew R.; Kessler, Linda V.; Kucharski, Jeff M.; Li, Lian-Sheng; Martin, Michael B.; Ren, Pingda; Jessen, Katti A.; Liu, Yi; Rommel, Christian; Fruman, David A.

    2013-01-01

    Class IA phosphoinositide 3-kinase (PI3K) is essential for clonal expansion, differentiation, and effector function of B and T lymphocytes. The p110δ catalytic isoform of PI3K is highly expressed in lymphocytes and plays a prominent role in B and T cell responses. Another class IA PI3K catalytic isoform, p110α, is a promising drug target in cancer but little is known about its function in lymphocytes. Here we used highly selective inhibitors to probe the function of p110α in lymphocyte responses in vitro and in vivo. p110α inhibition partially reduced B cell receptor (BCR)-dependent AKT activation and proliferation, and diminished survival supported by the cytokines BAFF and IL-4. Selective p110δ inhibition suppressed B cell responses much more strongly, yet maximal suppression was achieved by targeting multiple PI3K isoforms. In mouse and human T cells, inhibition of single class IA isoforms had little effect on proliferation, whereas pan-class I inhibition did suppress T cell expansion. In mice, selective p110α inhibition using the investigational agent MLN1117 (previously known as INK1117) did not disrupt the marginal zone B cell compartment and did not block T cell-dependent germinal center formation. In contrast, the selective p110δ inhibitor IC87114 strongly suppressed germinal center formation and reduced marginal zone B cell numbers, similar to a pan-class I inhibitor. These findings show that although acute p110α inhibition partially diminishes AKT activation, selective p110α inhibitors are likely to be less immunosuppressive in vivo compared with p110δ or pan-class I inhibitors. PMID:23275335

  8. Control of neurite outgrowth and growth cone motility by phosphatidylinositol-3-kinase.

    PubMed

    Tornieri, Karine; Welshhans, Kristy; Geddis, Matthew S; Rehder, Vincent

    2006-04-01

    Phosphatidylinositol-3-kinase (PI-3K) has been reported to affect neurite outgrowth both in vivo and in vitro. Here we investigated the signaling pathways by which PI-3K affects neurite outgrowth and growth cone motility in identified snail neurons in vitro. Inhibition of PI-3K with wortmannin (2 microM) or LY 294002 (25 microM) resulted in a significant elongation of filopodia and in a slow-down of neurite outgrowth. Experiments using cytochalasin and blebbistatin, drugs that interfere with actin polymerization and myosin II activity, respectively, demonstrated that filopodial elongation resulting from PI-3K inhibition was dependent on actin polymerization. Inhibition of strategic kinases located downstream of PI-3K, such as Akt, ROCK, and MEK, also caused significant filopodial elongation and a slow-down in neurite outgrowth. Another growth cone parameter, filopodial number, was not affected by inhibition of PI-3K, Akt, ROCK, or MEK. A detailed study of growth cone behavior showed that the filopodial elongation induced by inhibiting PI-3K, Akt, ROCK, and MEK was achieved by increasing two motility parameters: the rate with which filopodia extend (extension rate) and the time that filopodia spend elongating. Whereas the inhibition of ROCK or Akt (both activated by the lipid kinase activity of PI-3K) and MEK (activated by the protein kinase activity of PI-3K) had additive effects, simultaneous inhibition of Akt and ROCK showed no additive effect. We further demonstrate that the effects on filopodial dynamics investigated were calcium-independent. Taken together, our results suggest that inhibition of PI-3K signaling results in filopodial elongation and a slow-down of neurite advance, reminiscent of growth cone searching behavior.

  9. Phosphatidylinositol 3-kinase pathway regulates sperm viability but not capacitation on boar spermatozoa.

    PubMed

    Aparicio, I M; Bragado, M J; Gil, M C; Garcia-Herreros, M; Gonzalez-Fernandez, L; Tapia, J A; Garcia-Marin, L J

    2007-08-01

    Phosphatidylinositol 3-kinase (PI3-K) plays an important role in cell survival in somatic cells and recent data pointed out a role for this kinase in sperm capacitation and acrosome reaction (AR). This study was undertaken to evaluate the role of PI3-K pathway on porcine spermatozoa capacitation, AR, and viability using two unrelated PI3-K inhibitors, LY294002 and wortmannin. In boar spermatozoa, we have identified the presence of PDK1, PKB/Akt, and PTEN, three of the main key components of the PI3-K pathway. Incubation of boar sperm in a capacitating medium (TCM) caused a significant increase in the percentage of capacitated (25 +/- 2 to 34 +/- 1% P < 0.05, n = 6) and acrosome reacted (1 +/- 1 to 11 +/- 1% P < 0.01, n = 6) spermatozoa compared with sperm in basal medium (TBM). Inhibition of PI3-K did affect neither the capacitation status nor AR nor protein p32 tyrosine phosphorylation of boar spermatozoa incubated in TBM or TCM. Boar sperm viability in TBM was significantly decreased by 40 and 20% after pretreatment with LY294002 or wortmannin, respectively. Similar results were observed after incubation of boar spermatozoa in TCM. Treatment of boar spermatozoa with the analog of cAMP, 8Br-cAMP significantly prevented the reduction on sperm viability. Our results provide evidence for an important role of the PI3-K pathway in the regulation of boar sperm viability and suggests that other signaling pathways different from PI3-K must be activated downstream of cAMP to contribute to regulation of sperm viability. Finally, in our conditions the PI3-K pathway seems not related with boar sperm capacitation or AR.

  10. Assessing the subcellular distribution of oncogenic phosphoinositide 3-kinase using microinjection into live cells

    PubMed Central

    Layton, Meredith J.; Rynkiewicz, Natalie K.; Ivetac, Ivan; Horan, Kristy A.; Mitchell, Christina A.; Phillips, Wayne A.

    2014-01-01

    Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3Kα (phosphoinositide 3-kinase α) to its PM (plasma membrane) localized substrate is also required for increased levels of downstream PIP3/Akt [phosphoinositide-3,4,5-trisphosphate/also called PKB (protein kinase B)] signalling. We have studied the subcellular localization of wild-type and the two most common oncogenic mutants of PI3Kα in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3Kα is pre-formed as a 1:1 p110α:p85α complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipids in vitro or in cells maintained in 10% (v/v) FBS. Following stimulation of RTKs (receptor tyrosine kinases), microinjected PI3Kα was recruited to the PM, but oncogenic forms of PI3Kα were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3Kα. Instead, the E545K mutant specifically bound activated Cdc42 in vitro and microinjection of E545K was associated with the formation of cellular protrusions, providing some preliminary evidence that changes in protein–protein interactions may play a role in the oncogenicity of the E545K mutant in addition to the well-known changes in lipid kinase activity. PMID:27919038

  11. Involvement of Phosphatidylinositol 3-kinase in the regulation of proline catabolism in Arabidopsis thaliana

    PubMed Central

    Leprince, Anne-Sophie; Magalhaes, Nelly; De Vos, Delphine; Bordenave, Marianne; Crilat, Emilie; Clément, Gilles; Meyer, Christian; Munnik, Teun; Savouré, Arnould

    2015-01-01

    Plant adaptation to abiotic stresses such as drought and salinity involves complex regulatory processes. Deciphering the signaling components that are involved in stress signal transduction and cellular responses is of importance to understand how plants cope with salt stress. Accumulation of osmolytes such as proline is considered to participate in the osmotic adjustment of plant cells to salinity. Proline accumulation results from a tight regulation between its biosynthesis and catabolism. Lipid signal components such as phospholipases C and D have previously been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. In this study, we demonstrate that proline metabolism is also regulated by class-III Phosphatidylinositol 3-kinase (PI3K), VPS34, which catalyses the formation of phosphatidylinositol 3-phosphate (PI3P) from phosphatidylinositol. Using pharmacological and biochemical approaches, we show that the PI3K inhibitor, LY294002, affects PI3P levels in vivo and that it triggers a decrease in proline accumulation in response to salt treatment of A. thaliana seedlings. The lower proline accumulation is correlated with a lower transcript level of Pyrroline-5-carboxylate synthetase 1 (P5CS1) biosynthetic enzyme and higher transcript and protein levels of Proline dehydrogenase 1 (ProDH1), a key-enzyme in proline catabolism. We also found that the ProDH1 expression is induced in a pi3k-hemizygous mutant, further demonstrating that PI3K is involved in the regulation of proline catabolism through transcriptional regulation of ProDH1. A broader metabolomic analysis indicates that LY294002 also reduced other metabolites, such as hydrophobic and aromatic amino acids and sugars like raffinose. PMID:25628629

  12. Oxidative stress stimulates skeletal muscle glucose uptake through a phosphatidylinositol 3-kinase-dependent pathway

    PubMed Central

    Higaki, Yasuki; Mikami, Toshio; Fujii, Nobuharu; Hirshman, Michael F.; Koyama, Katsuhiro; Seino, Tetsuya; Tanaka, Keitaro; Goodyear, Laurie J.

    2010-01-01

    We determined the acute effects of oxidative stress on glucose uptake and intracellular signaling in skeletal muscle by incubating muscles with reactive oxygen species (ROS). Xanthine oxidase (XO) is a superoxide-generating enzyme that increases ROS. Exposure of isolated rat extensor digitorum longus (EDL) muscles to Hx/XO (Hx/XO) for 20 min resulted in a dose-dependent increase in glucose uptake. To determine whether the mechanism leading to Hx/XO-stimulated glucose uptake is associated with the production of H2O2, EDL muscles from rats were preincubated with the H2O2 scavenger catalase or the superoxide scavenger superoxide dismutase (SOD) prior to incubation with Hx/XO. Catalase treatment, but not SOD, completely inhibited the increase in Hx/XO-stimulated 2-deoxyglucose (2-DG) uptake, suggesting that H2O2 is an intermediary leading to Hx/XO-stimulated glucose uptake with incubation. Direct H2O2 also resulted in a dose-dependent increase in 2-DG uptake in isolated EDL muscles, and the maximal increase was threefold over basal levels at a concentration of 600 μmol/l H2O2. H2O2-stimulated 2-DG uptake was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, but not the nitric oxide inhibitor NG-monomethyl-L-arginine. H2O2 stimulated the phosphorylation of Akt Ser473 (7-fold) and Thr308 (2-fold) in isolated EDL muscles. H2O2 at 600 μmol/l had no effect on ATP concentrations and did not increase the activities of either the α1 or α2 catalytic isoforms of AMP-activated protein kinase. These results demonstrate that acute exposure of muscle to ROS is a potent stimulator of skeletal muscle glucose uptake and that this occurs through a PI3K-dependent mechanism. PMID:18303121

  13. Selective inhibition of phosphoinositide 3-kinase p110α preserves lymphocyte function.

    PubMed

    So, Lomon; Yea, Sung Su; Oak, Jean S; Lu, Mengrou; Manmadhan, Arun; Ke, Qiao Han; Janes, Matthew R; Kessler, Linda V; Kucharski, Jeff M; Li, Lian-Sheng; Martin, Michael B; Ren, Pingda; Jessen, Katti A; Liu, Yi; Rommel, Christian; Fruman, David A

    2013-02-22

    Class IA phosphoinositide 3-kinase (PI3K) is essential for clonal expansion, differentiation, and effector function of B and T lymphocytes. The p110δ catalytic isoform of PI3K is highly expressed in lymphocytes and plays a prominent role in B and T cell responses. Another class IA PI3K catalytic isoform, p110α, is a promising drug target in cancer but little is known about its function in lymphocytes. Here we used highly selective inhibitors to probe the function of p110α in lymphocyte responses in vitro and in vivo. p110α inhibition partially reduced B cell receptor (BCR)-dependent AKT activation and proliferation, and diminished survival supported by the cytokines BAFF and IL-4. Selective p110δ inhibition suppressed B cell responses much more strongly, yet maximal suppression was achieved by targeting multiple PI3K isoforms. In mouse and human T cells, inhibition of single class IA isoforms had little effect on proliferation, whereas pan-class I inhibition did suppress T cell expansion. In mice, selective p110α inhibition using the investigational agent MLN1117 (previously known as INK1117) did not disrupt the marginal zone B cell compartment and did not block T cell-dependent germinal center formation. In contrast, the selective p110δ inhibitor IC87114 strongly suppressed germinal center formation and reduced marginal zone B cell numbers, similar to a pan-class I inhibitor. These findings show that although acute p110α inhibition partially diminishes AKT activation, selective p110α inhibitors are likely to be less immunosuppressive in vivo compared with p110δ or pan-class I inhibitors.

  14. The Association of PI3 Kinase Signaling and Chemoresistance in Advanced Ovarian Cancer

    PubMed Central

    Carden, Craig P.; Stewart, Adam; Thavasu, Parames; Kipps, Emma; Pope, Lorna; Crespo, Mateus; Miranda, Susana; Attard, Gerhardt; Garrett, Michelle D.; Clarke, Paul A.; Workman, Paul; de Bono, Johann S.; Gore, Martin; Kaye, Stan B; Banerji, Udai

    2015-01-01

    Evidence that the phosphoinositide 3-kinase (PI3K) pathway is deregulated in ovarian cancer is largely based on the analysis of surgical specimens sampled at diagnosis and may not reflect the biology of advanced ovarian cancer. We aimed to investigate PI3K signaling in cancer cells isolated from patients with advanced ovarian cancer. Ascites samples were analyzed from 88 patients, of whom 61 received further treatment. Cancer cells were immunomagnetically separated from ascites, and the signaling output of the PI3K pathway was studied by quantifying p-AKT, p-p70S6K, and p-GSK3β by ELISA. Relevant oncogenes, such as PIK3CA and AKT, were sequenced by PCR-amplified mass spectroscopy detection methods. In addition, PIK3CA and AKT2 amplifications and PTEN deletions were analyzed by FISH. p-p70S6K levels were significantly higher in cells from 37 of 61 patients who did not respond to subsequent chemotherapy (0.7184 vs. 0.3496; P = 0.0100), and this difference was greater in patients who had not received previous chemotherapy. PIK3CA and AKT mutations were present in 5% and 0% of samples, respectively. Amplification of PIK3CA and AKT2 and deletion of PTEN was seen in 10%, 10%, and 27% of samples, respectively. Mutations of PIK3CA and amplification of PIK3CA/AKT2 or deletion of PTEN did not correlate with levels of p-AKT, p-p70S6K, and p-GSK3β. In patients with advanced ovarian cancer, there is an association between levels of p-p70S6K and response to subsequent chemotherapy. There is no clear evidence that this is driven specifically by PIK3CA or AKT mutations or by amplifications or deletion of PTEN. PMID:22556379

  15. Enterococcus faecalis infection activates phosphatidylinositol 3-kinase signaling to block apoptotic cell death in macrophages.

    PubMed

    Zou, Jun; Shankar, Nathan

    2014-12-01

    Apoptosis is an intrinsic immune defense mechanism in the host response to microbial infection. Not surprisingly, many pathogens have evolved various strategies to manipulate this important pathway to benefit their own survival and dissemination in the host during infection. To our knowledge, no attempts have been made to explore the host cell survival signals modulated by the bacterium Enterococcus faecalis. Here, we show for the first time that during early stages of infection, internalized enterococci can prevent host cell (RAW264.7 cells, primary macrophages, and mouse embryonic fibroblasts [MEFs]) apoptosis induced by a wide spectrum of proapoptotic stimuli. Activation of caspase 3 and cleavage of the caspase 3 substrate poly(ADP-ribose) polymerase were inhibited in E. faecalis-infected cells, indicating that E. faecalis protects macrophages from apoptosis by inhibiting caspase 3 activation. This antiapoptotic activity in E. faecalis-infected cells was dependent on the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, which resulted in the increased expression of the antiapoptotic factor Bcl-2 and decreased expression of the proapoptotic factor Bax. Further analysis revealed that active E. faecalis physiology was important for inhibition of host cell apoptosis, and this feature seemed to be a strain-independent trait among E. faecalis isolates. Employing a mouse peritonitis model, we also determined that cells collected from the peritoneal lavage fluid of E. faecalis-infected mice showed reduced levels of apoptosis compared to cells from uninfected mice. These results show early modulation of apoptosis during infection and have important implications for enterococcal pathogenesis.

  16. 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.

  17. Phosphoinositide 3-kinase signaling is critical for ErbB3-driven breast cancer cell motility and metastasis

    PubMed Central

    Smirnova, Tatiana; Zhou, Zhen Ni; Flinn, Rory J.; Wyckoff, Jeffrey; Boimel, Pamela J.; Pozzuto, Maria; Coniglio, Salvatore J.; Backer, Jonathan M.; Bresnick, Anne R.; Condeelis, John S.; Hynes, Nancy E.; Segall, Jeffrey E.

    2011-01-01

    Many malignancies show increased expression of the EGF receptor family member ErbB3 (HER3). ErbB3 binds beta-1 (HRGβ1), and forms a heterodimer with other ErbB family members, such as ErbB2 (HER2) or EGFR (HER1), enhancing phosphorylation of specific C terminal tyrosine residues and activation of downstream signaling pathways. ErbB3 contains six YXXM motifs that bind the p85 subunit of PI3-kinase. Previous studies demonstrated that overexpression of ErbB3 in mammary tumor cells can significantly enhance chemotaxis to HRGβ1 and overall metastatic potential. We tested the hypothesis that ErbB3-mediated PI3-kinase signaling is critical for heregulin-induced motility, and therefore crucial for ErbB3-mediated invasion, intravasation and metastasis. The tyrosines in the six YXXM motifs on the ErbB3 C-terminus were replaced with phenylalanine. In contrast to overexpression of the wild-type ErbB3, overexpression of the mutant ErbB3 did not enhance chemotaxis towards HRGβ1 in vitro or in vivo. We also observed reduced tumor cell motility in the primary tumor by multiphoton microscopy, as well as a dramatically reduced ability of these cells to cross the endothelium and intravasate into the circulation. Moreover, while mutation of the ErbB3 C-terminus had no effect on tumor growth, it had a dramatic effect on spontaneous metastatic potential. Treatment with the PI3-kinase inhibitor PIK-75 similarly inhibited motility and invasion in vitro and in vivo. Our results indicate that stimulation of the early metastatic steps of motility and invasion by ErbB3 requires activation of the PI3-kinase pathway by the ErbB3 receptor. PMID:21725367

  18. 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

  19. PI3 kinase is involved in cocaine behavioral sensitization and its reversal with brain area specificity

    SciTech Connect

    Zhang Xiuwu . E-mail: xwzhang@duke.edu; Mi Jing; Wetsel, William C.; Davidson, Colin; Xiong Xieying; Chen Qiang; Ellinwood, Everett H.; Lee, Tong H.

    2006-02-24

    Phosphatidylinositol 3-kinase (PI3K) is an important signaling molecule involved in cell differentiation, proliferation, survival, and phagocytosis, and may participate in various brain functions. To determine whether it is also involved in cocaine sensitization, we measured the p85{alpha}/p110 PI3K activity in the nuclear accumbens (NAc) shell, NAc core, and prefrontal cortex (PFC) following establishment of cocaine sensitization and its subsequent reversal. Naive rats were rank-ordered and split into either daily cocaine or saline pretreatment group based on their locomotor responses to an acute cocaine injection (7.5 mg/kg, i.p.). These two groups were then injected with cocaine (40 mg/kg, s.c.) or saline for 4 consecutive days followed by 9-day withdrawal. Cocaine sensitization was subsequently reversed by 5 daily injections of the D{sub 1}/D{sub 2} agonist pergolide (0.1 mg/kg, s.c.) in combination with the 5-HT{sub 3} antagonist ondansetron (0.2 mg/kg, s.c., 3.5 h after pergolide injection). After another 9-day withdrawal, behavioral cocaine sensitization and its reversal were confirmed with an acute cocaine challenge (7.5 mg/kg, i.p.), and animals were sacrificed the next day for measurement of p85{alpha}/p110 PI3K activity. Cocaine-sensitized animals exhibited increased PI3K activity in the NAc shell, and this increase was reversed by combined pergolide/ondansetron treatment, which also reversed behavioral sensitization. In the NAc core and PFC, cocaine sensitization decreased and increased the PI3K activity, respectively. These changes, in contrast to that in the NAc shell, were not normalized following the reversal of cocaine-sensitization. Interestingly, daily injections of pergolide alone in saline-pretreated animals induced PI3K changes that were similar to the cocaine sensitization-associated changes in the NAc core and PFC but not the NAc shell; furthermore, these changes in saline-pretreated animals were prevented by ondansetron given 3.5 h after

  20. Phosphatidylinositol-3 kinase-dependent translational regulation of Id1 involves the PPM1G phosphatase

    PubMed Central

    Xu, Kaiming; Wang, Lanfang; Feng, Wei; Feng, Yue; Shu, Hui-Kuo G.

    2016-01-01

    Id1 is a helix-loop-helix transcriptional modulator that increases the aggressiveness of malignant glial neoplasms. Since most glioblastomas (GBMs) show increased phosphatidylinositol-3 kinase (PI-3K) signaling, we sought to determine whether this pathway regulates Id1 expression. Higher basal Id1 expression correlates with dysregulated PI-3K signaling in multiple established GBM cell lines. Further characterization of PI-3K-dependent Id1 regulation reveals that chemical or genetic inhibition of PI-3K signaling reduces Id1 protein but not mRNA expression. Overall, PI-3K signaling appears to enhance Id1 translation with no significant effect on its stability. PI-3K signaling is known to regulate protein translation through mTORC1-dependent phosphorylation of 4E-BP1, which reduces its association with and inhibition of the translation initiation factor eIF4E. Interestingly, while inhibition of PI-3K and AKT lowers 4E-BP1 phosphorylation and expression of Id1 in all cases, inhibition of TORC1 with rapamycin does not consistently have a similar effect suggesting an alternative mechanism for PI-3K-dependent regulation of Id1 translation. We now identify a potential role for the serine-threonine phosphatase PPM1G in translational regulation of Id1 protein expression. PPM1G knockdown by siRNA increase both 4E-BP1 phosphorylation and Id1 expression and PPM1G and 4E-BP1 co-associates in GBM cells. Furthermore, PPM1G is a phosphoprotein and this phosphorylation appears to be regulated by PI-3K activity. Finally, PI-3K inhibition increases PPM1G activity when assessed by an in vitro phosphatase assay. Our findings provide the first evidence that the PI-3K/AKT signaling pathway modulates PPM1G activity resulting in a shift in the balance between hyper- and hypo-phosphorylated 4E-BP1 and translational regulation of Id1 expression. PMID:27065332

  1. 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.

  2. Insulin-regulated expression of Egr-1 and Krox20: dependence on ERK1/2 and interaction with p38 and PI3-kinase pathways.

    PubMed

    Keeton, Adam B; Bortoff, Katherine D; Bennett, William L; Franklin, J Lee; Venable, Derwei Y; Messina, Joseph L

    2003-12-01

    In addition to its ability to rapidly alter metabolism, insulin is also able to regulate the expression of numerous genes via activation of the PI3-kinase (PI3-K), MAPK kinase (MEK)-ERK, or p38 pathways. Using differential screening of H4IIE cells, we have identified two members of the Egr zinc-finger transcription factor family of early response genes, Egr-1 and Krox20, whose transcription is induced by insulin treatment. Egr-1 may be involved in insulin's regulation of hepatic gene expression. Krox20 regulation and expression have been primarily studied in neural cells and tissues, but little has been previously reported on the presence of Krox20 in cells of hepatic origin or its regulation by insulin. In the present studies, insulin treatment rapidly increased transcription of both Egr-1 and Krox20. In cells pretreated with a PI3-K inhibitor, there was no reduction in the effect of insulin on Egr-1 and Krox20, but an increase in Egr-1 transcription. The rapid induction of ERK1/2 phosphorylation was completely blocked by pretreatment with a MEK1 inhibitor and was associated with a nearly complete inhibition of insulin-stimulated induction of both Egr-1and Krox20, indicating this pathway is necessary for insulin's effect on these genes. Finally, inhibition of the p38 pathway, followed by insulin addition, caused an additive induction of both Egr-1and Krox20. In conclusion, these genes are induced by insulin via coordinated regulation of the MEK-ERK and p38 pathways and, in the case of Egr-1, the PI3-K pathway.

  3. A Functional Genetic Screen Identifies the Phosphoinositide 3-kinase Pathway as a Determinant of Resistance to Fibroblast Growth Factor Receptor Inhibitors in FGFR Mutant Urothelial Cell Carcinoma.

    PubMed

    Wang, Liqin; Šuštić, Tonći; Leite de Oliveira, Rodrigo; Lieftink, Cor; Halonen, Pasi; van de Ven, Marieke; Beijersbergen, Roderick L; van den Heuvel, Michel M; Bernards, René; van der Heijden, Michiel S

    2017-01-17

    Activating mutations and translocations of the FGFR3 gene are commonly seen in urothelial cell carcinoma (UCC) of the bladder and urinary tract. Several fibroblast growth factor receptor (FGFR) inhibitors are currently in clinical development and response rates appear promising for advanced UCC. A common problem with targeted therapeutics is intrinsic or acquired resistance of the cancer cells. To find potential drug targets that can act synergistically with FGFR inhibition, we performed a synthetic lethality screen for the FGFR inhibitor AZD4547 using a short hairpin RNA library targeting the human kinome in the UCC cell line RT112 (FGFR3-TACC3 translocation). We identified multiple members of the phosphoinositide 3-kinase (PI3K) pathway and found that inhibition of PIK3CA acts synergistically with FGFR inhibitors. The PI3K inhibitor BKM120 acted synergistically with inhibition of FGFR in multiple UCC and lung cancer cell lines having FGFR mutations. Consistently, we observed an elevated PI3K-protein kinase B pathway activity resulting from epidermal growth factor receptor or Erb-B2 receptor tyrosine kinase 3 reactivation caused by FGFR inhibition as the underlying molecular mechanism of the synergy. Our data show that feedback pathways activated by FGFR inhibition converge on the PI3K pathway. These findings provide a strong rationale to test FGFR inhibitors in combination with PI3K inhibitors in cancers harboring genetic activation of FGFR genes.

  4. Transformation of Rat-1 fibroblasts with the v-src oncogene induces inositol 1,4,5-trisphosphate 3-kinase expression.

    PubMed Central

    Woodring, P J; Garrison, J C

    1996-01-01

    Transformation of Rat-1 fibroblasts with the v-src oncogene leads to a 6- to 8-fold enhancement of the activity of the Ins(1,4,5)P3 3-kinase in cytosolic extracts [Johnson, Wasilenko, Mattingly, Weber and Garrison (1989) Science 246, 121-124]. This study confirms these results using another v-src-transformed Rat-1 cell line (B31 cells) and investigates the molecular mechanism by which pp60v-src activates Ins(1,4,5)P3 3-kinase. The mRNA and protein levels for two rat isoforms of Ins(1,4,5)P3 3-kinase were determined in the v-src-transformed cell line. Both the mRNA and protein levels for isoform A were elevated in v-src-transformed Rat-1 cells while those for isoform B were not significantly affected. Moreover, stable expression of either form of Ins(1,4,5)P3 3-kinase in the B31 v-src-transformed Rat-1 cell line did not result in tyrosine phosphorylation of Ins(1,4,5)P3 3-kinase A or B. These results suggest that at least one mechanism by which the v-src oncogene increases the activity of the Ins(1,4,5)P3 3-kinase in the Rat-1 transformed fibroblast is by increasing the level of expression of Ins(1,4,5)P3 3-kinase A. PMID:8870651

  5. Heregulin-dependent activation of phosphoinositide 3-kinase and Akt via the ErbB2/ErbB3 co-receptor.

    PubMed

    Hellyer, N J; Kim, M S; Koland, J G

    2001-11-09

    The ErbB2/ErbB3 heregulin co-receptor has been shown to couple to phosphoinositide (PI) 3-kinase in a heregulin-dependent manner. The recruitment and activation of PI 3-kinase by this co-receptor is presumed to occur via its interaction with phosphorylated Tyr-Xaa-Xaa-Met (YXXM) motifs occurring in the ErbB3 C terminus. In this study, mutant ErbB3 receptor proteins expressed in COS7 cells were used to investigate PI 3-kinase-dependent signaling pathways activated by the ErbB2/ErbB3 co-receptor. We observed that a mutant ErbB3 protein with each of its six YXXM motifs containing a Tyr --> Phe substitution was unable to bind either the p85 regulatory or p110 catalytic subunit of PI 3-kinase. However, restoration of a single YXXM motif was sufficient to mediate association with the PI 3-kinase holoenzyme, although at a lower level than wild-type ErbB3. When ErbB3 YXXM motifs were restored in pairs, evidence for cooperativity between two, those incorporating Tyr-1273 and Tyr-1286, was observed. Interestingly, we have shown that an apparent association of PI 3-kinase activity with ErbB2/Neu was due to the residual presence of ErbB3 in ErbB2 immunoprecipitates. The necessity of ErbB3 association with PI 3-kinase for downstream signaling to the effector kinase Akt was also investigated. Here, the heregulin-dependent translocation of Akt to the plasma membrane and its subsequent activation was observed in intact NIH-3T3 fibroblasts. Recruitment of PI 3-kinase to ErbB3 was required for both activities, and it appeared that ErbB2 activation alone was not sufficient to activate PI 3-kinase signaling in these cells.

  6. Contraction inhibits insulin-stimulated insulin receptor substrate-1/2-associated phosphoinositide 3-kinase activity, but not protein kinase B activation or glucose uptake, in rat muscle.

    PubMed Central

    Whitehead, J P; Soos, M A; Aslesen, R; O'rahilly, S; Jensen, J

    2000-01-01

    The initial stages of insulin-stimulated glucose uptake are thought to involve tyrosine phosphorylation of insulin receptor substrates (IRSs), which recruit and activate phosphoinositide 3-kinase (PI 3-kinase), leading to the activation of protein kinase B (PKB) and other downstream effectors. In contrast, contraction stimulates glucose uptake via a PI 3-kinase-independent mechanism. The combined effects of insulin and contraction on glucose uptake are additive. However, it has been reported that contraction causes a decrease in insulin-stimulated IRS-1-associated PI 3-kinase activity. To investigate this paradox, we have examined the effects of contraction on insulin-stimulated glucose uptake and proximal insulin-signalling events in isolated rat epitrochlearis muscle. Stimulation by insulin or contraction produced a 3-fold increase in glucose uptake, with the effects of simultaneous treatment by insulin and contraction being additive. Wortmannin completely blocked the additive effect of insulin in contracting skeletal muscle, indicating that this is a PI 3-kinase-dependent effect. Insulin-stimulated recruitment of PI 3-kinase to IRS-1 was unaffected by contraction; however, insulin produced no discernible increase in PI 3-kinase activity in IRS-1 or IRS-2 immunocomplexes in contracting skeletal muscle. Consistent with this, contraction inhibited insulin-stimulated p70(S6K) activation. In contrast, insulin-stimulated activation of PKB was unaffected by contraction. Thus, in contracting skeletal muscle, insulin stimulates glucose uptake and activates PKB, but not p70(S6K), by a PI 3-kinase-dependent mechanism that is independent of changes in IRS-1- and IRS-2-associated PI 3-kinase activity. PMID:10903138

  7. Endurance exercise training increases insulin responsiveness in isolated adipocytes through IRS/PI3-kinase/Akt pathway.

    PubMed

    Peres, Sidney B; de Moraes, Solange M Franzói; Costa, Cecilia E M; Brito, Luciana C; Takada, Julie; Andreotti, Sandra; Machado, Magaly A; Alonso-Vale, Maria Isabel C; Borges-Silva, Cristina N; Lima, Fabio B

    2005-03-01

    Endurance exercise training promotes important metabolic adaptations, and the adipose tissue is particularly affected. The aim of this study was to investigate how endurance exercise training modulates some aspects of insulin action in isolated adipocytes and in intact adipose tissue. Male Wistar rats were submitted to daily treadmill running (1 h/day) for 7 wk. Sedentary age-matched rats were used as controls. Final body weight, body weight gain, and epididymal fat pad weight did not show any statistical differences between groups. Adipocytes from trained rats were smaller than those from sedentary rats (205 +/- 16.8 vs. 286 +/- 26.4 pl; P < 0.05). Trained rats showed decreased plasma glucose (4.9 +/- 0.13 vs. 5.3 +/- 0.07 mM; P < 0.05) and insulin levels (0.24 +/- 0.012 vs. 0.41 +/- 0.049 mM; P < 0.05) and increased insulin-stimulated glucose uptake (23.1 +/- 3.1 vs. 12.1 +/- 2.9 pmol/cm(2); P < 0.05) compared with sedentary rats. The number of insulin receptors and the insulin-induced tyrosine phosphorylation of insulin receptor-beta subunit did not change between groups. Insulin-induced tyrosine phosphorylation insulin receptor substrates (IRS)-1 and -2 increased significantly (1.57- and 2.38-fold, respectively) in trained rats. Insulin-induced IRS-1/phosphatidylinositol 3 (PI3)-kinase (but not IRS-2/PI3-kinase) association and serine Akt phosphorylation also increased (2.06- and 3.15-fold, respectively) after training. The protein content of insulin receptor-beta subunit, IRS-1 and -2, did not differ between groups. Taken together, these data support the hypothesis that the increased adipocyte responsiveness to insulin observed after endurance exercise training is modulated by IRS/PI3-kinase/Akt pathway.

  8. Human Placental Lactogen Induces CYP2E1 Expression via PI 3-Kinase Pathway in Female Human Hepatocytes

    PubMed Central

    Lee, Jin Kyung; Chung, Hye Jin; Fischer, Liam; Fischer, James; Gonzalez, Frank J.

    2014-01-01

    The state of pregnancy is known to alter hepatic drug metabolism. Hormones that rise during pregnancy are potentially responsible for the changes. Here we report the effects of prolactin (PRL), placental lactogen (PL), and growth hormone variant (GH-v) on expression of major hepatic cytochromes P450 expression and a potential molecular mechanism underlying CYP2E1 induction by PL. In female human hepatocytes, PRL and GH-v showed either no effect or small and variable effects on mRNA expression of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5. On the other hand, PL increased expression level of CYP2E1 mRNA with corresponding increases in CYP2E1 protein and activity levels. Results from hepatocytes and HepaRG cells indicate that PL does not affect the expression or activity of HNF1α, the known transcriptional activator of basal CYP2E1 expression. Furthermore, transient transfection studies and Western blot results showed that STAT signaling, the previously known mediator of PL actions in certain tissues, does not play a role in CYP2E1 induction by PL. A chemical inhibitor of PI3-kinase signaling significantly repressed the CYP2E1 induction by PL in human hepatocytes, suggesting involvement of PI3-kinase pathway in CYP2E1 regulation by PL. CYP2E1-humanized mice did not exhibit enhanced CYP2E1 expression during pregnancy, potentially because of interspecies differences in PL physiology. Taken together, these results indicate that PL induces CYP2E1 expression via PI3-kinase pathway in human hepatocytes. PMID:24408518

  9. The Development of Novel Small Molecule Inhibitors of the Phosphoinositide-3-Kinase Pathway Through High-Throughput Cell-Based Screens

    DTIC Science & Technology

    2005-02-01

    cells. Psycho- Calmodulin antagonists inhibit insulin -stimulated GLUT4 ( glucose trans- pharmacology (Berl.) 150, 383-390. porter 4) translocation by...AD Award Number: W81XWH-04-1-0169 TITLE: The Development of Novel Small Molecule Inhibitors of the Phosphoinositide-3-Kinase Pathway Through High ...Phosphoinositide-3-Kinase Pathway Through High -Throughput Cell-Based Screens 6. AUTHOR(S) William R. Sellers, M.D. 7. PERFORMING ORGANIZA TION NAME(S) AND

  10. Phosphatidylinositol 3-kinase and NF-kappa B/Rel are at the divergence of CD40-mediated proliferation and survival pathways.

    PubMed

    Andjelic, S; Hsia, C; Suzuki, H; Kadowaki, T; Koyasu, S; Liou, H C

    2000-10-01

    CD40 receptor ligation evokes several crucial outcomes for the fate of an activated B cell, including proliferation and survival. Although multiple signaling molecules in the CD40 pathways have been identified, their specific roles in regulating proliferation and maintaining cell viability are still obscure. In this report, we demonstrate that the activation of both phosphatidylinositol 3-kinase (PI-3K) and NF-kappaB/Rel transcription factors is crucial for CD40-mediated proliferation. Furthermore, our data indicate that PI-3K is indispensable for CD40-mediated NF-kappaB/Rel activation. This is achieved via activation of AKT and the degradation of IkappaBalpha. Furthermore, we show that PI-3K activity is necessary for the degradation of cyclin-dependent kinase inhibitor p27kip. Therefore, both of these events comprise the mechanism by which PI-3K controls cell proliferation. In contrast to the absolute requirement of PI-3K and NF-kappaB/Rel for proliferation, these signaling molecules are only partially responsible for CD40-mediated survival, as blocking of PI-3K activity did not lead to apoptosis of anti-CD40-treated cells. However, the PI-3K/NF-kappaB pathway is still required for CD40-induced Bcl-X gene expression. Taken together, our data indicate that multiple survival pathways are triggered via this receptor, whereas NF-kappaB/Rel and PI-3K are crucial for CD40-induced proliferation.

  11. VEGF induces proliferation, migration, and TGF-{beta}1 expression in mouse glomerular endothelial cells via mitogen-activated protein kinase and phosphatidylinositol 3-kinase

    SciTech Connect

    Li Zhaodong; Bork, Jens Peter; Krueger, Bettina; Patsenker, Eleonora; Schulze-Krebs, Anja; Hahn, Eckhart G.; Schuppan, Detlef; E-mail: dschuppa@bidmc.harvard.edu

    2005-09-09

    The role of glomerular endothelial cells in kidney fibrosis remains incompletely understood. While endothelia are indispensable for repair of acute damage, they can produce extracellular matrix proteins and profibrogenic cytokines that promote fibrogenesis. We used a murine cell line with all features of glomerular endothelial cells (glEND.2), which dissected the effects of vascular endothelial growth factor (VEGF) on cell migration, proliferation, and profibrogenic cytokine production. VEGF dose-dependently induced glEND.2 cell migration and proliferation, accompanied by up-regulation of VEGFR-2 phosphorylation and mRNA expression. VEGF induced a profibrogenic gene expression profile, including up-regulation of TGF-{beta}1 mRNA, enhanced TGF-{beta}1 secretion, and bioactivity. VEGF-induced endothelial cell migration and TGF-{beta}1 induction were mediated by the phosphatidyl-inositol-3 kinase pathway, while proliferation was dependent on the Erk1/2 MAP kinase pathway. This suggests that differential modulation of glomerular angiogenesis by selective inhibition of the two identified VEGF-induced signaling pathways could be a therapeutic approach to treat kidney fibrosis.

  12. PI3-kinase/Akt pathway-regulated membrane insertion of acid-sensing ion channel 1a underlies BDNF-induced pain hypersensitivity.

    PubMed

    Duan, Bo; Liu, Di-Shi; Huang, Yu; Zeng, Wei-Zheng; Wang, Xiang; Yu, Hui; Zhu, Michael X; Chen, Zhe-Yu; Xu, Tian-Le

    2012-05-02

    Central neural plasticity plays a key role in pain hypersensitivity. This process is modulated by brain-derived neurotrophic factor (BDNF) and also involves the type 1a acid-sensing ion channel (ASIC1a). However, the interactions between the BDNF receptor, tropomyosin-related kinase B (TrkB), and ASIC1a are unclear. Here, we show that deletion of ASIC1 gene suppressed the sustained mechanical hyperalgesia induced by intrathecal BDNF application in mice. In both rat spinal dorsal horn neurons and heterologous cell cultures, the BDNF/TrkB pathway enhanced ASIC1a currents via phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB/Akt) cascade and phosphorylation of cytoplasmic residue Ser-25 of ASIC1a, resulting in enhanced forward trafficking and increased surface expression. Moreover, in both rats and mice, this enhanced ASIC1a activity was required for BDNF-mediated hypersensitivity of spinal dorsal horn nociceptive neurons and central mechanical hyperalgesia, a process that was abolished by intrathecal application of a peptide representing the N-terminal region of ASIC1a encompassing Ser-25. Thus, our results reveal a novel mechanism underlying central sensitization and pain hypersensitivity, and reinforce the critical role of ASIC1a channels in these processes.

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

    PubMed

    Moroi, Alyssa J; Watson, Steve P

    2015-04-01

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

  14. Polycystin-1 Induces Cell Migration by Regulating Phosphatidylinositol 3-kinase-dependent Cytoskeletal Rearrangements and GSK3β-dependent Cell–Cell Mechanical Adhesion

    PubMed Central

    Boca, Manila; D'Amato, Lisa; Distefano, Gianfranco; Polishchuk, Roman S.; Germino, Gregory G.

    2007-01-01

    Polycystin-1 (PC-1) is a large plasma-membrane receptor encoded by the PKD1 gene mutated in autosomal dominant polycystic kidney disease (ADPKD). Although the disease is thought to be recessive on a molecular level, the precise mechanism of cystogenesis is unclear, although cytoarchitecture defects seem to be the most likely initiating events. Here we show that PC-1 regulates the actin cytoskeleton in renal epithelial cells (MDCK) and induces cell scattering and cell migration. All of these effects require phosphatidylinositol 3-kinase (PI3-K) activity. Consistent with these observations Pkd1−/− mouse embryonic fibroblasts (MEFs) have reduced capabilities to migrate compared with controls. PC-1 overexpressing MDCK cells are able to polarize normally with proper adherens and tight junctions formation, but show quick reabsorption of ZO-1, E-cadherin, and β-catenin upon wounding of a monolayer and a transient epithelial-to-mesenchymal transition (EMT) that favors a rapid closure of the wound and repolarization. Finally, we show that PC-1 is able to control the turnover of cytoskeletal-associated β-catenin through activation of GSK3β. Expression of a nondegradable form of β-catenin in PC-1 MDCK cells restores strong cell–cell mechanical adhesion. We propose that PC-1 might be a central regulator of epithelial plasticity and its loss results in impaired normal epithelial homeostasis. PMID:17671167

  15. 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.

  16. Arabidopsis inositol polyphosphate 6-/3-kinase is a nuclear protein that complements a yeast mutant lacking a functional ArgR-Mcm1 transcription complex.

    PubMed

    Xia, Hui-Jun; Brearley, Charles; Elge, Stephan; Kaplan, Boaz; Fromm, Hillel; Mueller-Roeber, Bernd

    2003-02-01

    Inositol 1,4,5-trisphosphate 3-kinase, and more generally inositol polyphosphate kinases (Ipk), play important roles in signal transduction in animal cells; however, their functions in plant cells remain to be elucidated. Here, we report the molecular cloning of a cDNA (AtIpk2beta) from a higher plant, Arabidopsis. Arabidopsis AtIpk2beta is a 33-kD protein that exhibits weak homology ( approximately 25% identical amino acids) with Ipk proteins from animals and yeast and lacks a calmodulin binding site, as revealed by sequence analysis and calmodulin binding assays. However, recombinant AtIpk2beta phosphorylates inositol 1,4,5-trisphosphate to inositol 1,4,5,6-tetrakisphosphate and also converts it to inositol 1,3,4,5,6-pentakisphosphate [Ins(1,3,4,5,6)P(5)]. AtIpk2beta also phosphorylates inositol 1,3,4,5-tetrakisphosphate to Ins(1,3,4,5,6)P(5). Thus, the enzyme is a D3/D6 dual-specificity inositol phosphate kinase. AtIpk2beta complements a yeast ARG82/IPK2 mutant lacking a functional ArgR-Mcm1 transcription complex. This complex is involved in regulating Arg metabolism-related gene expression and requires inositol polyphosphate kinase activity to function. AtIpk2beta was found to be located predominantly in the nucleus of plant cells, as demonstrated by immunolocalization and fusion to green fluorescent protein. RNA gel blot analysis and promoter-beta-glucuronidase reporter gene studies demonstrated AtIpk2beta gene expression in various organs tested. These data suggest a role for AtIpk2beta as a transcriptional control mediator in plants.

  17. Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growth factor

    PubMed Central

    Holgado-Madruga, Marina; Moscatello, David K.; Emlet, David R.; Dieterich, Rebekka; Wong, Albert J.

    1997-01-01

    Nerve growth factor (NGF) prevents apoptosis through stimulation of the TrkA receptor protein tyrosine kinase. The downstream activation of phosphatidylinositol 3-kinase (PI 3-kinase) is essential for the inhibition of apoptosis, although this enzyme does not bind to and is not directly activated by TrkA. We have found that the addition of NGF to PC-12 cells resulted in the phosphorylation of the Grb2-associated binder-1 (Gab1) docking protein and induced the association of several SH2 domain-containing proteins, including PI 3-kinase. A substantial fraction of the total cellular PI 3-kinase activity was associated with Gab1. PC-12 cells that overexpressed Gab1 show a decreased requirement for the amount of NGF necessary to inhibit apoptosis. The expression of a Gab1 mutant that lacked the binding sites for PI 3-kinase enhanced apoptosis and diminished the protective effect of NGF. Hence, Gab1 has a major role in connecting TrkA with PI 3-kinase activation and for the promotion of cell survival by NGF. PMID:9356464

  18. Restructuring of focal adhesion plaques by PI 3-kinase. Regulation by PtdIns (3,4,5)-p(3) binding to alpha-actinin.

    PubMed

    Greenwood, J A; Theibert, A B; Prestwich, G D; Murphy-Ullrich, J E

    2000-08-07

    Focal adhesions are an elaborate network of interconnecting proteins linking actin stress fibers to the extracellular matrix substrate. Modulation of the focal adhesion plaque provides a mechanism for the regulation of cellular adhesive strength. Using interference reflection microscopy, we found that activation of phosphoinositide 3-kinase (PI 3-kinase) by PDGF induces the dissipation of focal adhesions. Loss of this close apposition between the cell membrane and the extracellular matrix coincided with a redistribution of alpha-actinin and vinculin from the focal adhesion complex to the Triton X-100-soluble fraction. In contrast, talin and paxillin remained localized to focal adhesions, suggesting that activation of PI 3-kinase induced a restructuring of the plaque rather than complete dispersion. Furthermore, phosphatidylinositol (3,4, 5)-trisphosphate (PtdIns (3,4,5)-P(3)), a lipid product of PI 3-kinase, was sufficient to induce restructuring of the focal adhesion plaque. We also found that PtdIns (3,4,5)-P(3) binds to alpha-actinin in PDGF-treated cells. Further evidence demonstrated that activation of PI 3-kinase by PDGF induced a decrease in the association of alpha-actinin with the integrin beta subunit, and that PtdIns (3,4,5)-P(3) could disrupt this interaction in vitro. Modification of focal adhesion structure by PI 3-kinase and its lipid product, PtdIns (3,4,5)-P(3), has important implications for the regulation of cellular adhesive strength and motility.

  19. Acadesine Inhibits Tissue Factor Induction and Thrombus Formation by Activating the Phosphoinositide 3-Kinase/Akt Signaling Pathway

    PubMed Central

    Zhang, Weiyu; Wang, Jianguo; Wang, Huan; Tang, Rong; Belcher, John D.; Viollet, Benoit; Geng, Jian-Guo; Zhang, Chunxiang; Wu, Chaodong; Slungaard, Arne; Zhu, Chuhong; Huo, Yuqing

    2013-01-01

    Objective Acadesine, an adenosine-regulating agent and activator of AMP-activated protein kinase, has been shown to possess antiinflammatory activity. This study investigated whether and how acadesine inhibits tissue factor (TF) expression and thrombus formation. Methods and Results Human umbilical vein endothelial cells and human peripheral blood monocytes were stimulated with lipopolysaccharide to induce TF expression. Pretreatment with acadesine dramatically suppressed the clotting activity and expression of TF (protein and mRNA). These inhibitory effects of acadesine were unchanged for endothelial cells treated with ZM241385 (a specific adenosine A2A receptor antagonist) or AMP-activated protein kinase inhibitor compound C, and in macrophages lacking adenosine A2A receptor or α1–AMP-activated protein kinase. In endothelial cells and macrophages, acadesine activated the phosphoinositide 3-kinase/Akt signaling pathway, reduced the activity of mitogen-activated protein kinases, and consequently suppressed TF expression by inhibiting the activator protein-1 and NF-κB pathways. In mice, acadesine suppressed lipopolysaccharide-mediated increases in blood coagulation, decreased TF expression in atherosclerotic lesions, and reduced deep vein thrombus formation. Conclusion Acadesine inhibits TF expression and thrombus formation by activating the phosphoinositide 3-kinase/Akt pathway. This novel finding implicates acadesine as a potentially useful treatment for many disorders associated with thrombotic pathology, such as angina pain, deep vein thrombosis, and sepsis. PMID:20185792

  20. Binding site identification and role of permanent water molecule of PIM-3 kinase: A molecular dynamics study.

    PubMed

    Ul-Haq, Zaheer; Gul, Sana; Usmani, Saman; Wadood, Abdul; Khan, Waqasuddin

    2015-11-01

    The kinome is a protein kinase complement of the human genome, categorized as serine/threonine and tyrosine kinases. These kinases catalyze phosphorylation reaction by using ATP as phosphoryl donor. Proviral Integration Site for Moloney Murine Leukemia Virus (PIM) kinase encodes serine/threonine protein kinases that recognized as proto-oncogene, responsible for rapid growth of cancerous cells. It is implicated in cell survival and function via cell cycle progression and its metabolism. PIM-3, sub-member of PIM kinases is a proto-oncogene, its overexpression inhibits apoptosis, and results in progression of hepatocellular carcinoma. PIM-3 is considered as a promising drug target but attempts to develop its specific inhibitors is slowed down due to the lack of 3D structure by any experimental technique. In silico techniques generally facilitate scientist to explore hidden structural features in order to improve drug discovery. In the present study, homology modeling, molecular docking and MD simulation techniques were utilized to explore the structure and dynamics of PIM-3 kinase. Induction of water molecules during molecular docking simulation explored differences in the hinge region between PIM-1 and PIM-3 kinases that may be responsible for specificity. Furthermore, role of water molecules in the active site was also explored via radial distribution function (RDF) after a 10 ns molecular dynamics (MD) simulations. Generated RDF plots exhibited the importance of water for inhibitor binding through their bridging capability that links the ligand with binding site residues.

  1. 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

  2. Phosphoinositide 3-kinase p110δ promotes lumen formation through enhancement of apico-basal polarity and basal membrane organization

    PubMed Central

    Sar, Sokhavuth; Komaiha, Ola Hamze; Moyano, Romina; Rayal, Amel; Samuel, Didier; Shewan, Annette; Vanhaesebroeck, Bart; Mostov, Keith; Gassama-Diagne, Ama

    2016-01-01

    Signaling triggered by adhesion to the extracellular matrix plays a key role in the spatial orientation of epithelial polarity and formation of lumens in glandular tissues. Phosphoinositide 3-kinase signaling in particular is known to influence the polarization process during epithelial cell morphogenesis. Here, using Madin-Darby canine kidney epithelial cells grown in 3D culture, we show that the p110δ isoform of phosphoinositide 3-kinase colocalizes with focal adhesion proteins at the basal surface of polarized cells. Pharmacological, siRNA- or kinase-dead mediated inhibition of p110δ impair the early stages of lumen formation, resulting in inverted polarized cysts, with no laminin or type IV collagen assembly at cell/extracellular matrix contacts. p110δ also regulates the organization of focal adhesions and membrane localization of dystroglycan. Thus, we uncover a previously unrecognized role for p110δ in epithelial cells in the orientation of the apico-basal axis and lumen formation. PMID:25583025

  3. 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.

  4. 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

  5. The Role of Phosphatidylinositol-3-Kinase and AMP-Activated Kinase in the Rapid Estrogenic Attenuation of Cannabinoid-Induced Changes in Energy Homeostasis

    PubMed Central

    Jeffery, Garrett S.; Peng, Kelly C.; Wagner, Edward J.

    2011-01-01

    We sought to determine the involvement of phosphatidyl inositol 3-kinase (PI3K) and AMP-activated protein kinase (AMPK) in the estrogenic antagonism of the cannabinoid regulation of energy homeostasis. Food intake and body weight were evaluated in ovariectomized female guinea pigs treated s.c. with estradiol benzoate (EB) or its sesame oil vehicle, or the CB1 receptor antagonist AM251 or its cremephor/ethanol/0.9% saline vehicle. AMPK catalytic subunit, PI3K p85α regulatory subunit and proopiomelanocortin (POMC) gene expression was assessed via quantitative RT-PCR in microdissected hypothalamic tissue. Whole-cell patch clamp recordings were performed in hypothalamic slices. Both EB and AM251 decreased food intake and weight gain, and increased AMPKα1, AMPKα2 and PI3K p85α gene expression in the mediobasal hypothalamus. 17β-Estradiol rapidly and markedly attenuated the decreases in glutamatergic miniature excitatory postsynaptic current (mEPSC) frequency caused by the cannabinoid receptor agonist WIN 55,212-2 in POMC neurons. This rapid estrogenic diminution of cannabinoid-induced decreases in mEPSC frequency was blocked by the estrogen receptor (ER) antagonist ICI 182,780 and the PI3K inhibitor PI 828, the latter of which also prevented the AM251-induced increase in mEPSC frequency. In addition, the AMPK activator metformin reversed the EB-induced decreases in food intake and weight gain and restored the ability of WIN 55,212-2 to reduce mEPSC frequency. These data reveal that estrogens physiologically antagonize cannabinoid-induced changes in appetite and POMC neuronal activity by activating PI3K and inhibiting AMPK. As such, they provide insight into the neuroanatomical substrates and signal transduction mechanisms upon which these counter-regulatory factors converge in the control of energy homeostasis.

  6. Inhibition of phosphoinositol 3 kinase contributes to nanoparticle-mediated exaggeration of endotoxin-induced leukocyte procoagulant activity

    PubMed Central

    Ilinskaya, Anna N; Man, Sonny; Patri, Anil K; Clogston, Jeffrey D; Crist, Rachael M; Cachau, Raul E; McNeil, Scott E; Dobrovolskaia, Marina A

    2014-01-01

    Aim Disseminated intravascular coagulation is an increasing concern for certain types of engineered nanomaterials. Recent studies have shed some light on the nanoparticle physicochemical properties contributing to this toxicity; however, the mechanisms are poorly understood. Leukocyte procoagulant activity (PCA) is a key factor contributing to the initiation of this toxicity. We have previously reported on the exaggeration of endotoxin-induced PCA by cationic dendrimers. Herein, we report an effort to discern the mechanism. Materials & methods Poly(amidoamine) dendrimers with various sizes and surface functionalities were studied in vitro by the recalcification test, flow cytometry and other relevant assays. Results & conclusion Cationic dendrimers exaggerated endotoxin-induced PCA, but their anionic or neutral counterparts did not; the cationic charge prompts this phenomenon, but different cationic surface chemistries do not influence it. Cationic dendrimers and endotoxin differentially affect the PCA complex. The inhibition of phosphoinositol 3 kinase by dendrimers contributes to the exaggeration of the endotoxin-induced PCA. PMID:24279459

  7. Regioselective synthesis of 5- and 6-methoxybenzimidazole-1,3,5-triazines as inhibitors of phosphoinositide 3-kinase.

    PubMed

    Miller, Michelle S; Pinson, Jo-Anne; Zheng, Zhaohua; Jennings, Ian G; Thompson, Philip E

    2013-02-01

    Phosphoinositide 3-kinases (PI3K) hold significant therapeutic potential as novel targets for the treatment of cancer. ZSTK474 (4a) is a potent, pan-PI3K inhibitor currently under clinical evaluation for the treatment of cancer. Structural studies have shown that derivatisation at the 5- or 6-position of the benzimidazole ring may influence potency and isoform selectivity. However, synthesis of these derivatives by the traditional route results in a mixture of the two regioisomers. We have developed a straightforward regioselective synthesis that gave convenient access to 5- and 6-methoxysubstituted benzimidazole derivatives of ZSTK474. While 5-methoxy substitution abolished activity at all isoforms, the 6-methoxy substitution is consistently 10-fold more potent. This synthesis will allow convenient access to further 6-position derivatives, thus allowing the full scope of the structure-activity relationships of ZSTK474 to be probed.

  8. 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.

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

    PubMed 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

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

    PubMed

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

    1998-07-01

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

  11. The phosphatidylinositol 3-kinase inhibitor, wortmannin, inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes.

    PubMed Central

    Standaert, M L; Avignon, A; Yamada, K; Bandyopadhyay, G; Farese, R V

    1996-01-01

    We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-alpha and PKC-beta in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulated, phospholipase D-dependent PC hydrolysis and subsequent translocation of PKC-alpha and PKC-beta to the plasma membrane. Wortmannin did not inhibit PKC directly in vitro, or the PKC-dependent effects of phorbol esters on glucose transport in intact adipocytes. The PKC inhibitor RO 31-8220 did not inhibit PI 3-kinase directly or its activation in situ by insulin, but inhibited both insulin-stimulated and phorbol ester-stimulated glucose transport. Our findings suggest that insulin acts through PI 3-kinase to activate a PC-specific phospholipase D and causes the translocative activation of PKC-alpha and PKC-beta in plasma membranes of rat adipocytes. PMID:8611143

  12. In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

    PubMed Central

    Cooper, J A; Kashishian, A

    1993-01-01

    We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8382774

  13. PI3 kinase inhibition improves vascular malformations in mouse models of hereditary haemorrhagic telangiectasia

    PubMed Central

    Ola, Roxana; Dubrac, Alexandre; Han, Jinah; Zhang, Feng; Fang, Jennifer S.; Larrivée, Bruno; Lee, Monica; Urarte, Ana A.; Kraehling, Jan R.; Genet, Gael; Hirschi, Karen K.; Sessa, William C.; Canals, Francesc V.; Graupera, Mariona; Yan, Minhong; Young, Lawrence H.; Oh, Paul S.; Eichmann, Anne

    2016-01-01

    Activin receptor-like kinase 1 (ALK1) is an endothelial serine–threonine kinase receptor for bone morphogenetic proteins (BMPs) 9 and 10. Inactivating mutations in the ALK1 gene cause hereditary haemorrhagic telangiectasia type 2 (HHT2), a disabling disease characterized by excessive angiogenesis with arteriovenous malformations (AVMs). Here we show that inducible, endothelial-specific homozygous Alk1 inactivation and BMP9/10 ligand blockade both lead to AVM formation in postnatal retinal vessels and internal organs including the gastrointestinal (GI) tract in mice. VEGF and PI3K/AKT signalling are increased on Alk1 deletion and BMP9/10 ligand blockade. Genetic deletion of the signal-transducing Vegfr2 receptor prevents excessive angiogenesis but does not fully revert AVM formation. In contrast, pharmacological PI3K inhibition efficiently prevents AVM formation and reverts established AVMs. Thus, Alk1 deletion leads to increased endothelial PI3K pathway activation that may be a novel target for the treatment of vascular lesions in HHT2. PMID:27897192

  14. Janus and PI3-kinases mediate glucocorticoid resistance in activated chronic leukemia cells

    PubMed Central

    Oppermann, Sina; Lam, Avery J.; Tung, Stephanie; Shi, Yonghong; McCaw, Lindsay; Wang, Guizhei; Ylanko, Jarkko; Leber, Brian; Andrews, David; Spaner, David E.

    2016-01-01

    Glucorticoids (GCs) such as dexamethasone (DEX) remain important treatments for Chronic Lymphocytic Leukemia (CLL) but the mechanisms are poorly understood and resistance is inevitable. Proliferation centers (PC) in lymph nodes and bone marrow offer protection against many cytotoxic drugs and circulating CLL cells were found to acquire resistance to DEX-mediated killing in conditions encountered in PCs including stimulation by toll-like receptor agonists and interactions with stromal cells. The resistant state was associated with impaired glucocorticoid receptor-mediated gene expression, autocrine activation of STAT3 through Janus Kinases (JAKs), and increased glycolysis. The JAK1/2 inhibitor ruxolitinib blocked STAT3-phosphorylation and partially improved DEX-mediated killing of stimulated CLL cells in vitro but not in CLL patients in vivo. An automated microscopy-based screen of a kinase inhibitor library implicated an additional protective role for the PI3K/AKT/FOXO pathway. Blocking this pathway with the glycolysis inhibitor 2-deoxyglucose (2-DG) or the PI3K-inhibitors idelalisib and buparlisib increased DEX-mediated killing but did not block STAT3-phosphorylation. Combining idelalisib or buparlisib with ruxolitinib greatly increased killing by DEX. These observations suggest that glucocorticoid resistance in CLL cells may be overcome by combining JAK and PI3K inhibitors. PMID:27579615

  15. PI3 kinase inhibition improves vascular malformations in mouse models of hereditary haemorrhagic telangiectasia.

    PubMed

    Ola, Roxana; Dubrac, Alexandre; Han, Jinah; Zhang, Feng; Fang, Jennifer S; Larrivée, Bruno; Lee, Monica; Urarte, Ana A; Kraehling, Jan R; Genet, Gael; Hirschi, Karen K; Sessa, William C; Canals, Francesc V; Graupera, Mariona; Yan, Minhong; Young, Lawrence H; Oh, Paul S; Eichmann, Anne

    2016-11-29

    Activin receptor-like kinase 1 (ALK1) is an endothelial serine-threonine kinase receptor for bone morphogenetic proteins (BMPs) 9 and 10. Inactivating mutations in the ALK1 gene cause hereditary haemorrhagic telangiectasia type 2 (HHT2), a disabling disease characterized by excessive angiogenesis with arteriovenous malformations (AVMs). Here we show that inducible, endothelial-specific homozygous Alk1 inactivation and BMP9/10 ligand blockade both lead to AVM formation in postnatal retinal vessels and internal organs including the gastrointestinal (GI) tract in mice. VEGF and PI3K/AKT signalling are increased on Alk1 deletion and BMP9/10 ligand blockade. Genetic deletion of the signal-transducing Vegfr2 receptor prevents excessive angiogenesis but does not fully revert AVM formation. In contrast, pharmacological PI3K inhibition efficiently prevents AVM formation and reverts established AVMs. Thus, Alk1 deletion leads to increased endothelial PI3K pathway activation that may be a novel target for the treatment of vascular lesions in HHT2.

  16. Synergistic inhibition of colon carcinoma cell growth by Hedgehog-Gli1 inhibitor arsenic trioxide and phosphoinositide 3-kinase inhibitor LY294002.

    PubMed

    Cai, Xinyi; Yu, Kun; Zhang, Lijuan; Li, Yunfeng; Li, Qiang; Yang, Zhibin; Shen, Tao; Duan, Lincan; Xiong, Wei; Wang, Weiya

    2015-01-01

    The Hedgehog (Hh) signaling pathway not only plays important roles in embryogenesis and adult tissue homeostasis, but also in tumorigenesis. Aberrant Hh pathway activation has been reported in a variety of malignant tumors including colon carcinoma. Here, we sought to investigate the regulation of the Hh pathway transcription factor Gli1 by arsenic trioxide and phosphoinositide 3-kinase (PI3K) inhibitor LY294002 in colon carcinoma cells. We transfected cells with siGli1 and observed a significant reduction of Gli1 expression in HCT116 and HT29 cells, which was confirmed by quantitative real-time polymerase chain reaction and Western blots. Knocking down endogenous Gli1 reduced colon carcinoma cell viability through inducing cell apoptosis. Similarly, knocking down Gli2 using short interfering RNA impaired colon carcinoma cell growth in vitro. To elucidate the regulation of Gli1 expression, we found that both Gli inhibitor arsenic trioxide and PI3K inhibitor LY294002 significantly reduced Gli1 protein expression and colon carcinoma cell proliferation. Arsenic trioxide treatment also reduced Gli1 downstream target gene expression, such as Bcl2 and CCND1. More importantly, the inhibition of Hedgehog-Gli1 by arsenic trioxide showed synergistic anticancer effect with the PI3K inhibitor LY294002 in colon carcinoma cells. Our findings suggest that the Hh pathway transcription factor Gli1 is involved in the regulation of colon carcinoma cell viability. Inhibition of Hedgehog-Gli1 expression by arsenic trioxide and PI3K inhibitor synergistically reduces colon cancer cell proliferation, indicating that they could be used as an effective anti-colon cancer combination therapy.

  17. Oncogenic mutations weaken the interactions that stabilize the p110α-p85α heterodimer in phosphatidylinositol 3-kinase α.

    PubMed

    Echeverria, Ignacia; Liu, Yunlong; Gabelli, Sandra B; Amzel, L Mario

    2015-09-01

    Phosphatidylinositol 3-kinase (PI3K) α is a heterodimeric lipid kinase that catalyzes the conversion of phosphoinositol-4,5-bisphosphate to phosphoinositol-3,4,5-trisphosphate. The PI3Kα signaling pathway plays an important role in cell growth, proliferation, and survival. This pathway is activated in numerous cancers, where the PI3KCA gene, which encodes for the p110α PI3Kα subunit, is mutated. Its mutation often results in gain of enzymatic activity; however, the mechanism of activation by oncogenic mutations remains unknown. Here, using computational methods, we show that oncogenic mutations that are far from the catalytic site and increase the enzymatic affinity destabilize the p110α-p85α dimer. By affecting the dynamics of the protein, these mutations favor the conformations that reduce the autoinhibitory effect of the p85α nSH2 domain. For example, we determined that, in all of the mutants, the nSH2 domain shows increased positional heterogeneity as compared with the wild-type, as demonstrated by changes in the fluctuation profiles computed by normal mode analysis of coarse-grained elastic network models. Analysis of the interdomain interactions of the wild-type and mutants at the p110α-p85α interface obtained with molecular dynamics simulations suggest that all of the tumor-associated mutations effectively weaken the interactions between p110α and p85α by disrupting key stabilizing interactions. These findings have important implications for understanding how oncogenic mutations change the conformational multiplicity of PI3Kα and lead to increased enzymatic activity. This mechanism may apply to other enzymes and/or macromolecular complexes that play a key role in cell signaling.

  18. Extended treatment with selective phosphatidylinositol 3-kinase and mTOR inhibitors has effects on metabolism, growth, behaviour and bone strength.

    PubMed

    Smith, Greg C; Ong, Wee-Kiat; Costa, Jessica L; Watson, Maureen; Cornish, Jillian; Grey, Andrew; Gamble, Greg D; Dickinson, Michelle; Leung, Sophie; Rewcastle, Gordon W; Han, Weiping; Shepherd, Peter R

    2013-11-01

    The class I phosphatidylinositol 3-kinases (PtdIns3Ks) mediate the effects of many hormones and growth factors on a wide range of cellular processes, and activating mutations or gene amplifications of class I PtdIns3K isoforms are known to contribute to oncogenic processes in a range of tumours. Consequently, a number of small-molecule PtdIns3K inhibitors are under development and in clinical trial. The central signalling role of PtdIns3K in many cellular processes suggests there will be on-target side effects associated with the use of these agents. To gain insights into what these might be we investigated the effect of extended daily dosing of eight small-molecule inhibitors of class Ia PtdIns3Ks. Animals were characterized in metabolic cages to analyse food intake, oxygen consumption and movement. Insulin tolerance and body composition were analysed at the end of the experiment, the latter using EchoMRI. Bone volume and strength was assessed by micro-CT and three-point bending, respectively. Surprisingly, after sustained dosing with pan-PtdIns3K inhibitors and selective inhibitors of the p110α isoform there was a resolution of the impairments in insulin tolerance observed in drug-naïve animals treated with the same drugs. However, pan-PtdIns3K inhibitors and selective inhibitors of the p110α have deleterious effects on animal growth, animal behaviour and bone volume and strength. Together, these findings identify a range of on target effects of PtdIns3K inhibitors and suggest use of these drugs in humans may have important adverse effects on metabolism, body composition, behaviour and skeletal health.

  19. Curcumin modulates nuclear factor kappaB (NF-kappaB)-mediated inflammation in human tenocytes in vitro: role of the phosphatidylinositol 3-kinase/Akt pathway.

    PubMed

    Buhrmann, Constanze; Mobasheri, Ali; Busch, Franziska; Aldinger, Constance; Stahlmann, Ralf; Montaseri, Azadeh; Shakibaei, Mehdi

    2011-08-12

    Inflammatory processes play essential roles in the pathogenesis of tendinitis and tendinopathy. These events are accompanied by catabolic processes initiated by pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). Pharmacological treatments for tendinitis are restricted to the use of non-steroidal anti-inflammatory drugs. Recent studies in various cell models have demonstrated that curcumin targets the NF-κB signaling pathway. However, its potential for the treatment of tendinitis has not been explored. Herein, we used an in vitro model of human tenocytes to study the mechanism of curcumin action on IL-1β-mediated inflammatory signaling. Curcumin at concentrations of 5-20 μm inhibited IL-1β-induced inflammation and apoptosis in cultures of human tenocytes. The anti-inflammatory effects of curcumin included down-regulation of gene products that mediate matrix degradation (matrix metalloproteinase-1, -9, and -13), prostanoid production (cyclooxygenase-2), apoptosis (Bax and activated caspase-3), and stimulation of cell survival (Bcl-2), all known to be regulated by NF-κB. Furthermore, curcumin suppressed IL-1β-induced NF-κB activation via inhibition of phosphorylation and degradation of inhibitor of κBα, inhibition of inhibitor of κB-kinase activity, and inhibition of nuclear translocation of NF-κB. Furthermore, the effects of IL-1β were abrogated by wortmannin, suggesting a role for the phosphatidylinositol 3-kinase (PI-3K) pathway in IL-1β signaling. Curcumin suppressed IL-1β-induced PI-3K p85/Akt activation and its association with IKK. These results demonstrate, for the first time, a potential role for curcumin in treating tendon inflammation through modulation of NF-κB signaling, which involves PI-3K/Akt and the tendon-specific transcription factor scleraxis in tenocytes.

  20. Effect of phosphatidylinositol-3 kinase inhibition on ovotoxicity caused by 4-vinylcyclohexene diepoxide and 7, 12-dimethylbenz[a]anthracene in neonatal rat ovaries

    SciTech Connect

    Keating, Aileen F.; Mark, Connie J.; Sen, Nivedita; Sipes, I. Glenn; Hoyer, Patricia B.

    2009-12-01

    4-vinylcyclohexene diepoxide (VCD) is an ovotoxicant that specifically destroys primordial and small primary follicles in the ovaries of mice and rats. In contrast, 7,12-dimethylbenz[a]anthracene (DMBA) is ovotoxic to all ovarian follicle classes. This study investigated phosphatidylinositol-3 kinase signaling involvement in VCD- and DMBA-induced ovotoxicity. Postnatal day (PND) 4 Fischer 344 (F344) rat whole ovaries were cultured for 2-12 days in vehicle control, VCD (30 muM), or DMBA (1 muM), +- PI3 kinase inhibitor LY294002 (20 muM) or its inactive analog LY303511 (20 muM). Following culture, ovaries were histologically evaluated, and healthy follicles were classified and counted. PI3 kinase inhibition had no effect on primordial follicle number, but reduced (P < 0.05) small primary and larger follicles beginning on day 4. VCD caused primordial and small primary follicle loss (P < 0.05) beginning on day 6. With PI3 kinase inhibition, VCD did not affect primordial follicles (P > 0.05) at any time, but did cause loss (P < 0.05) of small primary follicles. DMBA exposure caused primordial and small primary follicle loss (P < 0.05) on day 6. Further, DMBA-induced primordial and small primary follicle loss was greater with PI3 kinase inhibition (P < 0.05) than with DMBA alone. These results support that (1) PI3 kinase mediates primordial to small primary follicle recruitment, (2) VCD, but not DMBA, enhances ovotoxicity by increasing primordial to small primary follicle recruitment, and (3) in addition to xenobiotic-induced ovotoxicity, VCD is also a useful model chemical with which to elucidate signaling mechanisms involved in primordial follicle recruitment.

  1. Specific activation of p85-p110 phosphatidylinositol 3'-kinase stimulates DNA synthesis by ras- and p70 S6 kinase-dependent pathways.

    PubMed Central

    McIlroy, J; Chen, D; Wjasow, C; Michaeli, T; Backer, J M

    1997-01-01

    We have developed a polyclonal antibody that activates the heterodimeric p85-p110 phosphatidylinositol (PI) 3'-kinase in vitro and in microinjected cells. Affinity purification revealed that the activating antibody recognized the N-terminal SH2 (NSH2) domain of p85, and the antibody increased the catalytic activity of recombinant p85-p110 dimers threefold in vitro. To study the role of endogenous PI 3'-kinase in intact cells, the activating anti-NSH2 antibody was microinjected into GRC + LR73 cells, a CHO cell derivative selected for tight quiescence during serum withdrawal. Microinjection of anti-NSH2 antibodies increased bromodeoxyuridine (BrdU) incorporation fivefold in quiescent cells and enhanced the response to serum. These data reflect a specific activation of PI 3'-kinase, as the effect was blocked by coinjection of the appropriate antigen (glutathione S-transferase-NSH2 domains from p85 alpha), coinjection of inhibitory anti-p110 antibodies, or treatment of cells with wortmannin. We used the activating antibodies to study signals downstream from PI 3'-kinase. Although treatment of cells with 50 nM rapamycin only partially decreased anti-NSH2-stimulated BrdU incorporation, coinjection with an anti-p70 S6 kinase antibody effectively blocked anti-NSH2-stimulated DNA synthesis. We also found that coinjection of inhibitory anti-ras antibodies blocked both serum- and anti-NSH2-stimulated BrdU incorporation by approximately 60%, and treatment of cells with a specific inhibitor of MEK abolished antibody-stimulated BrdU incorporation. We conclude that selective activation of physiological levels of PI 3'-kinase is sufficient to stimulate DNA synthesis in quiescent cells. PI 3'-kinase-mediated DNA synthesis requires both p70 S6 kinase and the P21ras/MEK pathway. PMID:8972205

  2. Cudraflavone C Induces Tumor-Specific Apoptosis in Colorectal Cancer Cells through Inhibition of the Phosphoinositide 3-Kinase (PI3K)-AKT Pathway

    PubMed Central

    Soo, Hsien-Chuen; Chung, Felicia Fei-Lei; Lim, Kuan-Hon; Yap, Veronica Alicia; Bradshaw, Tracey D.; Hii, Ling-Wei; Tan, Si-Hoey; See, Sze-Jia; Tan, Yuen-Fen; Leong, Chee-Onn

    2017-01-01

    Cudraflavone C (Cud C) is a naturally-occurring flavonol with reported anti-proliferative activities. However, the mechanisms by which Cud C induced cytotoxicity have yet to be fully elucidated. Here, we investigated the effects of Cud C on cell proliferation, caspase activation andapoptosis induction in colorectal cancer cells (CRC). We show that Cud C inhibits cell proliferation in KM12, Caco-2, HT29, HCC2998, HCT116 and SW48 CRC but not in the non-transformed colorectal epithelial cells, CCD CoN 841. Cud C induces tumor-selective apoptosis via mitochondrial depolarization and activation of the intrinsic caspase pathway. Gene expression profiling by microarray analyses revealed that tumor suppressor genes EGR1, HUWE1 and SMG1 were significantly up-regulated while oncogenes such as MYB1, CCNB1 and GPX2 were down-regulated following treatment with Cud C. Further analyses using Connectivity Map revealed that Cud C induced a gene signature highly similar to that of protein synthesis inhibitors and phosphoinositide 3-kinase (PI3K)-AKT inhibitors, suggesting that Cud C might inhibit PI3K-AKT signaling. A luminescent cell free PI3K lipid kinase assay revealed that Cud C significantly inhibited p110β/p85α PI3K activity, followed by p120γ, p110δ/p85α, and p110α/p85α PI3K activities. The inhibition by Cud C on p110β/p85α PI3K activity was comparable to LY-294002, a known PI3K inhibitor. Cud C also inhibited phosphorylation of AKT independent of NFκB activity in CRC cells, while ectopic expression of myristoylated AKT completely abrogated the anti-proliferative effects, and apoptosis induced by Cud C in CRC. These findings demonstrate that Cud C induces tumor-selective cytotoxicity by targeting the PI3K-AKT pathway. These findings provide novel insights into the mechanism of action of Cud C, and indicate that Cud C further development of Cud C derivatives as potential therapeutic agents is warranted. PMID:28107519

  3. 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.

  4. 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

  5. Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

    PubMed

    Toubiana, Julie; Rossi, Anne-Lise; Belaidouni, Nadia; Grimaldi, David; Pene, Frederic; Chafey, Philippe; Comba, Béatrice; Camoin, Luc; Bismuth, Georges; Claessens, Yann-Erick; Mira, Jean-Paul; Chiche, Jean-Daniel

    2015-10-01

    TLR2 has a prominent role in host defense against a wide variety of pathogens. Stimulation of TLR2 triggers MyD88-dependent signaling to induce NF-κB translocation, and activates a Rac1-PI 3-kinase dependent pathway that leads to transactivation of NF-κB through phosphorylation of the P65 NF-κB subunit. This transactivation pathway involves tyrosine phosphorylations. The role of the tyrosine kinases in TLR signaling is controversial, with discrepancies between studies using only chemical inhibitors and knockout mice. Here, we show the involvement of the tyrosine-kinase Lyn in TLR2-dependent activation of NF-κB in human cellular models, by using complementary inhibition strategies. Stimulation of TLR2 induces the formation of an activation cluster involving TLR2, CD14, PI 3-kinase and Lyn, and leads to the activation of AKT. Lyn-dependent phosphorylation of the p110 catalytic subunit of PI 3-kinase is essential to the control of PI 3-kinase biological activity upstream of AKT and thereby to the transactivation of NF-κB. Thus, Lyn kinase activity is crucial in TLR2-mediated activation of the innate immune response in human mononuclear cells.

  6. Platelet-derived growth factor triggers translocation of the insulin-regulatable glucose transporter (type 4) predominantly through phosphatidylinositol 3-kinase binding sites on the receptor.

    PubMed Central

    Kamohara, S; Hayashi, H; Todaka, M; Kanai, F; Ishii, K; Imanaka, T; Escobedo, J A; Williams, L T; Ebina, Y

    1995-01-01

    Insulin is the only known hormone which rapidly stimulates glucose uptake in target tissues, mainly by translocation to the cell surface of the intracellular insulin-regulatable glucose transporter (glucose transporter type 4, GLUT4). We have developed a cell line for direct, sensitive detection of GLUT4 on the cell surface. We have suggested that insulin-activated phosphatidylinositol (PI) 3-kinase may be involved in the signaling pathway of insulin-stimulated GLUT4 translocation. We report that platelet-derived growth factor (PDGF), which stimulates PI 3-kinase activity, triggers GLUT4 translocation in Chinese hamster ovary (CHO) cells stably overexpressing the PDGF receptor and in 3T3-L1 mouse adipocytes. Using mutant PDGF receptors that cannot bind to Ras-GTPase-activating protein, phospholipase C-gamma, and PI 3-kinase, respectively, we obtained evidence that PI 3-kinase binding sites play a key role in the signaling pathway of PDGF-stimulated GLUT4 translocation in the CHO cell system. Images Fig. 1 Fig. 4 PMID:7862637

  7. In vitro glucose uptake activity of Aegles marmelos and Syzygium cumini by activation of Glut-4, PI3 kinase and PPARgamma in L6 myotubes.

    PubMed

    Anandharajan, R; Jaiganesh, S; Shankernarayanan, N P; Viswakarma, R A; Balakrishnan, A

    2006-06-01

    The purpose of the present study is to investigate the effect of methanolic extracts of Aegles marmelos and Syzygium cumini on a battery of targets glucose transporter (Glut-4), peroxisome proliferator activator receptor gamma (PPARgamma) and phosphatidylinositol 3' kinase (PI3 kinase) involved in glucose transport. A. marmelos and S. cumini are anti-diabetic medicinal plants being used in Indian traditional medicine. Different solvent extracts extracted sequentially were analysed for glucose uptake activity at each step and methanol extracts were found to be significantly active at 100ng/ml dose comparable with insulin and rosiglitazone. Elevation of Glut-4, PPARgamma and PI3 kinase by A. marmelos and S. cumini in association with glucose transport supported the up-regulation of glucose uptake. The inhibitory effect of cycloheximide on A. marmelos- and S. cumini-mediated glucose uptake suggested that new protein synthesis is required for the elevated glucose transport. Current observation concludes that methanolic extracts of A. marmelos and S. cumini activate glucose transport in a PI3 kinase-dependent fashion.

  8. PI-103, a dual inhibitor of Class IA phosphatidylinositide 3-kinase and mTOR, has antileukemic activity in AML.

    PubMed

    Park, S; Chapuis, N; Bardet, V; Tamburini, J; Gallay, N; Willems, L; Knight, Z A; Shokat, K M; Azar, N; Viguié, F; Ifrah, N; Dreyfus, F; Mayeux, P; Lacombe, C; Bouscary, D

    2008-09-01

    The phosphatidylinositol 3-kinase (PI3K)/Akt and mammalian target of rapamycin complex 1 (mTORC1) signaling pathways are frequently activated in acute myelogenous leukemia (AML). mTORC1 inhibition with RAD001 induces PI3K/Akt activation and both pathways are activated independently, providing a rationale for dual inhibition of both pathways. PI-103 is a new potent PI3K/Akt and mTOR inhibitor. In human leukemic cell lines and in primary blast cells from AML patients, PI-103 inhibited constitutive and growth factor-induced PI3K/Akt and mTORC1 activation. PI-103 was essentially cytostatic for cell lines and induced cell cycle arrest in the G1 phase. In blast cells, PI-103 inhibited leukemic proliferation, the clonogenicity of leukemic progenitors and induced mitochondrial apoptosis, especially in the compartment containing leukemic stem cells. In contrast, apoptosis was not induced with RAD001 and IC87114 association, which specifically inhibits mTORC1 and p110delta activity, respectively. PI-103 had additive proapoptotic effects with etoposide in blast cells and in immature leukemic cells. Interestingly, PI-103 did not induce apoptosis in normal CD34(+) cells and had moderate effects on their clonogenic and proliferative properties. Here, we demonstrate that multitargeted therapy against PI3K/Akt and mTOR with PI-103 may be of therapeutic value in AML.

  9. Targeting the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling network in cancer stem cells.

    PubMed

    Martelli, A M; Evangelisti, C; Follo, M Y; Ramazzotti, G; Fini, M; Giardino, R; Manzoli, L; McCubrey, J A; Cocco, L

    2011-01-01

    Cancer stem cells (CSCs) comprise a subset of hierarchically organized, rare cancer cells with the ability to initiate cancer in xenografts of genetically modified murine models. CSCs are thought to be responsible for tumor onset, self-renewal/maintenance, mutation accumulation, and metastasis. The existence of CSCs could explain the high frequency of neoplasia relapse and resistance to all of currently available therapies, including chemotherapy. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway is a key regulator of physiological cell processes which include proliferation, differentiation, apoptosis, motility, metabolism, and autophagy. Nevertheless, aberrantly upregulated PI3K/Akt/mTOR signaling characterizes many types of cancers where it negatively influences prognosis. Several lines of evidence indicate that this signaling system plays a key role also in CSC biology. Of note, CSCs are more sensitive to pathway inhibition with small molecules when compared to healthy stem cells. This observation provides the proof-of-principle that functional differences in signaling transduction pathways between CSCs and healthy stem cells can be identified. Here, we review the evidence which links the signals deriving from the PI3K/Akt/mTOR network with CSC biology, both in hematological and solid tumors. We then highlight how therapeutic targeting of PI3K/Akt/mTOR signaling with small molecule inhibitors could improve cancer patient outcome, by eliminating CSCs.

  10. Phosphatidylinositol 3-Kinase Mediates Bronchioalveolar Stem Cell Expansion in Mouse Models of Oncogenic K-ras-Induced Lung Cancer

    PubMed Central

    Yang, Yanan; Iwanaga, Kentaro; Raso, Maria Gabriela; Wislez, Marie; Hanna, Amy E.; Wieder, Eric D.; Molldrem, Jeffrey J.; Wistuba, Ignacio I.; Powis, Garth; Demayo, Francesco J.; Kim, Carla F.; Kurie, Jonathan M.

    2008-01-01

    Background Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in Western countries. Developing more effective NSCLC therapeutics will require the elucidation of the genetic and biochemical bases for this disease. Bronchioalveolar stem cells (BASCs) are a putative cancer stem cell population in mouse models of oncogenic K-ras-induced lung adenocarcinoma, an histologic subtype of NSCLC. The signals activated by oncogenic K-ras that mediate BASC expansion have not been fully defined. Methodology/Principal Findings We used genetic and pharmacologic approaches to modulate the activity of phosphatidylinositol 3-kinase (PI3K), a key mediator of oncogenic K-ras, in two genetic mouse models of lung adenocarcinoma. Oncogenic K-ras-induced BASC accumulation and tumor growth were blocked by treatment with a small molecule PI3K inhibitor and enhanced by inactivation of phosphatase and tensin homologue deleted from chromosome 10, a negative regulator of PI3K. Conclusions/Significance We conclude that PI3K is a critical regulator of BASC expansion, supporting treatment strategies to target PI3K in NSCLC patients. PMID:18493606

  11. MST3 Kinase Phosphorylates TAO1/2 to Enable Myosin Va Function in Promoting Spine Synapse Development

    PubMed Central

    Ultanir, Sila K.; Yadav, Smita; Hertz, Nicholas T.; Oses-Prieto, Juan A.; Claxton, Suzanne; Burlingame, Alma L.; Shokat, Kevan M.; Jan, Lily Y.; Jan, Yuh-Nung

    2014-01-01

    Summary Mammalian Sterile 20 (Ste20)-like kinase 3 (MST3) is a ubiquitously expressed kinase capable of enhancing axon outgrowth. Whether and how MST3 kinase signaling might regulate development of dendritic filopodia and spine synapses is unknown. Through shRNA-mediated depletion of MST3 and kinase-dead MST3 expression in developing hippocampal cultures, we found that MST3 is necessary for proper filopodia, dendritic spine, and excitatory synapse development. Knockdown of MST3 in layer 2/3 pyramidal neurons via in utero electroporation also reduced spine density in vivo. Using chemical genetics, we discovered thirteen candidate MST3 substrates and identified the phosphorylation sites. Among the identified MST3 substrates, TAO kinases regulate dendritic filopodia and spine development, similar to MST3. Furthermore, using stable isotope labeling by amino acids in culture (SILAC), we show that phosphorylated TAO1/2 associates with Myosin Va and is necessary for its dendritic localization, thus revealing a mechanism for excitatory synapse development in the mammalian CNS. PMID:25456499

  12. MST3 kinase phosphorylates TAO1/2 to enable Myosin Va function in promoting spine synapse development.

    PubMed

    Ultanir, Sila K; Yadav, Smita; Hertz, Nicholas T; Oses-Prieto, Juan A; Claxton, Suzanne; Burlingame, Alma L; Shokat, Kevan M; Jan, Lily Y; Jan, Yuh-Nung

    2014-12-03

    Mammalian Sterile 20 (Ste20)-like kinase 3 (MST3) is a ubiquitously expressed kinase capable of enhancing axon outgrowth. Whether and how MST3 kinase signaling might regulate development of dendritic filopodia and spine synapses is unknown. Through shRNA-mediated depletion of MST3 and kinase-dead MST3 expression in developing hippocampal cultures, we found that MST3 is necessary for proper filopodia, dendritic spine, and excitatory synapse development. Knockdown of MST3 in layer 2/3 pyramidal neurons via in utero electroporation also reduced spine density in vivo. Using chemical genetics, we discovered thirteen candidate MST3 substrates and identified the phosphorylation sites. Among the identified MST3 substrates, TAO kinases regulate dendritic filopodia and spine development, similar to MST3. Furthermore, using stable isotope labeling by amino acids in culture (SILAC), we show that phosphorylated TAO1/2 associates with Myosin Va and is necessary for its dendritic localization, thus revealing a mechanism for excitatory synapse development in the mammalian CNS.

  13. Distinct regulation of autophagic activity by Atg14L and Rubicon associated with Beclin 1-phosphatidylinositol-3-kinase complex.

    PubMed

    Zhong, Yun; Wang, Qing Jun; Li, Xianting; Yan, Ying; Backer, Jonathan M; Chait, Brian T; Heintz, Nathaniel; Yue, Zhenyu

    2009-04-01

    Beclin 1, a mammalian autophagy protein that has been implicated in development, tumour suppression, neurodegeneration and cell death, exists in a complex with Vps34, the class III phosphatidylinositol-3-kinase (PI(3)K) that mediates multiple vesicle-trafficking processes including endocytosis and autophagy. However, the precise role of the Beclin 1-Vps34 complex in autophagy regulation remains to be elucidated. Combining mouse genetics and biochemistry, we have identified a large in vivo Beclin 1 complex containing the known proteins Vps34, p150/Vps15 and UVRAG, as well as two newly identified proteins, Atg14L (yeast Atg14-like) and Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein). Characterization of the new proteins revealed that Atg14L enhances Vps34 lipid kinase activity and upregulates autophagy, whereas Rubicon reduces Vps34 activity and downregulates autophagy. We show that Beclin 1 and Atg14L synergistically promote the formation of double-membraned organelles that are associated with Atg5 and Atg12, whereas forced expression of Rubicon results in aberrant late endosomal/lysosomal structures and impaired autophagosome maturation. We hypothesize that by forming distinct protein complexes, Beclin 1 and its binding proteins orchestrate the precise function of the class III PI(3)K in regulating autophagy at multiple steps.

  14. Force engages vinculin and promotes tumor progression by enhancing PI3-kinase activation of phosphatidylinositol (3,4,5)-triphosphate

    PubMed Central

    Rubashkin, MG; Cassereau, L; Bainer, R; DuFort, CC; Yui, Y; Ou, G; Paszek, MJ; Davidson, MW; Chen, YY; Weaver, VM

    2014-01-01

    Extracellular matrix stiffness induces focal adhesion assembly to drive malignant transformation and tumor metastasis. Nevertheless, how force alters focal adhesions to promote tumor progression remains unclear. Here, we explored the role of the focal adhesion protein vinculin, a force-activated mechano-transducer, in mammary epithelial tissue transformation and invasion. We found that extracellular matrix stiffness stabilizes the assembly of a vinculin-talin-actin scaffolding complex that facilitates PI3-kinase mediated phosphatidylinositol (3,4,5)-triphosphate phosphorylation. Using defined two and three dimensional matrices, a mouse model of mammary tumorigenesis with vinculin mutants and a novel super resolution imaging approach, we established that ECM stiffness, per se, promotes the malignant progression of a mammary epithelium by activating and stabilizing vinculin and enhancing Akt signaling at focal adhesions. Our studies also revealed that vinculin strongly co-localizes with activated Akt at the invasive border of human breast tumors, where the ECM is stiffest and we detected elevated mechano-signaling. Thus, extracellular matrix stiffness could induce tumor progression by promoting the assembly of signaling scaffolds; a conclusion underscored by the significant association we observed between highly expressed focal adhesion plaque proteins and malignant transformation across multiple types of solid cancer. PMID:25183785

  15. SUMOylation of DNA topoisomerase IIα regulates histone H3 kinase Haspin and H3 phosphorylation in mitosis

    PubMed Central

    Yoshida, Makoto M.; Ting, Lily; Gygi, Steven P.

    2016-01-01

    DNA topoisomerase II (TOP2) plays a pivotal role in faithful chromosome separation through its strand-passaging activity that resolves tangled genomic DNA during mitosis. Additionally, TOP2 controls progression of mitosis by activating cell cycle checkpoints. Recent work showed that the enzymatically inert C-terminal domain (CTD) of TOP2 and its posttranslational modification are critical to this checkpoint regulation. However, the molecular mechanism has not yet been determined. By using Xenopus laevis egg extract, we found that SUMOylation of DNA topoisomerase IIα (TOP2A) CTD regulates the localization of the histone H3 kinase Haspin and phosphorylation of histone H3 at threonine 3 at the centromere, two steps known to be involved in the recruitment of the chromosomal passenger complex (CPC) to kinetochores in mitosis. Robust centromeric Haspin localization requires SUMOylated TOP2A CTD binding activity through SUMO-interaction motifs and the phosphorylation of Haspin. We propose a novel mechanism through which the TOP2 CTD regulates the CPC via direct interaction with Haspin at mitotic centromeres. PMID:27325792

  16. Novel Anti-Microbial Peptide SR-0379 Accelerates Wound Healing via the PI3 Kinase/Akt/mTOR Pathway

    PubMed Central

    Tomioka, Hideki; Nakagami, Hironori; Tenma, Akiko; Saito, Yoshimi; Kaga, Toshihiro; Kanamori, Toshihide; Tamura, Nao; Tomono, Kazunori; Kaneda, Yasufumi; Morishita, Ryuichi

    2014-01-01

    We developed a novel cationic antimicrobial peptide, AG30/5C, which demonstrates angiogenic properties similar to those of LL-37 or PR39. However, improvement of its stability and cost efficacy are required for clinical application. Therefore, we examined the metabolites of AG30/5C, which provided the further optimized compound, SR-0379. SR-0379 enhanced the proliferation of human dermal fibroblast cells (NHDFs) via the PI3 kinase-Akt-mTOR pathway through integrin-mediated interactions. Furthermore SR-0379 promoted the tube formation of human umbilical vein endothelial cells (HUVECs) in co-culture with NHDFs. This compound also displays antimicrobial activities against a number of bacteria, including drug-resistant microbes and fungi. We evaluated the effect of SR-0379 in two different would-healing models in rats, the full-thickness defects under a diabetic condition and an acutely infected wound with full-thickness defects and inoculation with Staphylococcus aureus. Treatment with SR-0379 significantly accelerated wound healing when compared to fibroblast growth factor 2 (FGF2). The beneficial effects of SR-0379 on wound healing can be explained by enhanced angiogenesis, granulation tissue formation, proliferation of endothelial cells and fibroblasts and antimicrobial activity. These results indicate that SR-0379 may have the potential for drug development in wound repair, even under especially critical colonization conditions. PMID:24675668

  17. Tannerella forsythia invasion in oral epithelial cells requires phosphoinositide 3-kinase activation and clathrin-mediated endocytosis.

    PubMed

    Mishima, Elina; Sharma, Ashu

    2011-08-01

    Tannerella forsythia, a Gram-negative anaerobe implicated in periodontitis, has been detected within human buccal epithelial cells and shown to invade oral epithelial cells in vitro. We have previously shown that this bacterium triggers host tyrosine kinase-dependent phosphorylation and actin-dependent cytoskeleton reorganization for invasion. On the bacterial side, the leucine-rich repeat cell-surface BspA protein is important for entry. The present study was undertaken to identify host signalling molecules during T. forsythia entry into human oral and cervical epithelial cells. Specifically, the roles of phosphatidylinositol 3-kinase (PI3K), Rho-family GTPases, cholesterol-rich membrane microdomains and the endocytic protein clathrin were investigated. For this purpose, cell lines were pretreated with chemical inhibitors or small interfering RNAs (siRNAs) that target PI3Ks, Rho GTPases, clathrin and cholesterol (a critical component of 'lipid rafts'), and the resulting effects on T. forsythia uptake were determined. Our studies revealed that T. forsythia entry is dependent on host PI3K signalling, and that purified BspA protein causes activation of this lipid kinase. Bacterial entry also requires the cooperation of host Rac1 GTPase. Finally, our findings indicate an important role for clathrin and cholesterol-rich lipid microdomains in the internalization process.

  18. Endoglin regulates PI3-kinase/Akt trafficking and signaling to alter endothelial capillary stability during angiogenesis

    PubMed Central

    Lee, Nam Y.; Golzio, Christelle; Gatza, Catherine E.; Sharma, Arun; Katsanis, Nicholas; Blobe, Gerard C.

    2012-01-01

    Endoglin (CD105) is an endothelial-specific transforming growth factor β (TGF-β) coreceptor essential for angiogenesis and vascular homeostasis. Although endoglin dysfunction contributes to numerous vascular conditions, the mechanism of endoglin action remains poorly understood. Here we report a novel mechanism in which endoglin and Gα-interacting protein C-terminus–interacting protein (GIPC)–mediated trafficking of phosphatidylinositol 3-kinase (PI3K) regulates endothelial signaling and function. We demonstrate that endoglin interacts with the PI3K subunits p110α and p85 via GIPC to recruit and activate PI3K and Akt at the cell membrane. Opposing ligand-induced effects are observed in which TGF-β1 attenuates, whereas bone morphogenetic protein-9 enhances, endoglin/GIPC-mediated membrane scaffolding of PI3K and Akt to alter endothelial capillary tube stability in vitro. Moreover, we employ the first transgenic zebrafish model for endoglin to demonstrate that GIPC is a critical component of endoglin function during developmental angiogenesis in vivo. These studies define a novel non-Smad function for endoglin and GIPC in regulating endothelial cell function during angiogenesis. PMID:22593212

  19. Andrographolide inhibits osteopontin expression and breast tumor growth through down regulation of PI3 kinase/Akt signaling pathway.

    PubMed

    Kumar, S; Patil, H S; Sharma, P; Kumar, D; Dasari, S; Puranik, V G; Thulasiram, H V; Kundu, G C

    2012-09-01

    Breast cancer is one of the most common cancers among women in India and around the world. Despite recent advancement in the treatment of breast cancer, the results of chemotherapy to date remain unsatisfactory, prompting a need to identify natural agents that could target cancer efficiently with least side effects. Andrographolide (Andro) is one such molecule which has been shown to possess inhibitory effect on cancer cell growth. In this study, Andro, a natural diterpenoid lactone isolated from Andrographis paniculata has been shown to inhibit breast cancer cell proliferation, migration and arrest cell cycle at G2/M phase and induces apoptosis through caspase independent pathway. Our experimental evidences suggest that Andro attenuates endothelial cell motility and tumor-endothelial cell interaction. Moreover, Andro suppresses breast tumor growth in orthotopic NOD/SCID mice model. The anti-tumor activity of Andro in both in vitro and in vivo model was correlated with down regulation of PI3 kinase/Akt activation and inhibition of pro-angiogenic molecules such as OPN and VEGF expressions. Collectively, these results demonstrate that Andro may act as an effective anti-tumor and anti-angiogenic agent for the treatment of breast cancer.

  20. Puquitinib mesylate, an inhibitor of phosphatidylinositol 3-kinase p110δ, for treating relapsed or refractory non-Hodgkin's lymphoma

    PubMed Central

    Zhan, Jing; Xia, Yi; Sun, Peng; Bi, Xi-Wen; Liu, Pan-Pan; Li, Zhi-Ming; Li, Su; Zou, Ben-Yan; Jiang, Wen-Qi

    2015-01-01

    Objectives To determine the safety of Puquitinib Mesylate (XC-302), an oral inhibitor of phosphatidylinositol 3-kinase, in treating relapsed or refractory non-Hodgkin's lymphoma (NHL). Methods Between October 2013 and July 2015, 21 patients from Sun Yat-sen University Cancer Center were treated twice daily on each day of a 28-day cycle (median number of cycles, 2; maximum, 20) with XC-302 at a post prandial dose of 25 mg, 37.5 mg, or 50 mg. Adverse events (AEs), AUClast and Cmax, response rates, and overall survival were assessed. Results Patients had received a median (range) of 1 (1 to 3) previous cancer treatments. At the latest follow-up, two patients were still benefitting from the study. The most common drug-related AEs were elevations in alanine transaminase (ALT, 14 of 21 patients) and aspartate transaminase (AST, 7 of 21 patients). Four patients, both in the-50-mg group, had dose-limiting toxicities, and therapy was discontinued in a fifth because of persistent abnormal liver function. The overall response rate was 2 of19. Serum concentrations of XC-302 increased in a dose-dependent pattern. Median progression-free survival in all patients was 1.9 (95% CI, 1.7 to 2.0) months. Conclusion XC-302 has an acceptable safety profile and offers potential therapeutic value to patients with relapsed or refractory non-Hodgkin lymphoma. PMID:26510909

  1. Icaritin requires Phosphatidylinositol 3 kinase (PI3K)/Akt signaling to counteract skeletal muscle atrophy following mechanical unloading

    PubMed Central

    ZHANG, Zong-Kang; LI, Jie; LIU, Jin; GUO, Baosheng; LEUNG, Albert; ZHANG, Ge; ZHANG, Bao-Ting

    2016-01-01

    Counteracting muscle atrophy induced by mechanical unloading/inactivity is of great clinical need and challenge. A therapeutic agent that could counteract muscle atrophy following mechanical unloading in safety is desired. This study showed that natural product Icaritin (ICT) could increase the phosphorylation level of Phosphatidylinositol 3 kinase (PI3K) at p110 catalytic subunit and promote PI3K/Akt signaling markers in C2C12 cells. This study further showed that the high dose ICT treatment could significantly attenuate the decreases in the phosphorylation level of PI3K at p110 catalytic subunit and its downstream markers related to protein synthesis, and inhibit the increases in protein degradation markers at mRNA and protein levels in rat soleus muscle following 28-day hindlimb unloading. In addition, the decreases in soleus muscle mass, muscle fiber cross-sectional area, twitch force, specific force, contraction time and half relaxation time could be significantly attenuated by the high dose ICT treatment. The low dose ICT treatment could moderately attenuate the above changes induced by unloading. Wortmannin, a specific inhibitor of PI3K at p110 catalytic subunit, could abolish the above effects of ICT in vitro and in vivo, indicating that PI3K/Akt signaling could be required by ICT to counteract skeletal muscle atrophy following mechanical unloading. PMID:26831566

  2. 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

  3. ERK and phosphoinositide 3-kinase temporally coordinate different modes of actin-based motility during embryonic wound healing.

    PubMed

    Li, Jingjing; Zhang, Siwei; Soto, Ximena; Woolner, Sarah; Amaya, Enrique

    2013-11-01

    Embryonic wound healing provides a perfect example of efficient recovery of tissue integrity and homeostasis, which is vital for survival. Tissue movement in embryonic wound healing requires two functionally distinct actin structures: a contractile actomyosin cable and actin protrusions at the leading edge. Here, we report that the discrete formation and function of these two structures is achieved by the temporal segregation of two intracellular upstream signals and distinct downstream targets. The sequential activation of ERK and phosphoinositide 3-kinase (PI3K) signalling divides Xenopus embryonic wound healing into two phases. In the first phase, activated ERK suppresses PI3K activity, and is responsible for the activation of Rho and myosin-2, which drives actomyosin cable formation and constriction. The second phase is dominated by restored PI3K signalling, which enhances Rac and Cdc42 activity, leading to the formation of actin protrusions that drive migration and zippering. These findings reveal a new mechanism for coordinating different modes of actin-based motility in a complex tissue setting, namely embryonic wound healing.

  4. Emergence of the PI3-kinase pathway as a central modulator of normal and aberrant B cell differentiation.

    PubMed

    Baracho, G V; Miletic, A V; Omori, S A; Cato, M H; Rickert, R C

    2011-04-01

    Phosphoinositide 3-kinase (PI3K) defines a family of lipid kinases that direct a wide range of cellular processes and cell fate decisions. Since its discovery, and that of its enzymatic antagonist PTEN, much of the focus on PI3K has been on its oncogenic potential. In recent years, studies on PI3K signaling in B lymphocytes have established the importance of this pathway in effecting B cell differentiation and associated molecular events such as V(D)J recombination and class switch recombination. Intriguing new findings also indicate that there is specificity in the PI3K pathway in B cells, including preferential expression or usage of particular PI3K isoforms and counter-regulation by the PTEN and SHIP phosphatases. The role of PI3K adaptor proteins (CD19, BCAP, and TC21) has also undergone revision to reflect both shared and unique properties. The emergence of Foxo1 as a critical PI3K regulatory target for B cell differentiation has united membrane proximal regulatory events orchestrated by PI3K/PTEN/SHIP with key transcriptional targets. Insights into the regulation and impact of PI3K signaling have been brought to bear in new treatments for B cell malignancies, and will also be an important topic of consideration for B cell-dependent autoimmune diseases.

  5. Cobalt chloride stimulates phosphoinositide 3-kinase/Akt signaling through the epidermal growth factor receptor in oral squamous cell carcinoma.

    PubMed

    Ryu, Mi Heon; Park, Jeong Hee; Park, Ji Eun; Chung, Jin; Lee, Chang Hun; Park, Hae Ryoun

    2010-04-01

    Tumor cells are often found under hypoxic conditions due to the rapid outgrowth of their vascular supply, and, in order to survive hypoxia, these cells induce numerous signaling factors. Akt is an important kinase in cell survival, and its activity is regulated by the upstream phosphoinositide 3-kinase (PI3K) and receptor tyrosine kinases (RTKs). In this study, we examined Akt activation and RTKs/PI3K/Akt signaling using the hypoxia-mimetic cobalt chloride in oral squamous carcinoma cells. Cobalt chloride increases Akt phosphorylation in both a dose- and time-dependent manner. Blocking the activation of the PI3K/Akt pathway using LY294002 abolished Akt activation in response to cobalt chloride, suggesting that Akt phosphorylation by cobalt chloride is dependent on PI3K. In addition, activation of the PI3K/Akt pathway seems to rely on the epidermal growth factor receptor (EGFR), since the inhibition of EGFR attenuated cobalt chloride-induced Akt activation. The results in this study also demonstrate that cobalt chloride increases EGFR protein levels and induces oral squamous cell carcinoma cells to enter S phase.

  6. 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

  7. Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration.

    PubMed

    Dise, Rebecca S; Frey, Mark R; Whitehead, Robert H; Polk, D Brent

    2008-01-01

    Regulated intestinal epithelial cell migration plays a key role in wound healing and maintenance of a healthy gastrointestinal tract. Epidermal growth factor (EGF) stimulates cell migration and wound closure in intestinal epithelial cells through incompletely understood mechanisms. In this study we investigated the role of the small GTPase Rac in EGF-induced cell migration using an in vitro wound-healing assay. In mouse colonic epithelial (MCE) cell lines, EGF-stimulated wound closure was accompanied by a doubling of the number of cells containing lamellipodial extensions at the wound margin, increased Rac membrane translocation in cells at the wound margin, and rapid Rac activation. Either Rac1 small interfering (si)RNA or a Rac1 inhibitor completely blocked EGF-stimulated wound closure. Whereas EGF failed to activate Rac in colon cells from EGF receptor (EGFR) knockout mice, stable expression of wild-type EGFR restored EGF-stimulated Rac activation and migration. Pharmacological inhibition of either phosphatidylinositol 3-kinase (PI3K) or Src family kinases reduced EGF-stimulated Rac activation. Cotreatment of cells with both inhibitors completely blocked EGF-stimulated Rac activation and localization to the leading edge of cells and lamellipodial extension. Our results present a novel mechanism by which the PI3K and Src signaling cascades cooperate to activate Rac and promote intestinal epithelial cell migration downstream of EGFR.

  8. Phosphoinositide-3-kinase and mitogen activated protein kinase signaling pathways mediate acute NGF sensitization of TRPV1.

    PubMed

    Zhu, Weiguo; Oxford, Gerry S

    2007-04-01

    Nerve growth factor (NGF) induces an acute sensitization of nociceptive DRG neurons, in part, through sensitization of the capsaicin receptor TRPV1 via the high affinity trkA receptor. The mechanisms linking trkA and TRPV1 remain controversial with several candidate signaling pathways proposed. Utilizing adult rat and mouse DRG neurons and CHO cells co-expressing trkA and TRPV1, we have investigated the signaling events underlying acute TRPV1 sensitization by NGF combining biochemical, electrophysiological, pharmacological, mutational and genetic knockout approaches. Pharmacological interference with p42/p44 mitogen activated protein kinase (MAPK) or phosphoinositide-3-kinase (PI3K), but not PLC abrogated sensitization of capsaicin responses. Co-expression of TRPV1 with wild-type or Y785F (PLC signal deficient) mutant human trkA reconstituted NGF sensitization. In contrast, TRPV1 co-expressed with MAPK signaling deficient Y490A or PI3K signaling deficient Y751F trkA mutants exhibited weaker sensitization. Biochemical analysis of p42/p44 and Akt phosphorylation confirmed the specificity of pharmacological agents and trkA mutants. Finally, NGF sensitization of capsaicin responses was greatly reduced in neurons from p85alpha (regulatory subunit of PI3K) null mice. These data strongly suggest that PI3K and MAPK pathways, but not the PLC pathway underlie the acute sensitization of TRPV1 by NGF.

  9. The novel PI3 kinase inhibitor, BAY 80-6946, impairs melanoma growth in vivo and in vitro.

    PubMed

    Schneider, Philine; Schön, Margarete; Pletz, Nadin; Seitz, Cornelia S; Liu, Ningshu; Ziegelbauer, Karl; Zachmann, Karolin; Emmert, Steffen; Schön, Michael P

    2014-08-01

    Due to its almost universal resistance to chemotherapy, metastasized melanoma remains a major challenge in clinical oncology. Given that phosphatidyl inositol-3 kinase (PI3K) activation in melanoma cells is associated with poor prognosis, disease progression and resistance to chemotherapy, the PI3K-Akt signalling pathway is a promising therapeutic target for melanoma treatment. We analysed six human melanoma cell lines for their constitutive activation of Akt and then tested two representative lines, A375 and LOX, for their susceptibility to PI3K-inhibition by the highly specific small molecule inhibitor, BAY 80-6946. In addition, the effect of BAY 80-6946 on A375 and LOX melanoma cells was assessed in vivo in a xenotransplantation mouse model. We provide experimental evidence that specifically inhibiting the PI3K pathway and phosphorylation of Akt by this novel compound results in antitumoral activities including inhibition of proliferation, induction of apoptosis and cell cycle arrest in vitro and in vivo. However, the susceptibility did not show a clear-cut pattern and differed between the melanoma cell lines tested, resulting in in vivo growth inhibition of A375 but not LOX melanoma cells. Thus, in some cases BAY 80-6946 or related compounds may be a valuable addition to the therapeutic armamentarium.

  10. Arecoline-induced phosphorylated p53 and p21(WAF1) protein expression is dependent on ATM/ATR and phosphatidylinositol-3-kinase in clone-9 cells.

    PubMed

    Chou, Wen-Wen; Guh, Jinn-Yuh; Tsai, Jung-Fa; Hwang, Chi-Ching; Chiou, Shean-Jaw; Chuang, Lea-Yea

    2009-06-01

    Betel-quid use is associated with liver cancer whereas its constituent arecoline is cytotoxic, genotoxic, and induces p53-dependent p21(WAF1) protein expression in Clone-9 cells (rat hepatocytes). The ataxia telangiectasia mutated (ATM)/rad3-related (ATR)-p53-p21(WAF1) and the phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathways are involved in the DNA damage response and the pathogenesis of cancers. Thus, we studied the role of ATM/ATR and PI3K in arecoline-induced p53 and p21(WAF1) protein expression in Clone-9 cells. We found that arecoline (0.5 mM) activated the ATM/ATR kinase at 30 min. The arecoline-activated ATM/ATR substrate contained p-p53Ser15. Moreover, arecoline only increased the levels of the p-p53Ser6, p-p53Ser15, and p-p53Ser392 phosphorylated p53 isoforms among the known isoforms. ATM shRNA attenuated arecoline-induced p-p53Ser15 and p21(WAF1) at 24 h. Arecoline (0.5 mM) increased phosphorylation levels of p-AktSer473 and p-mTORSer2448 at 30-60 min. Dominant-negative PI3K plasmids attenuated arecoline-induced p21(WAF1), but not p-p53Ser15, at 24 h. Rapamycin attenuated arecoline-induced phosphrylated p-p53Ser15, but not p21(WAF1), at 24 h. ATM shRNA, but not dominant-negative PI3K plasmids, attenuated arecoline-induced p21(WAF1) gene transcription. We conclude that arecoline activates the ATM/ATR-p53-p21(WAF1) and the PI3K/Akt-mTOR-p53 pathways in Clone-9 cells. Arecoline-induced phosphorylated p-p53Ser15 expression is dependent on ATM whereas arecoline-induced p21(WAF1) protein expression is dependent on ATM and PI3K. Moreover, p21(WAF1) gene is transcriptionally induced by arecoline-activated ATM.

  11. PI3 Kinase Disease

    MedlinePlus

    ... investigate other treatment options, such as PI3K-specific drugs. Featured Research Treating PASLI Disease Watch an NIAID video about ... Translational Research Other Resources Clinical Agents Repository ... NIAID Contributes to New TB Drug With Broad Antibiotic Potential NIAID-Developed Technology Helps ...

  12. Roles of mitogen-activated protein kinase and phosphoinositide 3'-kinase in ErbB2/ErbB3 coreceptor-mediated heregulin signaling.

    PubMed

    Vijapurkar, Ulka; Kim, Myong-Soo; Koland, John G

    2003-04-01

    ErbB2/HER2 and ErbB3/HER3, two members of the ErbB/HER family, together constitute a heregulin coreceptor complex that elicits a potent mitogenic and transforming signal. Among known intracellular effectors of the ErbB2/ErbB3 heregulin coreceptor are mitogen-activated protein kinase (MAPK) and phosphoinositide (PI) 3-kinase. Activation of the distinct MAPK and PI 3-kinase signaling pathways by the ErbB2/ErbB3 coreceptor in response to heregulin and their relative contributions to the mitogenic and transformation potentials of the activated coreceptor were investigated here. To this end, cDNAs encoding the wild-type ErbB3 protein (ErbB3-WT) and ErbB3 proteins with amino acid substitutions in either the Shc-binding site (ErbB3-Y1325F), the six putative PI 3-kinase-binding sites (ErbB3-6F), or both (ErbB3-7F) were generated and expressed in NIH-3T3 cells to form functional ErbB2/ErbB3 heregulin coreceptors. While the coreceptor incorporating ErbB3-WT activated both the MAPK and the PI 3-kinase signaling pathways, those incorporating ErbB3-Y1325F or ErbB3-6F activated either PI 3-kinase or MAPK, respectively. The ErbB2/ErbB3-7F coreceptor activated neither. Elimination of either signaling pathway lowered basal and eliminated heregulin-dependent expression of cyclin D1, which was in each case accompanied by an attenuated mitogenic response. Selective elimination of the PI 3-kinase pathway severely impaired the ability of heregulin to transform cells expressing the coreceptor, whereas attenuation of the MAPK pathway had a lesser effect. Thus, while both pathways contributed in a roughly additive manner to the mitogenic response elicited by the activated ErbB2/ErbB3 coreceptor, the PI 3-kinase pathway predominated in the induction of cellular transformation.

  13. 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

  14. Impaired activation of phosphoinositide 3-kinase by insulin in fibroblasts from patients with severe insulin resistance and pseudoacromegaly. A disorder characterized by selective postreceptor insulin resistance.

    PubMed Central

    Dib, K; Whitehead, J P; Humphreys, P J; Soos, M A; Baynes, K C; Kumar, S; Harvey, T; O'Rahilly, S

    1998-01-01

    Some patients with severe insulin resistance develop pathological tissue growth reminiscent of acromegaly. Previous studies of such patients have suggested the presence of a selective postreceptor defect of insulin signaling, resulting in the impairment of metabolic but preservation of mitogenic signaling. As the activation of phosphoinositide 3-kinase (PI 3-kinase) is considered essential for insulin's metabolic signaling, we have examined insulin-stimulated PI 3-kinase activity in anti-insulin receptor substrate (IRS)-1 immunoprecipitates from cultured dermal fibroblasts obtained from pseudoacromegalic (PA) patients and controls. At a concentration of insulin (1 nM) similar to that seen in vivo in PA patients, the activation of IRS-1-associated PI 3-kinase was reduced markedly in fibroblasts from the PA patients (32+/-7% of the activity of normal controls, P < 0.01). Genetic and biochemical studies indicated that this impairment was not secondary to a defect in the structure, expression, or activation of the insulin receptor, IRS-1, or p85alpha. Insulin stimulation of mitogenesis in PA fibroblasts, as determined by thymidine incorporation, was indistinguishable from controls, as was mitogen-activated protein kinase phosphorylation, confirming the integrity of insulin's mitogenic signaling pathways in this condition. These findings support the existence of an intrinsic defect of postreceptor insulin signaling in the PA subtype of insulin resistance, which involves impairment of the activation of PI 3-kinase. The PA tissue growth seen in such patients is likely to result from severe in vivo hyperinsulinemia activating intact mitogenic signaling pathways emanating from the insulin receptor. PMID:9486982

  15. Phosphatidylinositol 3-Kinase Plays a Vital Role in Regulation of Rice Seed Vigor via Altering NADPH Oxidase Activity

    PubMed Central

    Liu, Jian; Zhou, Jun; Xing, Da

    2012-01-01

    Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was also evaluated. Diphenylene iodonium (DPI), an NADPH oxidase inhibitor, suppressed most of ROS generation in rice seed germination, which suggested that NADPH oxidase was the main source of ROS in this process. Pharmacological experiment and RT-PCR demonstrated that PI3K promoted the expression of Os rboh9. Moreover, functional analysis by native PAGE and the measurement of the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-lium-5- carboxanilide (XTT) formazan concentration both showed that PI3K promoted the activity of NADPH oxidase. Furthermore, the western blot analysis of OsRac-1 demonstrated that the translocation of Rac-1 from cytoplasm to plasma membrane, which was known as a key factor in the assembly of NADPH oxidase, was suppressed by treatment with PI3K inhibitors, resulting in the decreased activity of NADPH oxidase. Taken together, these data favored the novel conclusion that PI3K regulated NADPH oxidase activity through modulating the recruitment of Rac-1 to plasma membrane and accelerated the process of rice seed germination. PMID:22448275

  16. Platelet-derived growth factor-dependent cellular transformation requires either phospholipase Cgamma or phosphatidylinositol 3 kinase.

    PubMed

    DeMali, K A; Whiteford, C C; Ulug, E T; Kazlauskas, A

    1997-04-04

    Although it has been well established that constitutive activation of receptor tyrosine kinases leads to cellular transformation, the signal relay pathways involved have not been systematically investigated. In this study we used a panel of platelet-derived growth factor (PDGF) beta receptor mutants (beta-PDGFR), which selectively activate various signal relay enzymes to define which signaling pathways are required for PDGF-dependent growth of cells in soft agar. The host cell line for these studies was Ph cells, a 3T3-like cell that expresses normal levels of the beta-PDGFR but no PDGF-alpha receptor (alpha-PDGFR). Hence, this cell system can be used to study signaling of mutant alphaPDGFRs or alpha/beta chimeras. We constructed chimeric receptors containing the alphaPDGFR extracellular domain and the betaPDGFR cytoplasmic domain harboring various phosphorylation site mutations. The mutants were expressed in Ph cells, and their ability to drive PDGF-dependent cellular transformation (growth in soft agar) was assayed. Cells infected with an empty expression vector failed to grow in soft agar, whereas introduction of the chimera with a wild-type beta-PDGFR cytoplasmic domain gave rise to a large number of colonies. In contrast, the N2F5 chimera, in which the binding sites for phospholipase Cgamma (PLC-gamma), RasGTPase-activating protein, phosphatidylinositol 3 kinase (PI3K), and SHP-2 were eliminated, failed to trigger proliferation. Restoring the binding sites for RasGTPase-activating protein or SHP-2 did not rescue the PDGF-dependent response. In contrast, receptors capable of associating with either PLC-gamma or PI3K relayed a growth signal that was comparable to wild-type receptors in the soft agar growth assay. These findings indicate that the PDGF receptor activates multiple signaling pathways that lead to cellular transformation, and that either PI3K or PLC-gamma are key initiators of such signal relay cascades.

  17. Promotion of melanoma cell invasion and tumor metastasis by microcystin-LR via phosphatidylinositol 3-kinase/AKT pathway.

    PubMed

    Xu, Pengfei; Zhang, Xu-Xiang; Miao, Chen; Fu, Ziyi; Li, Zhengrong; Zhang, Gen; Zheng, Maqing; Liu, Yuefang; Yang, Liuyan; Wang, Ting

    2013-08-06

    Recently, we have indicated that microcystin-LR, a cyanobacterial toxin produced in eutrophic lakes or reservoirs, can increase invasive ability of melanoma MDA-MB-435 cells; however, the stimulatory effect needs identification by in vivo experiment and the related molecular mechanism is poorly understood. In this study, in vitro and in vivo experiments were conducted to investigate the effect of microcystin-LR on invasion and metastasis of human melanoma cells, and the underlying molecular mechanism was also explored. MDA-MB-435 xenograft model assay showed that oral administration of nude mice with microcystin-LR at 0.001-0.1 mg/kg/d posed no significant effect on tumor weight. Histological examination demonstrated that microcystin-LR could promote lung metastasis, which is confirmed by Matrigel chamber assay suggesting that microcystin-LR treatment at 25 nM can increase the invasiveness of MDA-MB-435 cells. In vitro and in vivo experiments consistently showed that microcystin-LR exposure increased mRNA and protein levels of matrix metalloproteinases (MMP-2/-9) by activating phosphatidylinositol 3-kinase (PI3-K)/AKT. Additionally, microcystin-LR treatment at low doses (≤25 nM) decreased lipid phosphatase PTEN expression, and the microcystin-induced invasiveness enhancement and MMP-2/-9 overexpression were reversed by the PI3-K/AKT chemical inhibitor LY294002 and AKT siRNA, indicating that microcystin-LR promotes invasion and metastasis of MDA-MB-435 cells via the PI3-K/AKT pathway.

  18. Investigation into the Role of PI3K and JAK3 Kinase Inhibitors in Murine Models of Asthma.

    PubMed

    Wagh, Akshaya D; Sharma, Manoranjan; Mahapatra, Jogeshwar; Chatterjee, Abhijeet; Jain, Mukul; Addepalli, Veeranjaneyulu

    2017-01-01

    Asthma is a clinical disorder commonly characterized by chronic eosinophilic inflammation, remodeling and hyper responsiveness of the airways. However, the kinases like Phosphoinositide 3 kinase (PI3K) and Janus kinase 3 (JAK3) are involved in mast cell proliferation, activation, recruitment, migration, and prolonged survival of inflammatory cells. The present study was designed to evaluate the in-vivo comparative effects of two kinase inhibitors on airway inflammation and airway remodeling in acute and chronic models of asthma. Mice were sensitized twice intra-peritoneally and then challenged by inhalation with ovalbumin (OVA). They developed an extensive inflammatory response, goblet cell hyperplasia, collagen deposition, airway smooth muscle thickening similar to pathologies observed in human asthma. The effects of PI3K inhibitor (30 mg/kg, p.o), JAK3 inhibitor (30 mg/kg, p.o) and Dexamethasone (0.3 mg/kg) on airway inflammation and remodeling in OVA sensitized/challenged BALB/c mice were evaluated. Twenty-four hours after the final antigen challenge, bronchoalveolar lavage (BAL) and histological examinations were carried out. It was observed that kinase inhibitors significantly reduced airway inflammation as evidenced by the decrease in pro inflammatory cytokines in BALF and lung homogenate and inflammatory cell count in sensitized mice after allergen challenge. Lung histological analysis showed increased infiltration of inflammatory cells, hyperplasia of goblet cells and the collagen deposition, which were significantly reduced with kinase inhibitor. In conclusion, our data suggest that PI3K and JAK3 inhibitors showed promising alternative therapeutic activity in asthma, which might significantly counteract the airway inflammation in patients with allergic asthma.

  19. Identification of upregulated phosphoinositide 3-kinase γ as a target to suppress breast cancer cell migration and invasion

    PubMed Central

    Xie, Yan; Abel, Peter W.; Kirui, Joseph K.; Deng, Caishu; Sharma, Poonam; Wolff, Dennis W.; Toews, Myron L.; Tu, Yaping

    2013-01-01

    Metastasis is the major cause of breast cancer mortality. We recently reported that aberrant G-protein coupled receptor (GPCR) signaling promotes breast cancer metastasis by enhancing cancer cell migration and invasion. Phosphatidylinositol 3-kinase γ (PI3Kγ) is specifically activated by GPCRs. The goal of the present study was to determine the role of PI3Kγ in breast cancer cell migration and invasion. Immunohistochemical staining showed that the expression of PI3Kγ protein was significantly increased in invasive human breast carcinoma when compared to adjacent benign breast tissue or ductal carcinoma in situ. PI3Kγ was also detected in metastatic breast cancer cells, but not in normal breast epithelial cell line or in non-metastatic breast cancer cells. In contrast, PI3K isoforms α, β and δ were ubiquitously expressed in these cell lines. Overexpression of recombinant PI3Kγ enhanced the metastatic ability of non-metastatic breast cancer cells. Conversely, migration and invasion of metastatic breast cancer cells were inhibited by a PI3Kγ inhibitor or by siRNA knockdown of PI3Kγ but not by inhibitors or siRNAs of PI3Kα or PI3Kβ. Lamellipodia formation is a key step in cancer metastasis, and PI3Kγ blockade disrupted lamellipodia formation induced by the activation of GPCRs such as CXC chemokine receptor 4 and protease-activated receptor 1, but not by the epidermal growth factor tyrosine kinase receptor. Taken together, these results indicate that upregulated PI3Kγ conveys the metastatic signal initiated by GPCRs in breast cancer cells, and suggest that PI3Kγ may be a novel therapeutic target for development of chemotherapeutic agents to prevent breast cancer metastasis. PMID:23500535

  20. Regulation of PI-3-Kinase and Akt Signaling in T Lymphocytes and Other Cells by TNFR Family Molecules

    PubMed Central

    So, Takanori; Croft, Michael

    2013-01-01

    Activation of phosphoinositide 3-kinase (PI3K) and Akt (protein kinase B) is a common response triggered by a range of membrane-bound receptors on many cell types. In T lymphocytes, the PI3K-Akt pathway promotes clonal expansion, differentiation, and survival of effector cells and suppresses the generation of regulatory T cells. PI3K activation is tightly controlled by signals through the T cell receptor (TCR) and the co-stimulatory receptor CD28, however sustained and periodic signals from additional co-receptors are now being recognized as critical contributors to the activation of this pathway. Accumulating evidence suggests that many members of the Tumor Necrosis Factor receptor (TNFR) superfamily, TNFR2 (TNFRSF1B), OX40 (TNFRSF4), 4-1BB (TNFRSF9), HVEM (TNFRSF14), and DR3 (TNFRSF25), that are constitutive or inducible on T cells, can directly or indirectly promote activity in the PI3K-Akt pathway. We discuss recent data which suggests that ligation of one TNFR family molecule organizes a signalosome, via TNFR-associated factor (TRAF) adapter proteins in T cell membrane lipid microdomains, that results in the subsequent accumulation of highly concentrated depots of PI3K and Akt in close proximity to TCR signaling units. We propose this may be a generalizable mechanism applicable to other TNFR family molecules that will result in a quantitative contribution of these signalosomes to enhancing and sustaining PI3K and Akt activation triggered by the TCR. We also review data that other TNFR molecules, such as CD40 (TNFRSF5), RANK (TNFRSF11A), FN14 (TNFRSF12A), TACI (TNFRSF13B), BAFFR (TNFRSF13C), and NGFR (TNFRSF16), contribute to the activation of this pathway in diverse cell types through a similar ability to recruit PI3K or Akt into their signaling complexes. PMID:23760533

  1. Phosphatidylinositol-3-kinase pathway aberrations in gastric and colorectal cancer: meta-analysis, co-occurrence and ethnic variation.

    PubMed

    Chong, Mei-Ling; Loh, Marie; Thakkar, Bhavin; Pang, Brendan; Iacopetta, Barry; Soong, Richie

    2014-03-01

    Inhibition of the phosphatidylinositol-3-kinase (PI3K) signaling pathway is a cancer treatment strategy that has entered into clinical trials. We performed a meta-analysis on the frequency of prominent genetic (PIK3CA mutation, PIK3CA amplification and PTEN deletion) and protein expression (high PI3K, PTEN loss and high pAkt) aberrations in the PI3K pathway in gastric cancer (GC) and colorectal cancer (CRC). We also performed laboratory analysis to investigate the co-occurrence of these aberrations. The meta-analysis indicated that East Asian and Caucasian GC patients differ significantly for the frequencies of PIK3CA Exon 9 and 20 mutations (7% vs. 15%, respectively), PTEN deletion (21% vs. 4%) and PTEN loss (47% vs. 78%), while CRC patients differed for PTEN loss (57% vs. 26%). High study heterogeneity (I(2) > 80) was observed for all aberrations except PIK3CA mutations. Laboratory analysis of tumors from East Asian patients revealed significant differences between GC (n = 79) and CRC (n = 116) for the frequencies of PIK3CA amplification (46% vs. 4%) and PTEN loss (54% vs. 78%). The incidence of GC cases with 0, 1, 2 and 3 concurrent aberrations was 14%, 52%, 27% and 8%, respectively, while for CRC it was 10%, 60%, 25% and 4%, respectively. Our study consolidates knowledge on the frequency, co-occurrence and clinical relevance of PI3K pathway aberrations in GC and CRC. Up to 86% of GC and 90% of CRC have at least one aberration in the PI3K pathway, and there are significant differences in the frequencies of these aberrations according to cancer type and ethnicity.

  2. Signals controlling un-differentiated states in embryonic stem and cancer cells: role of the phosphatidylinositol 3' kinase pathway.

    PubMed

    Voskas, Daniel; Ling, Ling Sunny; Woodgett, James Robert

    2014-10-01

    The capacity of embryonic stem (ES) cells to differentiate into cell lineages comprising the three germ layers makes them powerful tools for studying mammalian early embryonic development in vitro. The human body consists of approximately 210 different somatic cell types, the majority of which have limited proliferative capacity. However, both stem cells and cancer cells bypass this replicative barrier and undergo symmetric division indefinitely when cultured under defined conditions. Several signal transduction pathways play important roles in regulating stem cell development, and aberrant expression of components of these pathways is linked to cancer. Among signaling systems, the critical role of leukemia inhibitory factor (LIF) coupled to the Jak/STAT3 (signal transduction and activation of transcription-3) pathway in maintaining stem cell self-renewal has been extensively reviewed. This pathway additionally plays multiple roles in tumorigenesis. Likewise, the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathway has been determined to play an important role in both stem cell maintenance and tumor development. This pathway is often induced in cancer with frequent mutational activation of the catalytic subunit of PI3K or loss of a primary PI3K antagonist, phosphatase and tensin homolog deleted on chromosome ten (PTEN). This review focusses on roles of the PI3K signal transduction pathway components, with emphasis on functions in stem cell maintenance and cancer. Since the PI3K pathway impinges on and collaborates with other signaling pathways in regulating stem cell development and/or cancer, aspects of the canonical Wnt, Ras/mitogen-activated protein kinase (MAPK), and TGF-β signaling pathways are also discussed.

  3. 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.

  4. Euphorbia fischeriana Steud inhibits malignant melanoma via modulation of the phosphoinositide-3-kinase/Akt signaling pathway

    PubMed Central

    DONG, MENG-HUA; ZHANG, QIAN; WANG, YUAN-YUAN; ZHOU, BAI-SUI; SUN, YU-FEI; FU, QIANG

    2016-01-01

    Euphorbia fischeriana Steud, a traditional Chinese medicine, has been shown to inhibit the growth of various cancers by the induction of apoptosis and cell cycle arrest. The purpose of the present study was to investigate the association between the phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway and the inhibitory effect of Euphorbia fischeriana Steud on the growth and metastasis of melanoma B16 cells in vitro, and the underlying mechanisms. MTT assay results indicated that Euphorbia fischeriana Steud inhibited the growth of B16 cells in a time- and dose-dependent manner. Flow cytometric analysis revealed that Euphorbia fischeriana Steud markedly induced apoptosis of the B16 cells, with arrest at the G0/G1 phase of the cell cycle. In addition, in a Transwell assay Euphorbia fischeriana Steud significantly suppressed the migration of B16 cells. Western blot analysis revealed that the expression levels of phosphatase and tensin homolog (PTEN) were upregulated, and the phosphorylation of Akt was downregulated, which resulted in inhibition of the PI3K/Akt signaling pathway and the eventual suppression of its downstream targets, such as matrix metalloproteinase-2 mRNA, in B16 cells. The results demonstrated that Euphorbia fischeriana Steud inhibited the growth and migration of B16 cells, possibly via modulation of the PI3K/Akt signaling pathway and upregulation of PTEN expression levels, in addition to downregulation of p-Akt expression. The aforementioned findings suggest that Euphorbia fischeriana Steud may have broad therapeutic applications in the treatment of malignant melanoma. PMID:27073468

  5. Phosphatidylinositol 3-kinase plays a vital role in regulation of rice seed vigor via altering NADPH oxidase activity.

    PubMed

    Liu, Jian; Zhou, Jun; Xing, Da

    2012-01-01

    Phosphatidylinositol 3-kinase (PI3K) has been reported to be important in normal plant growth and stress responses. In this study, it was verified that PI3K played a vital role in rice seed germination through regulating NADPH oxidase activity. Suppression of PI3K activity by inhibitors wortmannin or LY294002 could abate the reactive oxygen species (ROS) formation, which resulted in disturbance to the seed germination. And then, the signal cascades that PI3K promoted the ROS liberation was also evaluated. Diphenylene iodonium (DPI), an NADPH oxidase inhibitor, suppressed most of ROS generation in rice seed germination, which suggested that NADPH oxidase was the main source of ROS in this process. Pharmacological experiment and RT-PCR demonstrated that PI3K promoted the expression of Os rboh9. Moreover, functional analysis by native PAGE and the measurement of the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazo-lium-5- carboxanilide (XTT) formazan concentration both showed that PI3K promoted the activity of NADPH oxidase. Furthermore, the western blot analysis of OsRac-1 demonstrated that the translocation of Rac-1 from cytoplasm to plasma membrane, which was known as a key factor in the assembly of NADPH oxidase, was suppressed by treatment with PI3K inhibitors, resulting in the decreased activity of NADPH oxidase. Taken together, these data favored the novel conclusion that PI3K regulated NADPH oxidase activity through modulating the recruitment of Rac-1 to plasma membrane and accelerated the process of rice seed germination.

  6. K-Ras promotes growth transformation and invasion of immortalized human pancreatic cells by Raf and phosphatidylinositol 3-kinase signaling.

    PubMed

    Campbell, Paul M; Groehler, Angela L; Lee, Kwang M; Ouellette, Michel M; Khazak, Vladimir; Der, Channing J

    2007-03-01

    Mutational activation of the K-Ras oncogene is well established as a key genetic step in the development and growth of pancreatic adenocarcinomas. However, the mechanism by which aberrant Ras signaling promotes uncontrolled pancreatic tumor cell growth remains to be fully elucidated. The recent use of primary human cells to study Ras-mediated oncogenesis provides important model cell systems to dissect this mechanism. We have used a model of telomerase-immortalized human pancreatic duct-derived cells (E6/E7/st) to study mechanisms of Ras growth transformation. First, we found that human papillomavirus E6 and E7 oncogenes, which block the function of the p53 and Rb tumor suppressors, respectively, and SV40 small t antigen were required to allow mutant K-Ras(12D) growth transformation. Second, K-Ras(12D) caused growth transformation in vitro, including enhanced growth rate and loss of density dependency for growth, anchorage independence, and invasion through reconstituted basement membrane proteins, and tumorigenic transformation in vivo. Third, we determined that the Raf, phosphatidylinositol 3-kinase (PI3K), and Ral guanine nucleotide exchange factor effector pathways were activated, although extracellular signal-regulated kinase (ERK) activity was not up-regulated persistently. Finally, pharmacologic inhibition of Raf/mitogen-activated protein kinase/ERK and PI3K signaling impaired K-Ras-induced anchorage-independent growth and invasion. In summary, our studies established, characterized, and validated E6/E7/st cells for the study of Ras-induced oncogenesis.

  7. Involvement of PI 3 kinase/Akt-dependent Bad phosphorylation in Toxoplasma gondii-mediated inhibition of host cell apoptosis.

    PubMed

    Quan, Juan-Hua; Cha, Guang-Ho; Zhou, Wei; Chu, Jia-Qi; Nishikawa, Yoshifumi; Lee, Young-Ha

    2013-04-01

    Toxoplasma gondii-infected cells are resistant to various apoptotic stimuli, however, the role of the pro-apoptotic BH3-only Bad protein in T. gondii-imposed inhibition of host cell apoptosis in connection with the phosphoinositide 3-kinase (PI3K)-PKB/Akt pathway was not well delineated. Here, we investigated the signaling patterns of Bad, Bax and PKB/Akt in T. gondii-infected and uninfected THP-1 cells treated with staurosporine (STS) or PI3K inhibitors. STS treatment, without T. gondii infection, reduced the viability of THP-1 cells in proportion to STS concentration and triggered many cellular death events such as caspase-3 and -9 activation, Bax translocation, cytochrome c release from host cell mitochondria into cytosol, and PARP cleavage in the host cell. However, T. gondii infection eliminated the STS-triggered mitochondrial apoptotic events described above. Additionally, T. gondii infection in vitro and in vivo induced the phosphorylation of PKB/Akt and Bad in a parasite-load-dependent manner which subsequently inhibited Bax translocation. The PI3K inhibitors, LY294002 and Wortmannin, both blocked parasite-induced phosphorylation of PKB/Akt and Bad. Furthermore, THP-1 cells pretreated with these PI3K inhibitors showed reduced phosphorylation of Bad in a dose-dependent manner and subsequently failed to inhibit the Bax translocation, also these cells also failed to overcome the T. gondii-imposed inhibition of host cell apoptosis. These data demonstrate that the PI3K-PKB/Akt pathway may be one of the major route for T. gondii in the prevention of host cell apoptosis and T. gondii phosphorylates the pro-apoptotic Bad protein to prevent apoptosis.

  8. Thrombopoietin enhances the alpha IIb beta 3-dependent adhesion of megakaryocytic cells to fibrinogen or fibronectin through PI 3 kinase.

    PubMed

    Zauli, G; Bassini, A; Vitale, M; Gibellini, D; Celeghini, C; Caramelli, E; Pierpaoli, S; Guidotti, L; Capitani, S

    1997-02-01

    The effect of thrombopoietin (TPO) on the functional activity of surface alpha IIb beta 3 (GPIIbIIIa) was investigated in both primary human megakaryocytic cells, derived from peripheral blood CD34+ cells, and HEL hematopoietic cell line. TPO (100 ng/mL) induced a sixfold to ninefold enhancement of adhesion of both primary megakaryocytic and HEL cells to plates coated with either fibrinogen or fibronectin and a parallel increase of immunoreactivity to the PAC1 monoclonal antibody (MoAb) and fluorescein isothiocyanate-fibrinogen, both of which recognize an activated state of alpha IIb beta 3. The enhanced adhesion to fibrinogen or fibronectin was mediated by the Arg-Gly-Asp (RGD) recognition sequence of alpha IIb beta 3, as it was abolished by pretreatment of cells with saturating concentrations of RGDS peptide. A MoAb specific for the alpha IIb beta subunit of alpha IIb beta 3 also inhibited cell attachment to fibrinogen or fibronectin, while MoAb to anti-alpha v beta 3 or anti-alpha 5 integrins were completely ineffective, clearly indicating that alpha IIb beta 3 participates in this association. A role for PI 3 kinase (PI 3-K) in the TPO-mediated increase in alpha IIb beta 3 function in megakaryocytic cells was suggested by the ability of the PI 3-K inhibitor wortmannin (100 nmol/L) and antisense oligonucleotides directed against the p85 regulatory subunit of PI 3-K to completely block the TPO-induced increase in alpha IIb beta 3 integrin activity upon TPO stimulation. The modulation of adhesiveness to extracellular matrix proteins containing the RGD motif mediated by TPO likely plays a physiologic role in megakaryocytopoiesis, as pretreatment of CD34+ cells with RGDS or anti-alpha IIb MoAb significantly reduced the number of megakaryocytic colonies obtained in a fibrinclot semisolid assay.

  9. 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.

  10. Investigation into the Role of PI3K and JAK3 Kinase Inhibitors in Murine Models of Asthma

    PubMed Central

    Wagh, Akshaya D.; Sharma, Manoranjan; Mahapatra, Jogeshwar; Chatterjee, Abhijeet; Jain, Mukul; Addepalli, Veeranjaneyulu

    2017-01-01

    Asthma is a clinical disorder commonly characterized by chronic eosinophilic inflammation, remodeling and hyper responsiveness of the airways. However, the kinases like Phosphoinositide 3 kinase (PI3K) and Janus kinase 3 (JAK3) are involved in mast cell proliferation, activation, recruitment, migration, and prolonged survival of inflammatory cells. The present study was designed to evaluate the in-vivo comparative effects of two kinase inhibitors on airway inflammation and airway remodeling in acute and chronic models of asthma. Mice were sensitized twice intra-peritoneally and then challenged by inhalation with ovalbumin (OVA). They developed an extensive inflammatory response, goblet cell hyperplasia, collagen deposition, airway smooth muscle thickening similar to pathologies observed in human asthma. The effects of PI3K inhibitor (30 mg/kg, p.o), JAK3 inhibitor (30 mg/kg, p.o) and Dexamethasone (0.3 mg/kg) on airway inflammation and remodeling in OVA sensitized/challenged BALB/c mice were evaluated. Twenty-four hours after the final antigen challenge, bronchoalveolar lavage (BAL) and histological examinations were carried out. It was observed that kinase inhibitors significantly reduced airway inflammation as evidenced by the decrease in pro inflammatory cytokines in BALF and lung homogenate and inflammatory cell count in sensitized mice after allergen challenge. Lung histological analysis showed increased infiltration of inflammatory cells, hyperplasia of goblet cells and the collagen deposition, which were significantly reduced with kinase inhibitor. In conclusion, our data suggest that PI3K and JAK3 inhibitors showed promising alternative therapeutic activity in asthma, which might significantly counteract the airway inflammation in patients with allergic asthma. PMID:28293189

  11. 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.

  12. Phosphatidylinositol 3-kinase, MEK-1 and p38 mediate leptin/interferon-gamma synergistic NOS type II induction in chondrocytes.

    PubMed

    Otero, Miguel; Lago, Rocío; Gómez, Rodolfo; Lago, Francisca; Gomez-Reino, Juan Jesús; Gualillo, Oreste

    2007-10-27

    In a previous study, we established that leptin acts synergistically with interferon-gamma in inducing nitric oxide synthase type II in cultured chondrocytes via Janus kinase-2 activation. However, the exact molecular mechanism that accounts for this synergism is not completely understood. The aim of the present study was to further delineate the signalling pathway used by leptin/interferon-gamma in the nitric oxide synthase type II induction in chondrocytes. Consequently, the roles of PI-3 kinase, MEK1 and p38 kinase were investigated using specific pharmacological inhibitors (Wortmannin, LY 294002, PD 098,059 and SB 203580). For this purpose, the amount of stable nitrite, the end product of NO generation by activated chondrocytes, has been evaluated by Griess colorimetric reaction in culture medium of human primary chondrocytes and in the murine ATDC5 cell line stimulated with leptin (400 nM) and interferon-gamma (1 ng/ml), alone or in combination. Specific inhibitors for PI-3K, MEK1 and p38 were added 1 h before stimulation. Nitric oxide synthase type II mRNA was investigated by real-time RT-PCR and NOS type II protein expression has been evaluated by western blot analysis. Our results showed that, as expected, leptin synergizes with IFN-gamma in inducing NO accumulation in the supernatant of co-stimulated cells. Pre-treatment with Wortmannin, LY 294002, PD 098,059 and SB 203580 caused a significant decrease in nitrite production, NOS type II protein expression and NOS type II mRNA expression induced by leptin and interferon-gamma co-stimulation. These findings were confirmed in 15 and 21-day differentiated ATDC5 cells, and in normal human primary chondrocytes. This is the first report showing that NOS type II induction triggered by co-stimulation with leptin and interferon-gamma is mediated by a signaling pathway involving PI-3K, MEK1 and p38.

  13. Phosphatidylinositol 3-kinase activates ERK in primary sensory neurons and mediates inflammatory heat hyperalgesia through TRPV1 sensitization.

    PubMed

    Zhuang, Zhi-Ye; Xu, Haoxing; Clapham, David E; Ji, Ru-Rong

    2004-09-22

    Although the PI3K (phosphatidylinositol 3-kinase) pathway typically regulates cell growth and survival, increasing evidence indicates the involvement of this pathway in neural plasticity. It is unknown whether the PI3K pathway can mediate pain hypersensitivity. Intradermal injection of capsaicin and NGF produce heat hyperalgesia by activating their respective TRPV1 (transient receptor potential vanilloid receptor-1) and TrkA receptors on nociceptor sensory nerve terminals. We examined the activation of PI3K in primary sensory DRG neurons by these inflammatory agents and the contribution of PI3K activation to inflammatory pain. We further investigated the correlation between the PI3K and the ERK (extracellular signal-regulated protein kinase) pathway. Capsaicin and NGF induce phosphorylation of the PI3K downstream target AKT (protein kinase B), which is blocked by the PI3K inhibitors LY294002 and wortmannin, indicative of the activation of PI3K by both agents. ERK activation by capsaicin and NGF was also blocked by PI3K inhibitors. Similarly, intradermal capsaicin in rats activated PI3K and ERK in C-fiber DRG neurons and epidermal nerve fibers. Injection of PI3K or MEK (ERK kinase) inhibitors into the hindpaw attenuated capsaicin- and NGF-evoked heat hyperalgesia but did not change basal heat sensitivity. Furthermore, PI3K, but not ERK, inhibition blocked early induction of hyperalgesia. In acutely dissociated DRG neurons, the capsaicin-induced TRPV1 current was strikingly potentiated by NGF, and this potentiation was completely blocked by PI3K inhibitors and primarily suppressed by MEK inhibitors. Therefore, PI3K induces heat hyperalgesia, possibly by regulating TRPV1 activity, in an ERK-dependent manner. The PI3K pathway also appears to play a role that is distinct from ERK by regulating the early onset of inflammatory pain.

  14. PAQR3 modulates insulin signaling by shunting phosphoinositide 3-kinase p110α to the Golgi apparatus.

    PubMed

    Wang, Xiao; Wang, Lingdi; Zhu, Lu; Pan, Yi; Xiao, Fei; Liu, Weizhong; Wang, Zhenzhen; Guo, Feifan; Liu, Yong; Thomas, Walter G; Chen, Yan

    2013-02-01

    Phosphoinositide 3-kinase (PI3K) mediates insulin actions by relaying signals from insulin receptors (IRs) to downstream targets. The p110α catalytic subunit of class IA PI3K is the primary insulin-responsive PI3K implicated in insulin signaling. We demonstrate here a new mode of spatial regulation for the p110α subunit of PI3K by PAQR3 that is exclusively localized in the Golgi apparatus. PAQR3 interacts with p110α, and the intracellular targeting of p110α to the Golgi apparatus is reduced by PAQR3 downregulation and increased by PAQR3 overexpression. Insulin-stimulated PI3K activity and phosphoinositide (3,4,5)-triphosphate production are enhanced by Paqr3 deletion and reduced by PAQR3 overexpression in hepatocytes. Deletion of Paqr3 enhances insulin-stimulated phosphorylation of AKT and glycogen synthase kinase 3β, but not phosphorylation of IR and IR substrate-1 (IRS-1), in hepatocytes, mouse liver, and skeletal muscle. Insulin-stimulated GLUT4 translocation to the plasma membrane and glucose uptake are enhanced by Paqr3 ablation. Furthermore, PAQR3 interacts with the domain of p110α involved in its binding with p85, the regulatory subunit of PI3K. Overexpression of PAQR3 dose-dependently reduces the interaction of p85α with p110α. Thus, PAQR3 negatively regulates insulin signaling by shunting cytosolic p110α to the Golgi apparatus while competing with p85 subunit in forming a PI3K complex with p110α.

  15. PI3-kinase signaling contributes to orientation in shallow gradients and enhances speed in steep chemoattractant gradients.

    PubMed

    Bosgraaf, Leonard; Keizer-Gunnink, Ineke; Van Haastert, Peter J M

    2008-11-01

    Dictyostelium cells that chemotax towards cAMP produce phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] at the leading edge, which has been implicated in actin reorganization and pseudopod extension. However, in the absence of PtdIns(3,4,5)P(3) signaling, cells will chemotax via alternative pathways. Here we examined the potential contribution of PtdIns(3,4,5)P(3) to chemotaxis of wild-type cells. The results show that steep cAMP gradients (larger than 10% concentration difference across the cell) induce strong PtdIns(3,4,5)P(3) patches at the leading edge, which has little effect on the orientation but strongly enhances the speed of the cell. Using a new sensitive method for PtdIns(3,4,5)P(3) detection that corrects for the volume of cytosol in pixels at the boundary of the cell, we show that, in shallow cAMP gradient (less than 5% concentration difference across the cell), PtdIns(3,4,5)P(3) is still somewhat enriched at the leading edge. Cells lacking PI3-kinase (PI3K) activity exhibit poor chemotaxis in these shallow gradients. Owing to the reduced speed and diminished orientation of the cells in steep and shallow gradients, respectively, cells lacking PtdIns(3,4,5)P(3) signaling require two- to six-fold longer times to reach a point source of chemoattractant compared with wild-type cells. These results show that, although PI3K signaling is dispensable for chemotaxis, it gives the wild type an advantage over mutant cells.

  16. Class I PI3-kinase or Akt inhibition do not impair axonal polarization, but slow down axonal elongation.

    PubMed

    Diez, Héctor; Benitez, Ma José; Fernandez, Silvia; Torres-Aleman, Ignacio; Garrido, Juan José; Wandosell, Francisco

    2016-11-01

    PI3K proteins family have multiple and essential functions in most cellular events. This family is composed of class I, class II and class III PI3Ks, which upstream and downstream elements are not completely elucidated. Previous studies using the broad PI3K inhibitor, LY294002 allowed to propose that PI3 kinase>Akt pathway is a key element in the determination of axonal polarity in hippocampal neurons. Recently, new inhibitors with a higher selectivity for class I PI3K have been characterized. In the present study we have examined this widely accepted theory using a new class I PI3K inhibitor (GDC-0941), as well as Akt inhibitors, and PTEN phosphatase constructs to reduce PIP3 levels. Our present data show that both, class I PI3K inhibitor and Akt inhibitor did not alter axon specification in hippocampal neurons, but greatly reduced axon length. However, in the same experiments LY294002 effectively impeded axonal polarization, as previously reported. Our biochemical data show that both, class I PI3K and Akt inhibitors, effectively block downstream elements from Akt to S6K1 activity. Both inhibitors are stable in culture medium along the time period analysed, maintaining the inhibition better than LY294002. Besides, we found evidence that LY294002 directly inhibits mTORC1. However, further analysis using an mTORC1 inhibitor showed no change in neuron polarity. Same result was obtained using a general class III PI3K inhibitor. Interestingly, we found that either, wild-type PTEN, or a phosphatase-dead form of PTEN, disrupted axonal polarization, strongly suggesting that the role of PTEN in axonal polarity can be independent of PIP3.

  17. Efficacy of phosphatidylinositol-3 kinase inhibitors with diverse isoform selectivity profiles for inhibiting the survival of chronic lymphocytic leukemia cells.

    PubMed

    Göckeritz, Elisa; Kerwien, Susan; Baumann, Michael; Wigger, Marion; Vondey, Verena; Neumann, Lars; Landwehr, Thomas; Wendtner, Clemens M; Klein, Christian; Liu, Ningshu; Hallek, Michael; Frenzel, Lukas P; Krause, Günter

    2015-11-01

    Pharmacological inhibition of phosphatiylinositide-3-kinase (PI3K)-mediated signaling holds great promise for treating chronic lymphocytic leukemia (CLL). Therefore we assessed three structurally related PI3K inhibitors targeting the PI3K-δ isoform for their ability to inhibit the survival of freshly isolated CLL cells. The purely PI3K-δ-selective inhibitor idelalisib was compared to copanlisib (BAY 80-6946) and duvelisib (IPI-145), with isoform target profiles that additionally include PI3K-α or PI3K-γ, respectively. The concentrations leading to half-maximal reduction of the survival of CLL cells were more than ten-fold lower for copanlisib than for idelalisib and duvelisib. At concentrations reflecting the biological availability of the different inhibitors, high levels of apoptotic response among CLL samples were attained more consistently with copanlisib than with idelalisib. Copanlisib selectively reduced the survival of CLL cells compared to T cells and to B cells from healthy donors. In addition copanlisib and duvelisib impaired the migration of CLL cells towards CXCL12 to a greater extent than equimolar idelalisib. Similarly copanlisib and duvelisib reduced the survival of CLL cells in co-cultures with the bone marrow stroma cell line HS-5 more strongly than idelalisib. Survival inhibition by copanlisib and idelalisib was enhanced by the monoclonal CD20 antibodies rituximab and obinutuzumab (GA101), while antibody-dependent cellular cytotoxicity mediated by alemtuzumab and peripheral blood mononuclear cells was not substantially impaired by both PI3K inhibitors for the CLL-derived JVM-3 cell line as target cells. Taken together, targeting the α- and δ- p110 isoforms with copanlisib may be a useful strategy for the treatment of CLL and warrants further clinical investigation.

  18. Paclitaxel resistance in MCF-7/PTX cells is reversed by paeonol through suppression of the SET/phosphatidylinositol 3-kinase/Akt pathway.

    PubMed

    Zhang, Weipeng; Cai, Jiangxia; Chen, Siying; Zheng, Xiaowei; Hu, Sasa; Dong, Weihua; Lu, Jun; Xing, Jianfeng; Dong, Yalin

    2015-07-01

    Breast cancer is one of the most prevalent types of malignant tumor. Paclitaxel is widely used in the treatment of breast cancer; however, the major problem contributing to the failure of chemotherapy in breast cancer is the development of drug resistance. Therefore, it is necessary to identify novel therapeutic targets and reversal agents for breast cancer. In the present study, the protein expression levels of SET, protein phosphatase 2A (PP2A) and phosphatidylinositol 3-kinase (PI3K)/Akt pathway were determined in MCF-7/PTX human breast carcinoma paclitaxel-resistant cells using western blot analysis. Small interference RNAs (siRNAs) were used to knock down the gene expression of SET in MCF-7/PTX cells and the cell viability was assessed following treatment with paclitaxel, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays and flow cytometry. In addition, western blot analysis was used to determined PI3K/Akt pathway activity following SET knockdown. Furthermore, the reversal effects of paeonol on paclitaxel, and its underlying mechanisms of action, were investigated using western blot analysis and reverse transcription-quantitative polymerase chain reaction. The results demonstrated that increased levels of SET and PI3K/Akt pathway proteins were present in the MCF-7/PTX cells, compared with normal MCF-7 cells. Knockdown of SET significantly sensitized MCF-7/PTX cells to paclitaxel and induced cell apoptosis. In addition, the expression levels of the adenosine triphosphate binding cassette (ABC) transporter proteins were significantly reduced in the MCF-7/PTX cells compared with the normal MCF-7 cells. SET-induced paclitaxel resistance was found to be associated with the activation of the PI3K/Akt pathway. Paeonol significantly reduced the mRNA and protein expression levels of SET in the MCF-7/PTX cells. Furthermore, paeonol significantly sensitized the MCF-7/PTX to paclitaxel via regulation of ABC transporters, B cell lymphoma-2 (Bcl-2

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

    PubMed

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

    2006-01-01

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

  20. Multiple roles for PI 3-kinase in the regulation of PLCgamma activity and Ca2+ mobilization in antigen-stimulated mast cells.

    PubMed

    Barker, S A; Lujan, D; Wilson, B S

    1999-03-01

    Cross-linking the IgE-bound FcepsilonRI with polyvalent antigen leads to Ca2+-dependent degranulation from mast cells and basophils, initiating the allergic response. This overview addresses novel roles for PI 3-kinase in the regulation of signaling events that lie downstream of FcepsilonRI-mediated tyrosine kinase activation. The first novel role for PI 3-kinase is in the regulation of PLCgamma activity and is demonstrated by a dramatic inhibition of FcepsilonRI-induced Ins(1,4,5)P3 production after treatment of RBL-2H3 cells with wortmannin, a PI 3-kinase inhibitor. We show that PI 3-kinase lipid products support Ins(1,4,5)P3 production in at least two ways: by promoting translocation and phosphorylation of PLCgamma1 and by direct stimulation of both PLCgamma isoforms. In vitro stimulation of PLCgamma activity by PtdIns(3,4,5)P3 synergizes with activation by in vivo tyrosine phosphorylation for maximal enzymatic activity. A second novel role for PI 3-kinase is in the regulation of antigen-stimulated Ca2+ influx. Compared with control cells, Ca2+ responses are markedly diminished in antigen-stimulated cells after wortmannin pretreatment. Differences include both a longer lag time to the initial elevation in Ca2+ after antigen and an inhibition of the sustained Ca2+ influx phase. However, thapsigargin challenge during the sustained phase demonstrates no difference in the state of the Ca2+ stores in antigen-stimulated cells in the presence or absence of wortmannin. These data suggest that sufficient Ins(1,4,5)P3 is synthesized in wortmannin-treated cells to mobilize intracellular calcium stores and, furthermore, that the affected phase of Ca2+ influx is unlikely to be attributed to capacitative mechanisms. These data are consistent with a model where at least two pathways mediate Ca2+ influx in antigen-stimulated RBL-2H3 cells, one that is dependent on signals from empty stores (capacitative influx) and another that is downstream of PI 3-kinase.

  1. Molecular definition of a novel inositol polyphosphate metabolic pathway initiated by inositol 1,4,5-trisphosphate 3-kinase activity in Saccharomyces cerevisiae.

    PubMed

    Seeds, Andrew M; Bastidas, Robert J; York, John D

    2005-07-29

    The production of inositol polyphosphate (IPs) and pyrophosphates (PP-IPs) from inositol 1,4,5-trisphosphate (I(1,4,5)P3) requires the 6-/3-/5-kinase activity of Ipk2 (also known as Arg82 and inositol polyphosphate multikinase). Here, we probed the distinct roles for I(1,4,5)P3 6- versus 3-kinase activities in IP metabolism and cellular functions reported for Ipk2. Expression of either I(1,4,5)P3 6- or 3-kinase activity rescued growth of ipk2-deficient yeast at high temperatures, whereas only 6-kinase activity enabled growth on ornithine as the sole nitrogen source. Analysis of IP metabolism revealed that the 3-kinase initiated the synthesis of novel pathway consisting of over eleven IPs and PP-IPs. This pathway was present in wild-type and ipk2 null cells, albeit at low levels as compared with inositol hexakisphosphate synthesis. The primary route of synthesis was: I(1,4,5)P3 --> I(1,3,4,5)P4 --> I(1,2,3,4,5)P5 --> PP-IP4 --> PP2-IP3 and required Kcs1 (or possibly Ipk2), Ipk1, a novel inositol pyrophosphate synthase, and then Kcs1 again, respectively. Mutation of kcs1 ablated this pathway in ipk2 null cells and overexpression of Kcs1 in ipk2 mutant cells phenocopied IP3K expression, confirming it harbors a novel 3-kinase activity. Our work provides a revised genetic map of IP metabolism in yeast and evidence for dosage compensation between IPs and PP-IPs downstream of I(1,4,5)P3 in the regulation of nucleocytoplasmic processes.

  2. Combining TRAIL with PI3 Kinase or HSP90 inhibitors enhances apoptosis in colorectal cancer cells via suppression of survival signaling

    PubMed Central

    Saturno, Grazia; Valenti, Melanie; De Haven Brandon, Alexis; Thomas, George V.; Eccles, Suzanne; Clarke, Paul A.; Workman, Paul

    2013-01-01

    TRAIL has been shown to induce apoptosis in cancer cells, but in some cases they fail to respond to this ligand. We explored the ability of representative phosphatidylinositol-3-kinase (PI3 Kinase)/mTOR and HSP90 inhibitors to overcome TRAIL resistance by increasing apoptosis in colorectal cancer models. We determined the sensitivity of 27 human colorectal cancer and 2 non-transformed colon epithelial cell lines to TRAIL treatment. A subset of the cancer cell lines with a range of responses to TRAIL was selected from the panel for treatment with TRAIL combined with the PI3 Kinase/mTOR inhibitor PI-103 or the HSP90 inhibitor 17-AAG (tanespimycin). Two TRAIL-resistant cell lines were selected for in vivo combination studies with TRAIL and 17-AAG. We found that 13 colorectal cancer cell lines and the 2 non-transformed colon epithelial cell lines were resistant to TRAIL. We demonstrated that co-treatment of TRAIL and PI-103 or 17-AAG was synergistic or additive and significantly enhanced apoptosis in colorectal cancer cells. This was associated with decreased expression or activity of survival protein biomarkers such as ERBB2, AKT, IKKα and XIAP. In contrast, the effect of the combination treatments in non-transformed colon cells was minimal. We show here for the first time that co-treatment in vivo with TRAIL and 17-AAG in two TRAIL-resistant human colorectal cancer xenograft models resulted in significantly greater tumor growth inhibition compared to single treatments. We propose that combining TRAIL with PI3 Kinase/mTOR or HSP90 inhibitors has therapeutic potential in the treatment of TRAIL-resistant colorectal cancers. PMID:23852390

  3. 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

  4. ErbB3 (HER3) interaction with the p85 regulatory subunit of phosphoinositide 3-kinase.

    PubMed Central

    Hellyer, N J; Cheng, K; Koland, J G

    1998-01-01

    ErbB3 (HER3), a unique member of the ErbB receptor family, lacks intrinsic protein tyrosine kinase activity and contains six Tyr-Xaa-Xaa-Met (YXXM) consensus binding sites for the SH2 domains of the p85 regulatory subunit of phosphoinositide 3-kinase. ErbB3 also has a proline-rich sequence that forms a consensus binding site for the SH3 domain of p85. Here we have investigated the interacting domains of ErbB3 and p85 by a unique application of the yeast two-hybrid system. A chimaeric ErbB3 molecule containing the epidermal growth factor receptor protein tyrosine kinase domain was developed so that the C-terminal domain of ErbB3 could become phosphorylated in the yeast system. We also generated several ErbB3 deletion and Tyr-->Phe site-specific mutants, and observed that a single ErbB3 YXXM motif was necessary and sufficient for the association of ErbB3 with p85. The incorporation of multiple YXXM motifs into the ErbB3 C-terminus enabled a stronger ErbB3/p85 interaction. The proline-rich region of ErbB3 was not necessary for interaction with p85. However, either deletion or mutation of the p85 SH3 domain decreased the observed ErbB3/p85 association. Additionally an ErbB3/p85 SH3 domain interaction was detected by an assay in vitro. These results were consistent with a model in which pairs of phosphorylated ErbB3 YXXM motifs co-operate in binding to the tandem SH2 domains of p85. Although a contributing role for the p85 SH3 domain was suggested, the N- and C-terminal SH2 domains seemed to be primarily responsible for the high-affinity association of p85 and ErbB3. PMID:9677338

  5. ErbB3 (HER3) interaction with the p85 regulatory subunit of phosphoinositide 3-kinase.

    PubMed

    Hellyer, N J; Cheng, K; Koland, J G

    1998-08-01

    ErbB3 (HER3), a unique member of the ErbB receptor family, lacks intrinsic protein tyrosine kinase activity and contains six Tyr-Xaa-Xaa-Met (YXXM) consensus binding sites for the SH2 domains of the p85 regulatory subunit of phosphoinositide 3-kinase. ErbB3 also has a proline-rich sequence that forms a consensus binding site for the SH3 domain of p85. Here we have investigated the interacting domains of ErbB3 and p85 by a unique application of the yeast two-hybrid system. A chimaeric ErbB3 molecule containing the epidermal growth factor receptor protein tyrosine kinase domain was developed so that the C-terminal domain of ErbB3 could become phosphorylated in the yeast system. We also generated several ErbB3 deletion and Tyr-->Phe site-specific mutants, and observed that a single ErbB3 YXXM motif was necessary and sufficient for the association of ErbB3 with p85. The incorporation of multiple YXXM motifs into the ErbB3 C-terminus enabled a stronger ErbB3/p85 interaction. The proline-rich region of ErbB3 was not necessary for interaction with p85. However, either deletion or mutation of the p85 SH3 domain decreased the observed ErbB3/p85 association. Additionally an ErbB3/p85 SH3 domain interaction was detected by an assay in vitro. These results were consistent with a model in which pairs of phosphorylated ErbB3 YXXM motifs co-operate in binding to the tandem SH2 domains of p85. Although a contributing role for the p85 SH3 domain was suggested, the N- and C-terminal SH2 domains seemed to be primarily responsible for the high-affinity association of p85 and ErbB3.

  6. Ovarian expressed microsomal epoxide hydrolase: Role in detoxification of 4-vinylcyclohexene diepoxide and regulation by phosphatidylinositol-3 kinase signaling

    SciTech Connect

    Bhattacharya, Poulomi; Sen, Nivedita; Hoyer, Patricia B.; Keating, Aileen F.

    2012-01-01

    4-vinylcyclohexene diepoxide (VCD) is a metabolite of 4-vinylcyclohexene (VCH) which has the potential to be formed in the ovary through CYP2E1 activity. VCD specifically destroys primordial and small primary follicles in the rodent ovary. Mouse ovaries exposed to VCD demonstrate increased mRNA and protein expression of microsomal epoxide hydrolase (mEH), and an inactive tetrol metabolite (4-(1,2-dihydroxy)ethyl-1,2-dihydroxycyclohexane) can be formed in mouse ovarian follicles, potentially through detoxification action of mEH. In contrast, mEH can bioactivate another ovotoxic chemical, 7,12-dimethylbenz[a]anthracene (DMBA) to a more toxic compound, DMBA-3,4-diol-1,2-epoxide. Thus, the present study evaluated a functional role for mEH during detoxification of VCD. Additionally, because inhibition of the phosphatidyinositol-3 kinase (PI3K) signaling pathway in a previous study protected primordial follicles from VCD-induced destruction, but accelerated DMBA-induced ovotoxicity, a role for PI3K in ovarian mEH regulation was evaluated. Using a post-natal day (PND) 4 Fischer 344 rat whole ovary culture system inhibition of mEH using cyclohexene oxide during VCD exposure resulted in a greater (P < 0.05) loss of primordial and small primary follicles relative to VCD-treated ovaries. Also, relative to controls, meh mRNA was increased (P < 0.05) on day 4 of VCD (30 μM) exposure, followed by increased (P < 0.05) mEH protein after 6 days. Furthermore, inhibition of PI3K signaling increased mEH mRNA and protein expression. Thus, these results support a functional role for mEH in the rat ovary, and demonstrate the involvement of PI3K signaling in regulation of ovarian xenobiotic metabolism by mEH. -- Highlights: ► Ovarian mEH functions to metabolize VCD to a less toxic compound. ► mEH expression is increased in a temporal pattern in response to VCD exposure. ► PI3K signaling is involved in regulation of ovarian mEH expression.

  7. Inhibition of phosphatidylinositol 3-kinase stimulates activity of the small-conductance K channel in the CCD

    PubMed Central

    Li, Dimin; Wei, Yuan; Babilonia, Elisa; Wang, Zhijian; Wang, Wen-Hui

    2010-01-01

    We used Western blotting to examine the expression of phosphatidylinositol 3-kinase (PI3K) in the renal cortex and outer medulla and employed the patch-clamp technique to study the effect of PI3K on the ROMK-like small-conductance K (SK) channels in the cortical collecting duct (CCD). Low K intake increased the expression of the 110-kDa α-subunit (p110α) of PI3K compared with rats on a normal-K diet. Because low K intake increases superoxide levels (2), the possibility that increases in superoxide anions may be responsible for the effect of low K intake on the expression of PI3K is supported by finding that addition of H2O2 stimulates the expression of p110α in M1 cells. Inhibition of PI3K with either wortmannin or LY-294002 significantly increased channel activity in the CCD from rats on a K-deficient (KD) diet or on a normal-K diet. The stimulatory effect of wortmannin on ROMK channel activity cannot be mimicked by inhibition of phospholipase C with U-73122. This suggests that the effect of inhibiting PI3K was not the result of increasing the phosphatidylinositol 4,5-bisphosphate level. Moreover, application of the exogenous phosphatidylinositol 3,4,5-trisphosphate analog had no effect on channel activity in excised patches. Because low K intake has been shown to increase the activity of protein tyrosine kinase (PTK), we explored the role of the interaction between PTK and PI3K in the regulation of the SK channel activity. Inhibition of PTK increased SK channel activity in the CCD from rats on a KD diet. However, addition of wortmannin did not further increase ROMK channel activity. Also, the effect of wortmannin was abolished by treatment of CCD with phalloidin. We conclude that PI3K is involved in mediating the effect of low K intake on ROMK channel activity in the CCD and that the effect of PI3K on SK channels requires the involvement of PTK and the cytoskeleton. PMID:16204406

  8. Inhibition of phosphatidylinositol 3-kinase stimulates activity of the small-conductance K channel in the CCD.

    PubMed

    Li, Dimin; Wei, Yuan; Babilonia, Elisa; Wang, Zhijian; Wang, Wen-Hui

    2006-04-01

    We used Western blotting to examine the expression of phosphatidylinositol 3-kinase (PI3K) in the renal cortex and outer medulla and employed the patch-clamp technique to study the effect of PI3K on the ROMK-like small-conductance K (SK) channels in the cortical collecting duct (CCD). Low K intake increased the expression of the 110-kDa alpha-subunit (p110alpha) of PI3K compared with rats on a normal-K diet. Because low K intake increases superoxide levels (2), the possibility that increases in superoxide anions may be responsible for the effect of low K intake on the expression of PI3K is supported by finding that addition of H(2)O(2) stimulates the expression of p110alpha in M1 cells. Inhibition of PI3K with either wortmannin or LY-294002 significantly increased channel activity in the CCD from rats on a K-deficient (KD) diet or on a normal-K diet. The stimulatory effect of wortmannin on ROMK channel activity cannot be mimicked by inhibition of phospholipase C with U-73122. This suggests that the effect of inhibiting PI3K was not the result of increasing the phosphatidylinositol 4,5-bisphosphate level. Moreover, application of the exogenous phosphatidylinositol 3,4,5-trisphosphate analog had no effect on channel activity in excised patches. Because low K intake has been shown to increase the activity of protein tyrosine kinase (PTK), we explored the role of the interaction between PTK and PI3K in the regulation of the SK channel activity. Inhibition of PTK increased SK channel activity in the CCD from rats on a KD diet. However, addition of wortmannin did not further increase ROMK channel activity. Also, the effect of wortmannin was abolished by treatment of CCD with phalloidin. We conclude that PI3K is involved in mediating the effect of low K intake on ROMK channel activity in the CCD and that the effect of PI3K on SK channels requires the involvement of PTK and the cytoskeleton.

  9. Low [Mg2+]e enhances arterial spontaneous tone via phosphatidylinositol 3-kinase in DOCA-salt hypertension.

    PubMed

    Northcott, Carrie A; Watts, Stephanie W

    2004-01-01

    Phosphatidylinositol 3-kinase (PI3K) has been implicated in low extracellular Mg2+ concentration ( [Mg2+]e)-induced aortic contraction, and Mg2+ deficiency has been associated with hypertension. Moreover, arterial PI3K activity is increased in hypertensive deoxycorticosterone (DOCA)-salt rats. We hypothesized that low [Mg2+]e activates PI3K, eliciting enhanced vascular contraction, PI3K activity, and norepinephrine (NE)-induced contraction. Spontaneous tone was monitored in endothelium-denuded aortic strips from sham and DOCA-salt rats exposed to low Mg2+ (0.15 mmol/L), high Mg2+ (4.8 mmol/L), or normal (1.17 mmol/L) physiologic salt solution (PSS) in isolated tissue baths. LY294002 (20 micromol/L), a PI3K inhibitor, or vehicle was added (30 minutes), followed by NE (10(-9) to 3 x10(-5) mol/L). Low [Mg2+]e significantly enhanced tone in aortas from DOCA-salt and sham rats compared with normal PSS (DOCA-salt low [Mg2+]e, +51.5 +7.0 vs DOCA-salt normal PSS, +7.1 +1.4 % of initial phenylephrine [PE] contraction). LY294002 and incubation with high Mg2+ PSS decreased tone in aortas from DOCA-salt rats (low [Mg2+]e LY294002, --87.5 +8.8; normal PSS LY294002, -81.7 +13.7; and high [Mg2+]e, -31.2 +10.8 % of initial PE contraction). Low [Mg2+]e leftward-shifted NE-induced aortic contractions in sham and thus matched the shift observed with DOCA (-log EC50 mol/L: sham PSS, -7.7 +0.1; DOCA-salt PSS, -8.2 +0.1; sham low [Mg2+]e, -8.2 +0.1; and DOCA-salt low [Mg2+]e, -8.1 +0.1). Moreover, this shift was inhibited by LY294002. In conclusion, low [Mg2+]e might activate PI3K, leading to enhanced tone and agonist-induced contraction observed in aortas from DOCA-salt hypertensive rats.

  10. PTEN and PI-3 kinase inhibitors control LPS signaling and the lymphoproliferative response in the CD19+ B cell compartment.

    PubMed

    Singh, Alok R; Peirce, Susan K; Joshi, Shweta; Durden, Donald L

    2014-09-10

    Pattern recognition receptors (PRRs), e.g. toll receptors (TLRs) that bind ligands within the microbiome have been implicated in the pathogenesis of cancer. LPS is a ligand for two TLR family members, TLR4 and RP105 which mediate LPS signaling in B cell proliferation and migration. Although LPS/TLR/RP105 signaling is well-studied; our understanding of the underlying molecular mechanisms controlling these PRR signaling pathways remains incomplete. Previous studies have demonstrated a role for PTEN/PI-3K signaling in B cell selection and survival, however a role for PTEN/PI-3K in TLR4/RP105/LPS signaling in the B cell compartment has not been reported. Herein, we crossed a CD19cre and PTEN(fl/fl) mouse to generate a conditional PTEN knockout mouse in the CD19+ B cell compartment. These mice were further crossed with an IL-14α transgenic mouse to study the combined effect of PTEN deletion, PI-3K inhibition and expression of IL-14α (a cytokine originally identified as a B cell growth factor) in CD19+ B cell lymphoproliferation and response to LPS stimulation. Targeted deletion of PTEN and directed expression of IL-14α in the CD19+ B cell compartment (IL-14+PTEN-/-) lead to marked splenomegaly and altered spleen morphology at baseline due to expansion of marginal zone B cells, a phenotype that was exaggerated by treatment with the B cell mitogen and TLR4/RP105 ligand, LPS. Moreover, LPS stimulation of CD19+ cells isolated from these mice display increased proliferation, augmented AKT and NFκB activation as well as increased expression of c-myc and cyclinD1. Interestingly, treatment of LPS treated IL-14+PTEN-/- mice with a pan PI-3K inhibitor, SF1126, reduced splenomegaly, cell proliferation, c-myc and cyclin D1 expression in the CD19+ B cell compartment and normalized the splenic histopathologic architecture. These findings provide the direct evidence that PTEN and PI-3K inhibitors control TLR4/RP105/LPS signaling in the CD19+ B cell compartment and that pan PI-3

  11. Molecular modeling study of CP-690550 derivatives as JAK3 kinase inhibitors through combined 3D-QSAR, molecular docking, and dynamics simulation techniques.

    PubMed

    Wang, Jing Li; Cheng, Li Ping; Wang, Tian Chi; Deng, Wei; Wu, Fan Hong

    2017-03-01

    To develop more potent JAK3 kinase inhibitors, a series of CP-690550 derivatives were investigated using combined molecular modeling techniques, such as 3D-QSAR, molecular docking and molecular dynamics (MD). The leave-one-out correlation (q(2)) and non-cross-validated correlation coefficient (r(2)) of the best CoMFA model are 0.715 and 0.992, respectively. The q(2) and r(2) values of the best CoMSIA model are 0.739 and 0.995, respectively. The steric, electrostatic, and hydrophobic fields played important roles in determining the inhibitory activity of CP-690550 derivatives. Some new JAK3 kinase inhibitors were designed. Some of them have better inhibitory activity than the most potent Tofacitinib (CP-690550). Molecular docking was used to identify some key amino acid residues at the active site of JAK3 protein. 10ns MD simulations were successfully performed to confirm the detailed binding mode and validate the rationality of docking results. The calculation of the binding free energies by MMPBSA method gives a good correlation with the predicted biological activity. To our knowledge, this is the first report on MD simulations and free energy calculations for this series of compounds. The combination results of this study will be valuable for the development of potent and novel JAK3 kinase inhibitors.

  12. [Effects of phosphatidylinositol-3 kinase/protein kinase b/bone morphogenetic protein-15 pathway on the follicular development in the mammalian ovary].

    PubMed

    Wu, Yan-qing; Chen, Li-yun; Zhang, Zheng-hong; wang, Zheng-chao

    2013-04-01

    In mammals, ovarian follicle is made of an oocyte with its surrounding granulosa cells and theca cells. Follicular growth and development is a highly coordinated programmable process, which guarantees the normal oocyte maturation and makes it having the fertilizing capacity. The paracrine and autocrine between oocytes and granulosa cells are essential for the follicular development to provide a suitable microenvironment. Phosphatidylinositol-3 kinase /protein kinase B is one of these important regulatory signaling pathways during this developmental process, and bone morphogenetic protein-15 an oocyte-specific secreted signal molecule, which regulates the follicular development by paracrine in the mammalian ovary. The present article overviewed the role of phosphatidylinositol-3 kinase / protein kinase B signaling during the follicular development based on our previous investigation about protein kinase B /forkhead transcription factor forkhead family of transcription factors -3a, and then focused on the regulatory effects of bone morphogenetic protein-15, as a downstream signal molecule of phosphatidylinositol-3 kinase / forkhead family of transcription factors -3a pathway, on ovarian follicular development, which helped to further understand the molecular mechanism regulating the follicular development and to treat ovarian diseases like infertility.

  13. 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.

  14. Short-term low-protein diet during pregnancy alters islet area and protein content of phosphatidylinositol 3-kinase pathway in rats.

    PubMed

    Salvatierra, Cristiana S B; Reis, Sílvia R L; Pessoa, Ana F M; De Souza, Letícia M I; Stoppiglia, Luiz F; Veloso, Roberto V; Reis, Marise A B; Carneiro, Everardo M; Boschero, Antonio C; Colodel, Edson M; Arantes, Vanessa C; Latorraca, Márcia Q

    2015-01-01

    The phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways mediate β cell growth, proliferation, survival and death. We investigated whether protein restriction during pregnancy alters islet morphometry or the expression and phosphorylation of several proteins involved in the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways. As controls, adult pregnant and non-pregnant rats were fed a normal-protein diet (17%). Pregnant and non-pregnant rats in the experimental groups were fed a low-protein diet (6%) for 15 days. Low protein diet during pregnancy increased serum prolactin level, reduced serum corticosterone concentration and the expression of both protein kinase B/AKT1 (AKT1) and p70 ribosomal protein S6 kinase (p70S6K), as well as the islets area, but did not alter the insulin content of pancreatic islets. Pregnancy increased the expression of the Src homology/collagen (SHC) protein and the extracellular signal-regulated kinases 1/2 (ERK1/2) independent of diet. ERK1/2 phosphorylation (pERK1/2) was similar in islets from pregnant and non-pregnant rats fed a low-protein diet, and was higher in islets from pregnant rats than in islets from non-pregnant rats fed a normal-protein diet. Thus, a short-term, low-protein diet during pregnancy was sufficient to reduce the levels of proteins in the phosphatidylinositol 3-kinase pathway and affect islet morphometry.

  15. Role of erbB-2 and erbB-3 in the Activation of Phosphatidylinositol 3 -Kinase.

    DTIC Science & Technology

    1997-06-01

    34 induces full neoplastic transformation in human mammary epithelial cells still remains fragmentary. For example, although p18 5 erbB2 was originally...discovered as the neu transmembrane- mutated form of the gene in rat neuroblastoma cells (29), the c-erbB-2 gene found in human breast cancer cells has...not ever shown similar mutations (30). The level of c-erbB-2 gene expression seen in primary human mammary carcinomas in vivo always shows a direct

  16. Insulin receptor phosphorylation, insulin receptor substrate-1 phosphorylation, and phosphatidylinositol 3-kinase activity are decreased in intact skeletal muscle strips from obese subjects.

    PubMed Central

    Goodyear, L J; Giorgino, F; Sherman, L A; Carey, J; Smith, R J; Dohm, G L

    1995-01-01

    To determine whether the impaired insulin-stimulated glucose uptake in obese individuals is associated with altered insulin receptor signaling, we measured both glucose uptake and early steps in the insulin action pathway in intact strips of human skeletal muscle. Biopsies of rectus abdominus muscle were taken from eight obese and eight control subjects undergoing elective surgery (body mass index 52.9 +/- 3.6 vs 25.7 +/- 0.9). Insulin-stimulated 2-deoxyglucose uptake was 53% lower in muscle strips from obese subjects. Additional muscle strips were incubated in the basal state or with 10(-7) M insulin for 2, 15, or 30 min. In the lean subjects, tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 (IRS-1), measured by immunoblotting with anti-phosphotyrosine antibodies, was significantly increased by insulin at all time points. In the skeletal muscle from the obese subjects, insulin was less effective in stimulating tyrosine phosphorylation (maximum receptor and IRS-1 phosphorylation decreased by 35 and 38%, respectively). Insulin stimulation of IRS-1 immunoprecipitable phosphatidylinositol 3-kinase (PI 3-kinase) activity also was markedly lower in obese subjects compared with controls (10- vs 35-fold above basal, respectively). In addition, the obese subjects had a lower abundance of the insulin receptor, IRS-1, and the p85 subunit of PI 3-kinase (55, 54, and 64% of nonobese, respectively). We conclude that impaired insulin-stimulated glucose uptake in skeletal muscle from severely obese subjects is accompanied by a deficiency in insulin receptor signaling, which may contribute to decreased insulin action. Images PMID:7537758

  17. The novel orally bioavailable inhibitor of phosphoinositol-3-kinase and mammalian target of rapamycin, NVP-BEZ235, inhibits growth and proliferation in multiple myeloma

    SciTech Connect

    Baumann, Philipp Mandl-Weber, Sonja; Oduncu, Fuat; Schmidmaier, Ralf

    2009-02-01

    NVP-BEZ235 is a new inhibitor of phosphoinositol-3-kinase (PI3 kinase) and mammalian target of rapamycin (mTOR) whose efficacy in advanced solid tumours is currently being evaluated in a phase I/II clinical trial. Here we show that NVP-BEZ235 inhibits growth in common myeloma cell lines as well as primary myeloma cells at nanomolar concentrations in a time and dose dependent fashion. Further experiments revealed induction of apoptosis in three of four cell lines. Inhibition of cell growth was mainly due to inhibition of myeloma cell proliferation, as shown by the BrdU assay. Cell cycle analysis revealed induction of cell cycle arrest in the G1 phase, which was due to downregulation of cyclin D1, pRb and cdc25a. NVP-BEZ235 inhibited phosphorylation of protein kinase B (Akt), P70S6k and 4E-BP-1. Furthermore we show that the stimulatory effect of CD40-ligand (CD40L), insulin-like growth factor 1 (IGF-1), interleukin-6 (IL-6) and conditioned medium of HS-5 stromal cells on myeloma cell growth is completely abrogated by NVP-BEZ235. In addition, synergism studies revealed synergistic and additive activity of NVP-BEZ235 together with melphalan, doxorubicin and bortezomib. Taken together, inhibition of PI3 kinase/mTOR by NVP-BEZ235 is highly effective and NVP-BEZ235 represents a potential new candidate for targeted therapy in multiple myeloma.

  18. PSM/SH2-B distributes selected mitogenic receptor signals to distinct components in the PI3-kinase and MAP kinase signaling pathways.

    PubMed

    Deng, Youping; Xu, Hu; Riedel, Heimo

    2007-02-15

    The Pro-rich, PH, and SH2 domain containing mitogenic signaling adapter PSM/SH2-B has been implicated as a cellular partner of various mitogenic receptor tyrosine kinases and related signaling mechanisms. Here, we report in a direct comparison of three peptide hormones, that PSM participates in the assembly of distinct mitogenic signaling complexes in response to insulin or IGF-I when compared to PDGF in cultured normal fibroblasts. The complex formed in response to insulin or IGF-I involves the respective peptide hormone receptor and presumably the established components leading to MAP kinase activation. However, our data suggest an alternative link from the PDGF receptor via PSM directly to MEK1/2 and consequently also to p44/42 activation, possibly through a scaffold protein. At least two PSM domains participate, the SH2 domain anticipated to link PSM to the respective receptor and the Pro-rich region in an association with an unidentified downstream component resulting in direct MEK1/2 and p44/42 regulation. The PDGF receptor signaling complex formed in response to PDGF involves PI 3-kinase in addition to the same components and interactions as described for insulin or IGF-I. PSM associates with PI 3-kinase via p85 and in addition the PSM PH domain participates in the regulation of PI 3-kinase activity, presumably through membrane interaction. In contrast, the PSM Pro-rich region appears to participate only in the MAP kinase signal. Both pathways contribute to the mitogenic response as shown by cell proliferation, survival, and focus formation. PSM regulates p38 MAP kinase activity in a pathway unrelated to the mitogenic response.

  19. Artesunate Protected Blood-Brain Barrier via Sphingosine 1 Phosphate Receptor 1/Phosphatidylinositol 3 Kinase Pathway After Subarachnoid Hemorrhage in Rats.

    PubMed

    Zuo, Shilun; Ge, Hongfei; Li, Qiang; Zhang, Xuan; Hu, Rong; Hu, Shengli; Liu, Xin; Zhang, John H; Chen, Yujie; Feng, Hua

    2017-03-01

    Blood-brain barrier preservation plays an important role in attenuating vasogenic brain edema after subarachnoid hemorrhage (SAH). This study was designed to investigate the protective effect and mechanism of artesunate, a traditional anti-malaria drug, on blood-brain barrier after SAH. Three hundred and seventy-seven (377) male Sprague-Dawley rats were subjected to endovascular perforation model for SAH. The rats received artesunate alone or in combination with Sphingosine-1-phosphate receptor-1 (S1P1) small interfering RNA (siRNA), antagonist VPC23019, or phosphatidylinositol 3-kinase inhibitor wortmannin after SAH. Modified Garcia score, SAH grades, brain water content, Evans blue leakage, transmission electron microscope, immunohistochemistry staining, Western blot, and cultured endothelial cells were used to investigate the optimum concentration and the therapeutic mechanism of artesunate. We found that artesunate (200 mg/kg) could do better in raising modified Garcia score, reducing brain water content and Evans blue leakage than other groups after SAH. Moreover, artesunate elevated S1P1 expression, enhanced phosphatidylinositol 3-kinase activation, lowered GSK-3β activation, stabilized β-catenin, and improved the expression of Claudin-3 and Claudin-5 after SAH in rats. These effects were eliminated by S1P1 siRNA, VPC23019, and wortmannin. This study revealed that artesunate could preserve blood-brain barrier integrity and improve neurological outcome after SAH, possibly through activating S1P1, enhancing phosphatidylinositol 3-kinase activation, stabilizing β-catenin via GSK-3β inhibition, and then effectively raising the expression of Claudin-3 and Claudin-5. Therefore, artesunate may be favorable for the blood-brain barrier (BBB) protection after SAH and become a potential candidate for the treatment of SAH patients.

  20. Human SMG-1, a novel phosphatidylinositol 3-kinase-related protein kinase, associates with components of the mRNA surveillance complex and is involved in the regulation of nonsense-mediated mRNA decay

    PubMed Central

    Yamashita, Akio; Ohnishi, Tetsuo; Kashima, Isao; Taya, Yoichi; Ohno, Shigeo

    2001-01-01

    Nonsense-mediated mRNA decay (NMD) is a conserved surveillance mechanism that eliminates imperfect mRNAs that contain premature translation termination codons (PTCs) and code for nonfunctional or potentially harmful polypeptides. We show that a novel phosphatidylinositol 3-kinase-related protein kinase, hSMG-1, is a human ortholog of a product of Caenorhabditis elegans smg-1, one of seven smg genes involved in NMD. hSMG-1 phosphorylates hUPF1/SMG-2 in vivo and in vitro at specific serine residues in SQ motifs. hSMG-1 can associate with hUPF1/SMG-2 and other components of the surveillance complex. In particular, overexpression of a kinase-deficient point mutant of hSMG-1, hSMG-1-DA, results in a marked suppression of the PTC-dependent β-globin mRNA degradation; whereas that of wild-type hSMG-1 enhances it. We also show that inhibitors of hSMG-1 induce the accumulation of truncated p53 proteins in human cancer cell lines with p53 PTC mutation. Taken together, we conclude that hSMG-1 plays a critical role in NMD through the direct phosphorylation of hUPF1/SMG-2 in the evolutionally conserved mRNA surveillance complex. PMID:11544179

  1. Static magnetic field enhances the viability and proliferation rate of adipose tissue-derived mesenchymal stem cells potentially through activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) pathway.

    PubMed

    Marędziak, Monika; Tomaszewski, Krzysztof; Polinceusz, Paulina; Lewandowski, Daniel; Marycz, Krzysztof

    2017-01-01

    The aim of this work was to investigate the effects of 0.5T static magnetic field (sMF) on the viability and proliferation rate of human adipose-derived mesenchymal stromal stem cells (hASCs) via activation of the phosphoinositide 3-kinase/Akt (PI3K/Akt) signaling pathway. In a 7-d culture we examined cell growth kinetic and population doubling time (PDT). We also examined cell morphology and the cellular senescence markers level. Exposure to sMF enhanced the viability of these cells. However, the effect was blocked by treating the cells with LY294002, a P13K inhibitor. We compared this effect by Western Blot analysis of Akt protein expression. We also examined whether the cell response on sMF stimulation is dependent on integrin engagement and we measured integrin gene expression. Our results suggest that stimulation using sMF is a viable method to improve hASC viability. sMF is involved in mechanisms associated with controlling cell proliferative potential signaling events.

  2. Telencephalin protects PAJU cells from amyloid beta protein-induced apoptosis by activating the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway.

    PubMed

    Yang, Heping; Wu, Dapeng; Zhang, Xiaojie; Wang, Xiang; Peng, Yi; Hu, Zhiping

    2012-10-05

    Telencephalin is a neural glycoprotein that reduces apoptosis induced by amyloid beta protein in the human neural tumor cell line PAJU. In this study, we examined the role of the ezrin/radixin/moesin protein family/phosphatidylinositol-3-kinase/protein kinase B pathway in this process. Western blot analysis demonstrated that telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B were not expressed in PAJU cells transfected with empty plasmid, while they were expressed in PAJU cells transfected with a telencephalin expression plasmid. After treatment with 1.0 nM amyloid beta protein 42, expression of telencephalin and phosphorylated phosphatidylinositol-3-kinase/protein kinase B in the transfected cells gradually diminished, while levels of phosphorylated ezrin/radixin/moesin increased. In addition, the high levels of telencephalin, phosphorylated ezrin/radixin/moesin and phosphatidylinositol-3-kinase/protein kinase B expression in PAJU cells transfected with a telencephalin expression plasmid could be suppressed by the phosphatidylinositol-3-kinase inhibitor LY294002. These findings indicate that telencephalin activates the ezrin/radixin/moesin family/phosphatidylinositol-3-kinase/protein kinase B pathway and protects PAJU cells from amyloid beta protein-induced apoptosis.

  3. Phosphoinositide-3-kinases p110α and p110β mediate S phase entry in astroglial cells in the marginal zone of rat neocortex.

    PubMed

    Müller, Rabea; Fischer, Catharina; Wilmes, Thomas; Heimrich, Bernd; Distel, Vanessa; Klugbauer, Norbert; Meyer, Dieter K

    2013-01-01

    In cells cultured from neocortex of newborn rats, phosphoinositide-3-kinases of class I regulate the DNA synthesis in a subgroup of astroglial cells. We have studied the location of these cells as well as the kinase isoforms which facilitate the S phase entry. Using dominant negative (dn) isoforms as well as selective pharmacological inhibitors we quantified S phase entry by nuclear labeling with bromodeoxyuridine (BrdU). Only in astroglial cells harvested from the marginal zone (MZ) of the neocortex inhibition of phosphoinositide-3-kinases reduced the nuclear labeling with BrdU, indicating that neocortical astroglial cells differ in the regulation of proliferation. The two kinase isoforms p110α and p110β were essential for S phase entry. p110α diminished the level of the p27(Kip1) which inactivates the complex of cyclin E and CDK2 necessary for entry into the S phase. p110β phosphorylated and inhibited glycogen synthase kinase-3β which can prevent S-phase entry. Taken together, both isoforms mediated S phase in a subgroup of neocortical astroglial cells and acted via distinct pathways.

  4. Dynamic changes of connexin-43, gap junctional protein, in outer layers of cumulus cells are regulated by PKC and PI 3-kinase during meiotic resumption in porcine oocytes.

    PubMed

    Shimada, M; Maeda, T; Terada, T

    2001-04-01

    Mammalian oocytes are surrounded by numerous layers of cumulus cells, and the loss of gap junctional communication in the outer layers of cumulus cells induces meiotic resumption in oocytes. In this study, we investigated the dynamic changes in the gap junctional protein connexin-43 in cumulus cells during the meiotic resumption of porcine oocytes. The amount of connexin-43 in all layers of cumulus cells recovered from cumulus-oocyte complexes was increased after 4-h cultivation. However, at 12-h cultivation, the positive signal for connexin-43 immunoreactivity was markedly reduced in the outer layers of cumulus cells. When these reductions of connexin-43 were blocked by protein kinase C (PKC) or phosphatidylinositol (PI) 3-kinase inhibitor, networks of filamentous bivalents (i.e., advanced chromosomal status) were undetectable in the germinal vesicle of the oocyte. After 28-h cultivation, when the majority of oocytes were reaching the metaphase I (MI) stage, the connexin-43 in the inner layers of cumulus cells was phosphorylated, regardless of mitogen-activated protein (MAP) kinase activation. These results suggest that the initiation of meiotic resumption, namely, the formation of networks of filamentous bivalents in germinal vesicle, is associated with the reduction of gap junctional protein connexin-43 in the outer layers of cumulus cells via the PKC and/or PI 3-kinase pathway. Moreover, the connexin-43 in the inner layers of cumulus cells is phosphorylated during meiotic progression beyond the MI stage, regardless of MAP kinase activation in cumulus cells surrounding the oocyte.

  5. Structure of a specific peptide complex of the carboxy-terminal SH2 domain from the p85 alpha subunit of phosphatidylinositol 3-kinase.

    PubMed Central

    Breeze, A L; Kara, B V; Barratt, D G; Anderson, M; Smith, J C; Luke, R W; Best, J R; Cartlidge, S A

    1996-01-01

    We have determined the solution structure of the C-terminal SH2 domain of the p85 alpha subunit of human phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) in complex with a phosphorylated tyrosine pentapeptide sequence from the platelet-derived growth factor receptor using heteronuclear nuclear magnetic resonance spectroscopy. Overall, the structure is similar to other SH2 domain complexes, but displays different detail interactions within the phosphotyrosine binding site and in the recognition site for the +3 methionine residue of the peptide, the side chain of which inserts into a particularly deep and narrow pocket which is displaced relative to that of other SH2 domains. The contacts made within this +3 pocket provide the structural basis for the strong selection for methionine at this position which characterizes the SH2 domains of PI3-kinase. Comparison with spectral and structural features of the uncomplexed domain shows that the long BG loop becomes less mobile in the presence of the bound peptide. In contrast, extreme resonance broadening encountered for most residues in the beta D', beta E and beta F strands and associated connecting loops of the domain in the absence of peptide persists in the complex, implying conformational averaging in this part of the molecule on a microsecond-to-millisecond time scale. Images PMID:8670861

  6. Roles of phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase in the regulation of protein kinase C-alpha activation in interferon-gamma-stimulated macrophages.

    PubMed

    Hardy, Pierre-Olivier; Diallo, Tamsir O; Matte, Christine; Descoteaux, Albert

    2009-09-01

    Members of the protein kinase C (PKC) family are activated by interferon-gamma (IFN-gamma) and modulate IFN-gamma-induced cellular responses by regulating the activity of transcription factors. We previously reported that PKC-alpha enhances the ability of IFN regulatory factor-1 to transactivate the class II transactivator (CIITA) promoter IV in IFN-gamma-stimulated macrophages. In addition, we showed that IFN-gamma induces the nuclear translocation of PKC-alpha but the mechanisms for this remain to be elucidated. In this study, we sought to identify signalling pathways involved in IFN-gamma-induced activation of PKC-alpha and to characterize their potential roles in modulating IFN-gamma-induced responses in macrophages. IFN-gamma-mediated nuclear translocation of PKC-alpha was a Janus activated kinase 2 (JAK2)-independent process, which required phosphatidylinositol 3-kinase (PI3K) and p38 mitogen-activated protein kinase (MAPK). However, PKC-alpha phosphorylation was independent of PI3K and p38 MAPK, indicating that IFN-gamma-induced phosphorylation and nuclear translocation of PKC-alpha are mediated by distinct mechanisms. In addition, inhibition of PI3K, but not of p38 MAPK, strongly impaired IFN-gamma-induced CIITA and MHC II gene expression. Finally, PKC-alpha associated with signal transducer and activator of transcription 1 (STAT1) and was required for the phosphorylation of STAT1 on serine 727 in IFN-gamma-stimulated macrophages. Taken together, our data indicate that PI3K and p38 MAPK modulate IFN-gamma-stimulated PKC-alpha nuclear translocation independently of JAK2 activity and that both PI3K and PKC-alpha are required for type IV CIITA and MHC II gene expression in IFN-gamma-stimulated macrophages.

  7. A regulatory role for cAMP in phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase-mediated DNA synthesis in platelet-derived-growth-factor-stimulated bovine airway smooth-muscle cells.

    PubMed Central

    Scott, P H; Belham, C M; al-Hafidh, J; Chilvers, E R; Peacock, A J; Gould, G W; Plevin, R

    1996-01-01

    In bovine airway smooth-muscle cells platelet-derived growth factor (PDGF) and endothelin (Et-1) stimulate sustained and comparable activation of mitogen-activated protein kinase (MAP kinase) but display very different mitogenic efficacies, with PDGF inducing 50 times more DNA synthesis than Et-1. To examine additional signalling pathways which may be involved in this response, we investigated the role of phosphatidylinositol 3-kinase (PtdIns 3-kinase)/p70 ribosomal protein S6 kinase (p70s6k) in mediating PDGF- and Et-1-induced mitogenesis, and whether inhibition of this pathway may underly the ability of cAMP to inhibit cell proliferation. PDGF stimulated an increase in PtdIns 3-kinase activity and a sustained 15-fold increase in p70s6k activity that was abolished by both wortmannin and rapamycin. Et-1, however, stimulated only a 2-fold increase in p70s6k activity that was rapamycin-sensitive but wortmannin-insensitive. DNA synthesis stimulated by PDGF (50-fold) and Et-1 (2-fold) followed a similar pattern of inhibition. Pretreatment with phorbol ester did not affect p70s6k activation in response to PDGF. Raising intracellular cAMP levels using forskolin, however, resulted in a marked time-dependent inhibition of p70s6k activity, a decrease in the tyrosine phosphorylation of the PtdIns 3-kinase p85 subunit and reduced PtdIns 3-kinase activity. Forskolin also inhibited PDGF-stimulated DNA synthesis. These results suggest that PtdIns 3-kinase-dependent activation of p70s6k may determine mitogenic efficacy of agonists that generate comparable MAP kinase signals. Negative regulation of PtdIns 3-kinase by cAMP may play an important role in the inhibition of airway smooth-muscle cell proliferation. PMID:8836145

  8. The PI3-Kinase/mTOR-Targeting Drug NVP-BEZ235 Inhibits Growth and IgE-Dependent Activation of Human Mast Cells and Basophils

    PubMed Central

    Blatt, Katharina; Herrmann, Harald; Mirkina, Irina; Hadzijusufovic, Emir; Peter, Barbara; Strommer, Sabine; Hoermann, Gregor; Mayerhofer, Matthias; Hoetzenecker, Konrad; Klepetko, Walter; Ghanim, Viviane; Marth, Katharina; Füreder, Thorsten; Wacheck, Volker; Valenta, Rudolf; Valent, Peter

    2012-01-01

    The phosphoinositide 3-kinase (PI3-kinase) and the mammalian target of rapamycin (mTOR) are two major signaling molecules involved in growth and activation of mast cells (MC) and basophils (BA). We examined the effects of the dual PI3-kinase/mTOR blocker NVP-BEZ235 on growth of normal and neoplastic BA and MC as well as immunoglobulin E (IgE)-dependent cell activation. Growth of MC and BA were determined by measuring 3H-thymidine uptake and apoptosis. Cell activation was determined in histamine release experiments and by measuring upregulation of CD63 and CD203c after challenging with IgE plus anti-IgE or allergen. We found that NVP-BEZ235 exerts profound inhibitory effects on growth of primary and cloned neoplastic MC. In the MC leukemia cell line HMC-1, NVP-BEZ235 showed similar IC50 values in the HMC-1.1 subclone lacking KIT D816V (0.025 µM) and the HMC-1.2 subclone expressing KIT D816V (0.005 µM). Moreover, NVP-BEZ235 was found to exert strong growth-inhibitory effects on neoplastic MC in a xenotransplant-mouse model employing NMR1-Foxn1nu mice. NVP-BEZ235 also exerted inhibitory effects on cytokine-dependent differentiation of normal BA and MC, but did not induce growth inhibition or apoptosis in mature MC or normal bone marrow cells. Finally, NVP-BEZ235 was found to inhibit IgE-dependent histamine release in BA and MC (IC50 0.5–1 µM) as well as anti-IgE-induced upregulation of CD203c in BA and IgE-dependent upregulation of CD63 in MC. In summary, NVP-BEZ235 produces growth-inhibitory effects in immature neoplastic MC and inhibits IgE-dependent activation of mature BA and MC. Whether these potentially beneficial drug effects have clinical implications is currently under investigation. PMID:22299028

  9. Rumex acetosa L. induces vasorelaxation in rat aorta via activation of PI3-kinase/Akt- AND Ca(2+)-eNOS-NO signaling in endothelial cells.

    PubMed

    Sun, Y Y; Su, X H; Jin, J Y; Zhou, Z Q; Sun, S S; Wen, J F; Kang, D G; Lee, H S; Cho, K W; Jin, S N

    2015-12-01

    Rumex acetosa L. (RA) (Polygonaceae) is an important traditional Chinese medicine (TCM) commonly used in clinic for a long history in China and the aerial parts of RA has a wide variety of pharmacological actions such as diuretic, anti-hypertensive, anti-oxidative, and anti-cancer effects. However, the mechanisms involved are to be defined. The purpose of the present study was to evaluate the vasorelaxant effect and define the mechanism of action of the ethanol extract of Rumex acetosa L. (ERA) in rat aorta. ERA was examined for its vascular relaxant effect in isolated phenylephrine-precontracted rat thoracic aorta and its acute effects on arterial blood pressure. In addition, the roles of the nitric oxide synthase (NOS)-nitric oxide (NO) signaling in the ERA-induced effects were tested in human umbilical vein endothelial cells (HUVECs). The phosphorylation levels of Akt and eNOS were assessed by Western blot analysis in the cultured HUVECs. ERA induced endothelium-dependent vasorelaxation. The ERA-induced vasorelaxation was abolished by L-NAME (an NOS inhibitor) or ODQ (a sGC inhibitor), but not by indomethacin. Inhibition of PI3-kinase/Akt signaling pathway markedly reduced the ERA-induced vasorelaxation. In HUVECs, ERA increased NO formation in a dose-dependent manner, which was inhibited by L-NAME and by removing extracellular Ca(2+). In addition, ERA promoted phosphorylation of Akt and eNOS, which was prevented by wortmannin and LY294002, indicating that ERA induces eNOS phosphorylation through the PI3-kinase/Akt pathway. Further, in anesthetized rats, intravenously administered ERA decreased arterial blood pressure in a dose-dependent manner through an activation of the NOS-NO system. In summary, the ERA- induced vasorelaxation was dependent on endothelial integrity and NO production, and was mediated by activation of both the endothelial PI3-kinase/Akt- and Ca(2+)-eNOS-NO signaling and muscular NO-sGC-cGMP signaling.

  10. 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

  11. 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

  12. Neuroprotection of brain-derived neurotrophic factor against hypoxic injury in vitro requires activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase.

    PubMed

    Sun, Xiaomei; Zhou, Hui; Luo, Xiaoli; Li, Shengfu; Yu, Dan; Hua, Jiping; Mu, Dezhi; Mao, Meng

    2008-01-01

    Intrauterine asphyxia is one of the major contributors for perinatal death, mental and physical disorders of surviving children. Brain-derived neurotrophic factor (BDNF) provides a promising solution to hypoxic injury due to its survival-promoting effects. In an attempt to identify possible molecular mechanisms underlying the neuroprotective role of BDNF, we studied extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI-3-K) and p38 mitogen-activated protein kinase (MAPK) pathways. We demonstrated that BDNF protected cortical neurons against hypoxic injury in vitro via activation of both the ERK and PI-3-K pathways but not the p38 MAPK pathway. We also showed that both hypoxic stimuli and exogenous BDNF treatment phosphorylated the cyclic AMP response element-binding protein (CREB) and that CREB phosphorylation induced by BDNF was mediated via the ERK pathway in cultured cortical neurons.

  13. A retinoic acid receptor beta agonist (CD2019) overcomes inhibition of axonal outgrowth via phosphoinositide 3-kinase signalling in the injured adult spinal cord.

    PubMed

    Agudo, Marta; Yip, Ping; Davies, Meirion; Bradbury, Elizabeth; Doherty, Patrick; McMahon, Stephen; Maden, Malcolm; Corcoran, Jonathan P T

    2010-01-01

    After spinal cord injury in the adult mammal, axons do not normally regrow and this commonly leads to paralysis. Retinoic acid (RA) can stimulate neurite outgrowth in vitro of both the embryonic central and peripheral nervous system, via activation of the retinoic acid receptor (RAR) beta2. We show here that regions of the adult CNS, including the cerebellum and cerebral cortex, express RARbeta2. We show that when cerebellar neurons are grown in the presence of myelin-associated glycoprotein (MAG) which inhibits neurite outgrowth, RARbeta can be activated in a dose dependent manner by a RARbeta agonist (CD2019) and neurite outgrowth can occur via phosphoinositide 3-kinase (PI3K) signalling. In a model of spinal cord injury CD2019 also acts through PI3K signalling to induce axonal outgrowth of descending corticospinal fibres and promote functional recovery. Our data suggest that RARbeta agonists may be of therapeutic potential for human spinal cord injuries.

  14. Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

    SciTech Connect

    Hwang, Yong Pil; Kim, Hyung Gyun; Hien, Tran Thi; Jeong, Myung Ho; Jeong, Tae Cheon; Jeong, Hye Gwang

    2011-11-15

    The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-{alpha}-stimulated monocytes to endothelial cells and suppressed the TNF-{alpha} induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-{alpha}-induced nuclear factor-{kappa}B activation, which was attenuated by pretreatment with N{sup G}-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: Black-Right-Pointing-Pointer Puerarin induced the phosphorylation of eNOS and the production of NO. Black-Right-Pointing-Pointer Puerarin activated eNOS through ER-dependent PI3-kinase and Ca{sup 2+}-dependent AMPK. Black-Right-Pointing-Pointer Puerarin-induced NO was involved in the inhibition of NF-kB activation. Black-Right-Pointing-Pointer Puerarin may help for prevention of vascular dysfunction and diabetes.

  15. Redox-Sensitive Induction of Src/PI3-kinase/Akt and MAPKs Pathways Activate eNOS in Response to EPA:DHA 6:1

    PubMed Central

    Zgheel, Faraj; Alhosin, Mahmoud; Rashid, Sherzad; Burban, Mélanie; Auger, Cyril; Schini-Kerth, Valérie B.

    2014-01-01

    Aims Omega-3 fatty acid products containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have vasoprotective effects, in part, by stimulating the endothelial formation of nitric oxide (NO). This study determined the role of the EPA:DHA ratio and amount, and characterized the mechanism leading to endothelial NO synthase (eNOS) activation. Methods and Results EPA:DHA 6∶1 and 9∶1 caused significantly greater endothelium-dependent relaxations in porcine coronary artery rings than EPA:DHA 3∶1, 1∶1, 1∶3, 1∶6, 1∶9, EPA and DHA alone, and EPA:DHA 6∶1 with a reduced EPA + DHA amount, which were inhibited by an eNOS inhibitor. Relaxations to EPA:DHA 6∶1 were insensitive to cyclooxygenase inhibition, and reduced by inhibitors of either oxidative stress, Src kinase, PI3-kinase, p38 MAPK, MEK, or JNK. EPA:DHA 6∶1 induced phosphorylation of Src, Akt, p38 MAPK, ERK, JNK and eNOS; these effects were inhibited by MnTMPyP. EPA:DHA 6∶1 induced the endothelial formation of ROS in coronary artery sections as assessed by dihydroethidium, and of superoxide anions and hydrogen peroxide in cultured endothelial cells as assessed by electron spin resonance with the spin probe CMH, and the Amplex Red based assay, respectively. Conclusion Omega-3 fatty acids cause endothelium-dependent NO-mediated relaxations in coronary artery rings, which are dependent on the EPA:DHA ratio and amount, and involve an intracellular activation of the redox-sensitive PI3-kinase/Akt and MAPKs pathways to activate eNOS. PMID:25133540

  16. Hypoxia Up-regulates CD36 Expression and Function via Hypoxia-inducible Factor-1- and Phosphatidylinositol 3-Kinase-dependent Mechanisms*

    PubMed Central

    Mwaikambo, Bupe R.; Yang, Chun; Chemtob, Sylvain; Hardy, Pierre

    2009-01-01

    Neovascular and degenerative diseases of the eye are leading causes of impaired vision and blindness in the world. Hypoxia or reduced oxygen tension is considered central to the pathogenesis of these disorders. Although the CD36 scavenger receptor features prominently in ocular homeostasis and pathology, little is known regarding its modulation by hypoxia. Herein we investigated the role and regulation of CD36 by hypoxia and by the major hypoxia effector, hypoxia-inducible factor (HIF)-1. In vivo, hypoxia markedly induced CD36 mRNA in corneal and retinal tissue. Subsequent experiments on human retinal pigment epithelial cells revealed that hypoxia time-dependently increased CD36 mRNA, protein, and surface expression; these responses were reliant upon reactive oxygen species production. As an important novel finding, we demonstrate that hypoxic stimulation of CD36 is mediated by HIF-1; HIF-1α down-regulation abolished CD36 induction by both hypoxia and cobalt chloride. Sequence analysis of the human CD36 promoter region revealed a functional HIF-1 binding site. A luciferase reporter construct containing this promoter fragment was activated by hypoxia, whereas mutation at the HIF-1 consensus site decreased promoter activation. Specific binding of HIF-1 to this putative site in hypoxic cells was detected by a chromatin immunoprecipitation assay. Interestingly, inhibition of the phosphatidylinositol 3-kinase pathway blocked the hypoxia-dependent induction of CD36 expression and promoter activity. Functional ramifications of CD36 hypoxic accumulation were evinced by CD36-dependent increases in scavenging and anti-angiogenic activities. Together, our findings indicate a novel mechanism by which hypoxia induces CD36 expression via activation of HIF-1 and the phosphatidylinositol 3-kinase pathway. PMID:19640849

  17. Inhibition of constitutively activated phosphoinositide 3-kinase/AKT pathway enhances antitumor activity of chemotherapeutic agents in breast cancer susceptibility gene 1-defective breast cancer cells.

    PubMed

    Yi, Yong Weon; Kang, Hyo Jin; Kim, Hee Jeong; Hwang, Jae Seok; Wang, Antai; Bae, Insoo

    2013-09-01

    Loss or decrease of wild type BRCA1 function, by either mutation or reduced expression, has a role in hereditary and sporadic human breast and ovarian cancers. We report here that the PI3K/AKT pathway is constitutively active in BRCA1-defective human breast cancer cells. Levels of phospho-AKT are sustained even after serum starvation in breast cancer cells carrying deleterious BRCA1 mutations. Knockdown of BRCA1 in MCF7 cells increases the amount of phospho-AKT and sensitizes cells to small molecule protein kinase inhibitors (PKIs) targeting the PI3K/AKT pathway. Restoration of wild type BRCA1 inhibits the activated PI3K/AKT pathway and de-sensitizes cells to PKIs targeting this pathway in BRCA1 mutant breast cancer cells, regardless of PTEN mutations. In addition, clinical PI3K/mTOR inhibitors, PI-103, and BEZ235, showed anti-proliferative effects on BRCA1 mutant breast cancer cell lines and synergism in combination with chemotherapeutic drugs, cisplatin, doxorubicin, topotecan, and gemcitabine. BEZ235 synergizes with the anti-proliferative effects of gemcitabine by enhancing caspase-3/7 activity. Our results suggest that the PI3K/AKT pathway can be an important signaling pathway for the survival of BRCA1-defective breast cancer cells and pharmacological inhibition of this pathway is a plausible treatment for a subset of breast cancers.

  18. Involvement of p21racA, phosphoinositide 3-kinase, and vacuolar ATPase in phagocytosis of bacteria and erythrocytes by Entamoeba histolytica: suggestive evidence for coincidental evolution of amebic invasiveness.

    PubMed Central

    Ghosh, S K; Samuelson, J

    1997-01-01

    Trophozoites of Entamoeba histolytica, the protozoan parasite that causes amebic dysentery, phagocytose bacteria in the colonic lumen and erythrocytes (RBC) in host tissues. Because tissue invasion is an evolutionary dead end, it is likely that amebic pathogenicity is coincidentally selected, i.e., the same methods used to kill bacteria in the colonic lumen are used by parasites to damage host cells and cause disease. In support of this idea, the amebic lectin and pore-forming peptide are involved in binding and killing, respectively, bacteria and host epithelial cells. Here amebic phagocytosis of bacteria, RBC, and mucin-coated beads was disrupted by overexpression of E. histolytica p21(racA-V12), a ras-family protein involved in selection of sites of actin polymerization, which had been mutated to eliminate its GTPase activity. p21(racA-V12) transformants were also defective in capping and cytokinesis, while pinocytosis of fluorescent dextrans was not affected. Wortmannin, a fungal inhibitor of phosphoinositide 3-kinase, markedly inhibited phagocytosis of bacteria, RBC, and mucin-coated beads by wild-type amebae. In contrast to p21(racA-V12) overexpression, wortmannin abolished amebic pinocytosis of dextrans but had no inhibitory effects on capping. Inhibition of amebic vacuolar acidification by bafilomycin also decreased bacterial and RBC uptake. These results, which demonstrate similarities between mechanisms of phagocytosis of bacteria and RBC by amebae and macrophages, support the idea of coincidental selection of amebic genes encoding proteins that mediate destruction of host cells. PMID:9317033

  19. Anticarcinogenic action of quercetin by downregulation of phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) via induction of p53 in hepatocellular carcinoma (HepG2) cell line.

    PubMed

    Maurya, Akhilendra Kumar; Vinayak, Manjula

    2015-09-01

    Protein kinase C (PKC) is a key regulator of cell growth and differentiation in mammalian cells and hyperactivation of PKC is believed to play an important role in tumor progression. PKC is downstream to signaling protein of phosphatidylinositol 3-Kinase (PI3K), a known up-regulator of cell proliferation and survival. Accumulation of reactive oxygen species (ROS) triggers oxidative stress in the tumor microenvironment, leading to the hyperactivation of various oxidative stress-stimulated signaling molecules. Quercetin (QUE) is a naturally occurring dietary flavonoid having antioxidant properties. QUE is reported to show antitumor activity both in vitro and in vivo; however, the molecular mechanism is yet to be thoroughly explored. HepG2 cells display cellular functions similar to the normal hepatocytes with high degree of morphological and functional differentiation, therefore HepG2 cell line is chosen as the suitable model for drug targeting. Present study is aimed to establish the signaling pathway involved in the anticarcinogenic action of QUE in HepG2 cell line. HepG2 cells were treated with different doses of QUE. Protein level and gene expression were analysed by Western blotting and RT-PCR, respectively. PKC activity was measured by non-radioactive-tagged phosphorylation. Results showed downregulation of expression of PI3K, PKC, COX-2 and ROS caused by QUE. Additionally, QUE enhanced the expression of p53 and BAX in HepG2 cells. Overall, results of the current study suggested that QUE elicited anticarcinogenic action by upregulation of p53 and BAX in HepG2 cells via downregulation of ROS, PKC, PI3K and COX-2, confirming our earlier report on the animal model.

  20. Dihydrotestosterone differentially modulates the mitogen-activated protein kinase and the phosphoinositide 3-kinase/Akt pathways through the nuclear and novel membrane androgen receptor in C6 cells.

    PubMed

    Gatson, Joshua W; Kaur, Paramjit; Singh, Meharvan

    2006-04-01

    Androgens such as dihydrotestosterone (DHT) are known to exert their effects through the activation of intracellular receptors that regulate the transcription of target genes. Alternatively, nongenomic mechanisms, including the activation of such signaling pathways as the MAPK pathways, have been described. It is unclear, however, whether this latter mechanism of action is mediated by the classical androgen receptor (AR) or some alternative mechanism. In this study, using a glial cell model (C6 cells) that we found to express the AR, we identified that DHT increased the phosphorylation of both ERK and Akt, key effectors of the neuroprotection-associated MAPK and phosphoinositide 3-kinase signaling pathways, respectively, and ERK phosphorylation was blocked by the AR antagonist, flutamide. In contrast, the membrane-impermeable, BSA-conjugated androgen (DHT-BSA) caused a dose-dependent suppression of ERK and Akt phosphorylation, suggesting the existence of a novel membrane-associated AR that mediates this opposite effect on neuroprotective signaling. This is also supported by the observation of DHT-displaceable binding sites on the cell surface of live C6 cells. Collectively, these data support the existence of a novel membrane-associated AR in glial cells and argue for the existence of two, potentially competing, pathways in a given cell or tissue. This mutual antagonism was supported by the ability of DHT-BSA to attenuate DHT-induced ERK phosphorylation. Thus, depending on the predominance of one receptor mechanism over another, the outcome of androgen treatment may be very different and, as such, could help explain existing discrepancies as to whether androgens are protective or damage inducing.

  1. Gastrin induces sodium-hydrogen exchanger 3 phosphorylation and mTOR activation via a phosphoinositide 3-kinase-/protein kinase C-dependent but AKT-independent pathway in renal proximal tubule cells derived from a normotensive male human.

    PubMed

    Liu, Tianbing; Jose, Pedro A

    2013-02-01

    Gastrin is natriuretic, but its renal molecular targets and signal transduction pathways are not fully known. In this study, we confirmed the existence of CCKBR (a gastrin receptor) in male human renal proximal tubule cells and discovered that gastrin induced S6 phosphorylation, a downstream component of the phosphatidylinositol 3 kinase (PI3 kinase)-mammalian target of rapamycin pathway. Gastrin also increased the phosphorylation of sodium-hydrogen exchanger 3 (NHE3) at serine 552, caused its internalization, and decreased its expression at the cell surface and NHE activity. The phosphorylation of NHE3 and S6 was dependent on PI3 kinases because it was blocked by 2 different PI3-kinase inhibitors, wortmannin and LY294,002. The phosphorylation of NHE3 and S6 was not affected by the protein kinase A inhibitor H-89 but was blocked by a pan-PKC (chelerythrine) and a conventional PKC (cPKC) inhibitor (Gö6976) (10 μM) and an intracellular calcium chelator, 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester, suggesting the importance of cPKC and intracellular calcium in the gastrin signaling pathway. The cPKC involved was probably PKCα because it was phosphorylated by gastrin. The gastrin-mediated phosphorylation of NHE3, S6, and PKCα was via phospholipase C because it was blocked by a phospholipase C inhibitor, U73122 (10 μM). The phosphorylation (activation) of AKT, which is usually upstream of mammalian target of rapamycin in the classic PI3 kinase-AKT-p70S6K signaling pathway, was not affected, suggesting that the gastrin-induced phosphorylation of NHE3 and S6 is dependent on both PI3 kinase and PKCα but not AKT.

  2. Integrin-dependent translocation of phosphoinositide 3-kinase to the cytoskeleton of thrombin-activated platelets involves specific interactions of p85 alpha with actin filaments and focal adhesion kinase

    PubMed Central

    1995-01-01

    Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti- phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas

  3. High-Throughput Small Interfering RNA Screening Identifies Phosphatidylinositol 3-Kinase Class II Alpha as Important for Production of Human Cytomegalovirus Virions

    PubMed Central

    Polachek, William S.; Moshrif, Hanan F.; Franti, Michael; Coen, Donald M.; Sreenu, Vattipally B.

    2016-01-01

    ABSTRACT High-throughput small interfering RNA (siRNA) screening is a useful methodology to identify cellular factors required for virus replication. Here we utilized a high-throughput siRNA screen based on detection of a viral antigen by microscopy to interrogate cellular protein kinases and phosphatases for their importance during human cytomegalovirus (HCMV) replication and identified the class II phosphatidylinositol 3-kinase class II alpha (PI3K-C2A) as being involved in HCMV replication. Confirming this observation, infected cells treated with either pooled or individual siRNAs targeting PI3K-C2A mRNA produced approximately 10-fold less infectious virus than the controls. Western blotting and quantitative PCR analysis of infected cells treated with siRNAs indicated that depletion of PI3K-C2A slightly reduced the accumulation of late but not immediate early or early viral antigens and had no appreciable effect on viral DNA synthesis. Analysis of siRNA-treated cells by electron microscopy and Western blotting indicated that PI3K-C2A was not required for the production of viral capsids but did lead to increased numbers of enveloped capsids in the cytoplasm that had undergone secondary envelopment and a reduction in the amount of viral particles exiting the cell. Therefore, PI3K-C2A is a factor important for HCMV replication and has a role in the production of HCMV virions. IMPORTANCE There is limited information about the cellular factors required for human cytomegalovirus (HCMV) replication. Therefore, to identify proteins involved in HCMV replication, we developed a methodology to conduct a high-throughput siRNA screen of HCMV-infected cells. From our screening data, we focused our studies on the top hit from our screen, the lipid kinase phosphatidylinositol 3-kinase class II alpha (PI3K-C2A), as its role in HCMV replication was unknown. Interestingly, we found that PI3K-C2A is important for the production of HCMV virions and is involved in virion production

  4. The Putative Mevalonate Diphosphate Decarboxylase from Picrophilus torridus Is in Reality a Mevalonate-3-Kinase with High Potential for Bioproduction of Isobutene

    PubMed Central

    Hall, Stephen J.; Eastham, Graham; Licence, Peter; Stephens, Gill

    2015-01-01

    Mevalonate diphosphate decarboxylase (MVD) is an ATP-dependent enzyme that catalyzes the phosphorylation/decarboxylation of (R)-mevalonate-5-diphosphate to isopentenyl pyrophosphate in the mevalonate (MVA) pathway. MVD is a key enzyme in engineered metabolic pathways for bioproduction of isobutene, since it catalyzes the conversion of 3-hydroxyisovalerate (3-HIV) to isobutene, an important platform chemical. The putative homologue from Picrophilus torridus has been identified as a highly efficient variant in a number of patents, but its detailed characterization has not been reported. In this study, we have successfully purified and characterized the putative MVD from P. torridus. We discovered that it is not a decarboxylase per se but an ATP-dependent enzyme, mevalonate-3-kinase (M3K), which catalyzes the phosphorylation of MVA to mevalonate-3-phosphate. The enzyme's potential in isobutene formation is due to the conversion of 3-HIV to an unstable 3-phosphate intermediate that undergoes consequent spontaneous decarboxylation to form isobutene. Isobutene production rates were as high as 507 pmol min−1 g cells−1 using Escherichia coli cells expressing the enzyme and 2,880 pmol min−1 mg protein−1 with the purified histidine-tagged enzyme, significantly higher than reported previously. M3K is a key enzyme of the novel MVA pathway discovered very recently in Thermoplasma acidophilum. We suggest that P. torridus metabolizes MVA by the same pathway. PMID:25636853

  5. miR-362-5p inhibits proliferation and migration of neuroblastoma cells by targeting phosphatidylinositol 3-kinase-C2β.

    PubMed

    Wu, Kai; Yang, Liucheng; Chen, Jianfeng; Zhao, Haijun; Wang, Jianjun; Xu, Shuai; Huang, Zonghai

    2015-07-08

    miR-362-5p is down-regulated in high-risk neuroblastoma and can function as a tumor suppressor. However, its role remains poorly understood. We show that miR-362-5p is down-regulated in metastatic neuroblastoma compared with primary neuroblastoma. Overexpression of miR-362-5p inhibits cell proliferation, migration and invasion of neuroblastoma cells in vitro and suppresses tumor growth of neuroblastoma in vivo. Phosphatidylinositol 3-kinase (PI3K)-C2β is a target of miR-362-5p. Knockdown of PI3K-C2β by siRNA had a similar effect to overexpression of miR-362-5p on SH-SY5Y cells. Overexpression of PI3K-C2β partially reversed tumor-suppressive effects of miR-362-5p. We suggest that miR-362-5p suppresses neuroblastoma cell growth and motility, partially by targeting PI3K-C2β.

  6. Dual Inhibiton of Bruton's Tyrosine Kinase and Phosphoinositide-3-Kinase p110δ as a Therapeutic Approach to Treat non-Hodgkin's B cell Malignancies.

    PubMed

    Alfaro, Jennifer; Perez de Arce, Felipe; Belmar, Sebastian; Fuentealba, Glenda; Avila, Patricio; Ureta, Gonzalo; Flores, Camila; Acuna, Claudia; Delgado, Luz; Gaete, Diana; Pujala, Brahmam; Barde, Anup; Nayak, Anjan K; Upendra, Tvr; Patel, Dhananjay; Chauhan, Shailender; Sharma, Vijay K; Kanno, Stacy; Almirez, Ramona G; Hung, David T; Chakravarty, Sarvajit; Rai, Roopa; Bernales, Sebastian; Quinn, Kevin P; Pham, Son M; McCullagh, Emma

    2017-03-15

    Although new targeted therapies such as ibrutinib and idelalisib have made a large impact on non-Hodgkin's lymphoma (NHL) patients, the disease is often fatal because patients are initially resistant to these targeted therapies or because they eventually develop resistance. New drugs and treatments are necessary for these patients. One attractive approach is to inhibit multiple parallel pathways that drive the growth of these hematologic tumors and possibly prolonging the duration of the response and reducing resistance. Early clinical trials have tested this approach by dosing two drugs in combination in NHL patients. We have discovered a single molecule MDVN1003 that inhibits Bruton's tyrosine kinase (BTK) and phosphatidylinositol-3-kinase delta (PI3Kδ), two proteins regulated by the B cell receptor (BCR) that drive the growth of many NHLs. In this report, we show that this dual inhibitor prevents the activation of B cells and inhibits the phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK 1/2), two downstream mediators that are important for this process. Additionally, MDVN1003 induces cell death in a B cell lymphoma cell line but not in an irrelevant erythroblast cell line. Importantly, we found that this orally bioavailable dual inhibitor reduced tumor growth in a B cell lymphoma xenograft model more effectively than either ibrutinib or idelalisib. Taken together these results suggest that dual inhibition of these two key pathways by a single molecule could be a viable approach for treatment of NHL patients.

  7. Insulin dependent apolipoprotein B degradation and phosphatidylinositide 3-kinase activation with microsomal translocation are restored in McArdle RH7777 cells following serum deprivation

    PubMed Central

    Sparks, Janet D.; Magra, Amy L.; Chamberlain, Jeffrey M.; O’Dell, Colleen; Sparks, Charles E.

    2015-01-01

    Previous studies in rat hepatocytes demonstrated that insulin-dependent apolipoprotein (apo) B degradation (IDAD) is lost when cells are maintained for 3 d under enriched culture conditions. Loss of IDAD correlates with increased expression of protein tyrosine phosphatase 1B (PTP1B) known to be associated with resistance to insulin signaling in the liver. McArdle RH7777 hepatoma (McA) cells cultured in serum containing medium are resistant to IDAD; demonstrate a 30% increase in apo B secretion, and express increased levels of PTP1B protein and mRNA. In addition, insulin-stimulated Class I phosphatidylinositide 3-kinase (PI3K) activity of anti-pY immunoprecipitates is severely blunted. IDAD resistance in McA cells correlates with diminished translocation of insulin-stimulated pY-IRS1 to intracellular membranes. Incubation of McA cells with RK682, a protein tyrosine phosphatase inhibitor, is sufficient to restore IDAD in resistant McA cells. Overall, results further support the importance of Class I PI3K activity in IDAD, and suggest that loss of this activity is sufficient to cause resistance. Although other factors are involved in downstream events including sortilin binding to apo B, autophagy, and lysosomal degradation, loss of signal generation and reduced localization of Class I PI3K to intracellular membranes plays a significant role in IDAD resistance. PMID:26616056

  8. Vav1 transduces T cell receptor signals to the activation of phospholipase C-gamma1 via phosphoinositide 3-kinase-dependent and -independent pathways.

    PubMed

    Reynolds, Lucinda F; Smyth, Lesley A; Norton, Trisha; Freshney, Norman; Downward, Julian; Kioussis, Dimitris; Tybulewicz, Victor L J

    2002-05-06

    Vav1 is a signal transducing protein required for T cell receptor (TCR) signals that drive positive and negative selection in the thymus. Furthermore, Vav1-deficient thymocytes show greatly reduced TCR-induced intracellular calcium flux. Using a novel genetic system which allows the study of signaling in highly enriched populations of CD4(+)CD8(+) double positive thymocytes, we have studied the mechanism by which Vav1 regulates TCR-induced calcium flux. We show that in Vav1-deficient double positive thymocytes, phosphorylation, and activation of phospholipase C-gamma1 (PLCgamma1) is defective. Furthermore, we demonstrate that Vav1 regulates PLCgamma1 phosphorylation by at least two distinct pathways. First, in the absence of Vav1 the Tec-family kinases Itk and Tec are no longer activated, most likely as a result of a defect in phosphoinositide 3-kinase (PI3K) activation. Second, Vav1-deficient thymocytes show defective assembly of a signaling complex containing PLCgamma1 and the adaptor molecule Src homology 2 domain-containing leukocyte phosphoprotein 76. We show that this latter function is independent of PI3K.

  9. Vav1 Transduces T Cell Receptor Signals to the Activation of Phospholipase C-γ1 via Phosphoinositide 3-Kinase-dependent and -independent Pathways

    PubMed Central

    Reynolds, Lucinda F.; Smyth, Lesley A.; Norton, Trisha; Freshney, Norman; Downward, Julian; Kioussis, Dimitris; Tybulewicz, Victor L.J.

    2002-01-01

    Vav1 is a signal transducing protein required for T cell receptor (TCR) signals that drive positive and negative selection in the thymus. Furthermore, Vav1-deficient thymocytes show greatly reduced TCR-induced intracellular calcium flux. Using a novel genetic system which allows the study of signaling in highly enriched populations of CD4+CD8+ double positive thymocytes, we have studied the mechanism by which Vav1 regulates TCR-induced calcium flux. We show that in Vav1-deficient double positive thymocytes, phosphorylation, and activation of phospholipase C-γ1 (PLCγ1) is defective. Furthermore, we demonstrate that Vav1 regulates PLCγ1 phosphorylation by at least two distinct pathways. First, in the absence of Vav1 the Tec-family kinases Itk and Tec are no longer activated, most likely as a result of a defect in phosphoinositide 3-kinase (PI3K) activation. Second, Vav1-deficient thymocytes show defective assembly of a signaling complex containing PLCγ1 and the adaptor molecule Src homology 2 domain–containing leukocyte phosphoprotein 76. We show that this latter function is independent of PI3K. PMID:11994416

  10. 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.

  11. Putative Phosphatidylinositol 3-Kinase (PI3K) Binding Motifs in Ovine Betaretrovirus Env Proteins Are Not Essential for Rodent Fibroblast Transformation and PI3K/Akt Activation

    PubMed Central

    Liu, Shan-Lu; Lerman, Michael I.; Miller, A. Dusty

    2003-01-01

    Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are simple betaretroviruses that cause epithelial cell tumors in the lower and upper airways of sheep and goats. The envelope (Env) glycoproteins of both viruses can transform rodent and chicken fibroblasts, indicating that they play an essential role in oncogenesis. Previous studies found that a YXXM motif in the Env cytoplasmic tail, a putative docking site for phosphatidylinositol 3-kinase (PI3K) after tyrosine phosphorylation, was necessary for rodent cell transformation but was not required for transformation of DF-1 chicken fibroblasts. Here we show that JSRV and ENTV Env proteins with tyrosine or methionine mutations in the YXXM motif can still transform rodent fibroblasts, albeit with reduced efficiency. Akt was activated in cells transformed by JSRV or ENTV Env proteins and in cells transformed by the proteins with tyrosine mutations. Furthermore, the PI3K-specific inhibitor LY294002 could inhibit Akt activation and cell transformation in all cases, indicating that Akt activation and transformation is PI3K dependent. However, we could not detect tyrosine phosphorylation of JSRV or ENTV Env proteins or an interaction between the Env proteins and PI3K in the transformed cells. We found no evidence for mitogen-activated protein kinase activation in cells that were transformed by the JSRV or ENTV Env proteins. We conclude that ovine betaretrovirus Env proteins transform the rodent fibroblasts by indirectly activating the PI3K/Akt pathway. PMID:12829832

  12. Phosphatidylinositol 3-kinase-δ inhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals

    PubMed Central

    Herman, Sarah E. M.; Gordon, Amber L.; Wagner, Amy J.; Heerema, Nyla A.; Zhao, Weiqiang; Flynn, Joseph M.; Jones, Jeffrey; Andritsos, Leslie; Puri, Kamal D.; Lannutti, Brian J.; Giese, Neill A.; Zhang, Xiaoli; Wei, Lai; Byrd, John C.

    2010-01-01

    Targeted therapy with imatinib in chronic myeloid leukemia (CML) prompted a new treatment paradigm. Unlike CML, chronic lymphocytic leukemia (CLL) lacks an aberrant fusion protein kinase but instead displays increased phosphatidylinositol 3-kinase (PI3K) activity. To date, PI3K inhibitor development has been limited because of the requirement of this pathway for many essential cellular functions. Identification of the hematopoietic-selective isoform PI3K-δ unlocks a new therapeutic potential for B-cell malignancies. Herein, we demonstrate that PI3K has increased enzymatic activity and that PI3K-δ is expressed in CLL cells. A PI3K-δ selective inhibitor CAL-101 promoted apoptosis in primary CLL cells ex vivo in a dose- and time-dependent fashion that was independent of common prognostic markers. CAL-101–mediated cytotoxicity was caspase dependent and was not diminished by coculture on stromal cells. In addition, CAL-101 abrogated protection from spontaneous apoptosis induced by B cell–activating factors CD40L, TNF-α, and fibronectin. In contrast to malignant cells, CAL-101 does not promote apoptosis in normal T cells or natural killer cells, nor does it diminish antibody-dependent cellular cytotoxicity. However, CAL-101 did decrease activated T-cell production of various inflammatory and antiapoptotic cytokines. Collectively, these studies provide rationale for the clinical development of CAL-101 as a first-in-class targeted therapy for CLL and related B-cell lymphoproliferative disorders. PMID:20522708

  13. Phosphatidylinositol 3-kinase/Akt signaling pathway mediates acupuncture-induced dopaminergic neuron protection and motor function improvement in a mouse model of Parkinson's disease.

    PubMed

    Kim, Seung-Nam; Kim, Seung-Tae; Doo, Ah-Reum; Park, Ji-Yeun; Moon, Woongjoon; Chae, Younbyoung; Yin, Chang Shik; Lee, Hyejung; Park, Hi-Joon

    2011-10-01

    It has been reported that acupuncture treatment reduced dopaminergic neuron degeneration in Parkinson's disease (PD) models. However, the mechanistic pathways underlying, such neuroprotection, are poorly understood. Here, we investigated the effects and the underlying mechanism of acupuncture in a mouse model of PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). First, we observed that MPTP-induced impairment of Akt activation, but not MPTP-induced c-Jun activation, was effectively restored by acupuncture treatment in the substantia nigra. Furthermore, we demonstrated for the first time that the brain-specific blockade of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, by intranasal administration of LY294002, a specific inhibitor of PI3K/Akt signaling pathway, significantly blocked acupuncture-induced dopaminergic neuron protection and motor function improvement. Our results provide evidence that PI3K/Akt signaling pathway may play a central role in the mechanism underlying acupuncture-induced benefits in Parkinsonian mice.

  14. Structure of the iSH2 domain of Human phosphatidylinositol 3-kinase p85 beta Subunit Reveals Conformational Plasticity in the Interhelical Turn Region

    SciTech Connect

    C Schauder; L Ma; R Krug; G Montelione; R Guan

    2011-12-31

    Phosphatidylinositol 3-kinase (PI3K) proteins actively trigger signaling pathways leading to cell growth, proliferation and survival. These proteins have multiple isoforms and consist of a catalytic p110 subunit and a regulatory p85 subunit. The iSH2 domain of the p85 {beta} isoform has been implicated in the binding of nonstructural protein 1 (NS1) of influenza A viruses. Here, the crystal structure of human p85 {beta} iSH2 determined to 3.3 {angstrom} resolution is reported. The structure reveals that this domain mainly consists of a coiled-coil motif. Comparison with the published structure of the bovine p85 {beta} iSH2 domain bound to the influenza A virus nonstructural protein 1 indicates that little or no structural change occurs upon complex formation. By comparing this human p85 {beta} iSH2 structure with the bovine p85 {beta} iSH2 domain, which shares 99% sequence identity, and by comparing the multiple conformations observed within the asymmetric unit of the bovine iSH2 structure, it was found that this coiled-coil domain exhibits a certain degree of conformational variability or 'plasticity' in the interhelical turn region. It is speculated that this plasticity of p85 {beta} iSH2 may play a role in regulating its functional and molecular-recognition properties.

  15. Inhibition of p85, the non-catalytic subunit of phosphatidylinositol 3-kinase, exerts potent antitumor activity in human breast cancer cells

    PubMed Central

    Folgiero, V; Di Carlo, S E; Bon, G; Spugnini, E P; Di Benedetto, A; Germoni, S; Pia Gentileschi, M; Accardo, A; Milella, M; Morelli, G; Bossi, G; Mottolese, M; Falcioni, R

    2012-01-01

    The phosphoinositide 3-kinases (PI3Ks) are heterodimers consisting of the catalytic subunit p110 and the regulatory subunit p85. The PI3K/Akt pathway is strongly deregulated in breast cancer (BC) representing one of the mechanisms of resistance to therapies. Therefore, the identification of inhibitors of PI3K components represents one of the main goals to produce therapeutic agents. Here, we evaluated the efficacy of a phosphopeptide 1257 (P-1257) that targeting p85 strongly inhibits PI3K activity. We tested the effects of P-1257 administration in vitro and in vivo using BC cells expressing different levels of ErbB-2 and resistant or responsive to Trastuzumab. We demonstrated that inhibition of p85 activity by P-1257 induces cell death and sensitizes JIMT-1 and KPL-4 ErbB-2-overexpressing BC cells to Trastuzumab treatment. It is noteworthy that P-1257 delivery in vivo by electroporation or liposomes significantly inhibits the proliferation of tumor cells engrafted at subcutaneous and visceral sites. Overall, our data indicate that the p85 subunit is a valid target for therapeutic approaches and suggest that the structure of the peptide used in our study could be utilized for the development of novel drugs to apply in combination with therapies that fail to cure BCs with high PI3K activity. PMID:23222510

  16. 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

    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.

  17. 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

  18. SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation

    PubMed Central

    Hayashi, Moyuru; Kaneda, Mizuho; Iida, Kazuko; Shimonaka, Motoyuki; Hara, Takahiko; Arai, Morio; Koike, Yuichi; Yamamoto, Naomasa; Kasahara, Kohji

    2017-01-01

    Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation. PMID:28072855

  19. Pancreatic cell plasticity and cancer initiation induced by oncogenic Kras is completely dependent on wild-type PI 3-kinase p110α

    PubMed Central

    Baer, Romain; Cintas, Célia; Dufresne, Marlène; Cassant-Sourdy, Stéphanie; Schönhuber, Nina; Planque, Laetitia; Lulka, Hubert; Couderc, Bettina; Bousquet, Corinne; Garmy-Susini, Barbara; Vanhaesebroeck, Bart; Pyronnet, Stéphane; Saur, Dieter; Guillermet-Guibert, Julie

    2014-01-01

    Increased PI 3-kinase (PI3K) signaling in pancreatic ductal adenocarcinoma (PDAC) correlates with poor prognosis, but the role of class I PI3K isoforms during its induction remains unclear. Using genetically engineered mice and pharmacological isoform-selective inhibitors, we found that the p110α PI3K isoform is a major signaling enzyme for PDAC development induced by a combination of genetic and nongenetic factors. Inactivation of this single isoform blocked the irreversible transition of exocrine acinar cells into pancreatic preneoplastic ductal lesions by oncogenic Kras and/or pancreatic injury. Hitting the other ubiquitous isoform, p110β, did not prevent preneoplastic lesion initiation. p110α signaling through small GTPase Rho and actin cytoskeleton controls the reprogramming of acinar cells and regulates cell morphology in vivo and in vitro. Finally, p110α was necessary for pancreatic ductal cancers to arise from Kras-induced preneoplastic lesions by increasing epithelial cell proliferation in the context of mutated p53. Here we identify an in vivo context in which p110α cellular output differs depending on the epithelial transformation stage and demonstrate that the PI3K p110α is required for PDAC induced by oncogenic Kras, the key driver mutation of PDAC. These data are critical for a better understanding of the development of this lethal disease that is currently without efficient treatment. PMID:25452273

  20. Enhanced Efficacy of 5-Fluorouracil in Combination with a Dual Histone Deacetylase and Phosphatidylinositide 3-Kinase Inhibitor (CUDC-907) in Colorectal Cancer Cells

    PubMed Central

    Hamam, Rimi; Ali, Dalia; Vishnubalaji, Radhakrishnan; Alsaaran, Zaid F.; Chalisserry, Elna Paul; Alfayez, Musaad; Aldahmash, Abdullah; Alajez, Nehad M.

    2017-01-01

    Background/Aims: 5-Fluorouracil (5-FU) is widely used in the treatment of patients with colorectal cancer (CRC). However, the efficacy of 5-FU as a single agent is limited, with multiple undesired side effects. Therefore, the aim of the current study was to assess the efficacy of CUDC-907 (a dual inhibitor of histone deacetylase and phosphatidylinositide 3-kinase) in combination with 5-FU against CRC cells. Materials and Methods: Cell viability was determined using AlamarBlue and colony formation assays. Acridine orange/ethidium bromide staining and flow cytometry were used to measure apoptotic and necrotic events, as well as cell cycle progression. Immunoblotting was used to assess acetylation of histone H3 and phosphorylation of AKT. Results: Our data revealed enhanced toxicity of CUDC-907 against HCT116, RKO, COLO-205, and HT-29 CRC cells when combined with 5-FU. Similarly, the colony formation capability of HCT116 cells was suppressed by the combination treatment. Cells treated with CUDC-907 and 5-FU underwent apoptosis and necrosis, and exhibited increased polyploidy. Furthermore, CRC cells treated with CUDC-907 exhibited a higher degree of histone H3 lysine 9 acetylation (H3K9ac) and reduced AKT phosphorylation (Ser473). Conclusion: Our data revealed, for the first time, the enhanced inhibitory effect of CUDC-907 against CRC cells when combined with 5-FU, supporting the application of this combination as a potential therapeutic strategy in CRC treatment. PMID:28139498

  1. 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

  2. Mitogen-activated protein kinases and phosphatidylinositol 3-kinase are involved in Prevotella intermedia-induced proinflammatory cytokines expression in human periodontal ligament cells.

    PubMed

    Guan, Su-Min; Zhang, Ming; He, Jian-Jun; Wu, Jun-Zheng

    2009-08-28

    Chronic periodontitis is an inflammatory disease affecting periodontal connective tissues and alveolar bone. Proinflammatory mediators induced by periodontal pathogens play vital roles in the initiation and progression of the disease. In this study, we examined whether Prevotella intermedia induces proinflammatory cytokines expression in human periodontal ligament cells (hPDLs). The mRNA expression and protein production were determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbant assay (ELISA) respectively. P. intermedia treatment dose- and time-dependently increased IL-6, IL-8 and M-CSF, but not IL-1beta and TNF-alpha mRNA expression and protein secretion. Preincubation of hPDLs with extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 kinase and phosphatidylinositol 3-kinase (PI3K) inhibitors PD98059, SP600125, SB203580 and LY294002 resulted in significant reduction in P. intermedia-induced IL-6, IL-8 and M-CSF expression. Blocking the synthesis of prostaglandin E(2) (PGE(2)) by indomethacin also abolished the stimulatory effects of P. intermedia on cytokines expression. Our results indicate that P. intermedia induces proinflammatory cytokines through MAPKs and PI3K signaling pathways, and PGE(2) is involved in the P. intermedia-induced proinflammatory cytokines upregulation.

  3. SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation.

    PubMed

    Ohtsuka, Hiroko; Iguchi, Tomohiro; Hayashi, Moyuru; Kaneda, Mizuho; Iida, Kazuko; Shimonaka, Motoyuki; Hara, Takahiko; Arai, Morio; Koike, Yuichi; Yamamoto, Naomasa; Kasahara, Kohji

    2017-01-01

    Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation.

  4. 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.

  5. Inhibitory effect of capsaicin on B16-F10 melanoma cell migration via the phosphatidylinositol 3-kinase/Akt/Rac1 signal pathway

    PubMed Central

    Shin, Dong-Hoon; Kim, Ok-Hee; Jun, Hye-Seung

    2008-01-01

    Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the major pungent ingredient of red pepper, has been reported to possess anti-carcinogenic and anti-mutagenic activities. In this study, the anti-migration activity of capsaicin on highly metastatic B16-F10 melanoma cells was investigated. Capsaicin significantly inhibited the migration of melanoma cells without showing obvious cellular cytotoxicity at low doses. This effect correlated with the down-regulation of phosphatidylinositol 3-kinase (PI3-K) and its downstream target, Akt. Although B16-F10 cell migration was increased by the PI3-K activator through the activation of Akt, these PI3-K activator-induced phenomena were attenuated by capsaicin. Moreover, capsaicin was found to significantly inhibit Rac1 activity in a pull-down assay. These results demonstrate that capsaicin inhibits the migration of B16-F10 cells through the inhibition of the PI3-K/Akt/Rac1 signal pathway. The present investigation suggests that capsaicin targets PI3-K/Akt/Rac1-mediated cellular events in B16-F10 melanoma cells. Consequently, capsaicin administration should be considered an effective approach for the suppression of invasion and metastasis in malignant melanoma chemotherapy. PMID:18985006

  6. 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

  7. WAVE2, N-WASP, and Mena facilitate cell invasion via phosphatidylinositol 3-kinase-dependent local accumulation of actin filaments.

    PubMed

    Takahashi, Kazuhide; Suzuki, Katsuo

    2011-11-01

    Cell migration is accomplished by the formation of cellular protrusions such as lamellipodia and filopodia. These protrusions result from actin filament (F-actin) rearrangement at the cell cortex by WASP/WAVE family proteins and Drosophila enabled (Ena)/vasodilator-stimulated factor proteins. However, the role of each of these actin cytoskeletal regulatory proteins in the regulation of three-dimensional cell invasion remains to be clarified. We found that platelet-derived growth factor (PDGF) induces invasion of MDA-MB-231 human breast cancer cells through invasion chamber membrane pores. This invasion was accompanied by intensive F-actin accumulation at the sites of cell infiltration. After PDGF stimulation, WAVE2, N-WASP, and a mammalian Ena (Mena) colocalized with F-actin at the sites of cell infiltration in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. Depletion of WAVE2, N-WASP, or Mena by RNA interference (RNAi) abrogated both cell invasion and intensive F-actin accumulation at the invasion site. These results indicate that by mediating intensive F-actin accumulation at the sites of cell infiltration, WAVE2, N-WASP, and Mena are crucial for PI3K-dependent cell invasion induced by PDGF.

  8. 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

  9. Autophosphorylation of p110delta phosphoinositide 3-kinase: a new paradigm for the regulation of lipid kinases in vitro and in vivo.

    PubMed Central

    Vanhaesebroeck, B; Higashi, K; Raven, C; Welham, M; Anderson, S; Brennan, P; Ward, S G; Waterfield, M D

    1999-01-01

    Phosphoinositide 3-kinases (PI3Ks) are lipid kinases which also possess an in vitro protein kinase activity towards themselves or their adaptor proteins. The physiological relevance of these phosphorylations is unclear at present. Here, the protein kinase activity of the tyrosine kinase-linked PI3K, p110delta, is characterized and its functional impact assessed. In vitro autophosphorylation of p110delta completely down-regulates its lipid kinase activity. The single site of autophosphorylation was mapped to Ser1039 at the C-terminus of p110delta. Antisera specific for phospho-Ser1039 revealed a very low level of phosphorylation of this residue in cell lines. However, p110delta that is recruited to activated receptors (such as CD28 in T cells) shows a time-dependent increase in Ser1039 phosphorylation and a concomitant decrease in associated lipid kinase activity. Treatment of cells with okadaic acid, an inhibitor of Ser/Thr phosphatases, also dramatically increases the level of Ser1039-phosphorylated p110delta. LY294002 and wortmannin blocked these in vivo increases in Ser1039 phosphorylation, consistent with the notion that PI3Ks, and possibly p110delta itself, are involved in the in vivo phosphorylation of p110delta. In summary, we show that PI3Ks are subject to regulatory phosphorylations in vivo similar to those identified under in vitro conditions, identifying a new level of control of these signalling molecules. PMID:10064595

  10. Diacylglycerol kinase theta is translocated and phosphoinositide 3-kinase-dependently activated by noradrenaline but not angiotensin II in intact small arteries.

    PubMed Central

    Walker, A J; Draeger, A; Houssa, B; van Blitterswijk , W J; Ohanian, V; Ohanian, J

    2001-01-01

    Diacylglycerol (DG) kinase (DGK) phosphorylates the lipid second messenger DG to phosphatidic acid. We reported previously that noradrenaline (NA), but not angiotensin II (AII), increases membrane-associated DGK activity in rat small arteries [Ohanian and Heagerty (1994) Biochem. J. 300, 51-56]. Here, we have identified this DGK activity as DGKtheta, present in both smooth muscle and endothelial cells of these small vessels. Subcellular fractionation of artery homogenates revealed that DGKtheta was present in nuclear, plasma membrane (and/or Golgi) and cytosolic fractions. Upon NA stimulation, DGKtheta translocated towards the membrane and cytosol (155 and 153% increases relative to the control, respectively) at 30 s, followed by a return to near-basal levels at 5 min; AII was without effect. Translocation to the membrane was to both Triton-soluble and -insoluble fractions. NA, but not AII, transiently increased DGKtheta activity in immunoprecipitates (126% at 60 s). Membrane translocation and DGKtheta activation were regulated differently: NA-induced DGKtheta activation, but not translocation, was dependent on transient activation of phosphoinositide 3-kinase (PI 3-K). In addition, DGK activity co-immunoprecipitated with protein kinase B, a downstream effector of PI 3-K, and was increased greatly by NA stimulation. The rapid and agonist-specific activation of DGKtheta suggests that this pathway may have a physiological role in vascular smooth-muscle responses. PMID:11115406

  11. Vitamin E protected cultured cortical neurons from oxidative stress-induced cell death through the activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase.

    PubMed

    Numakawa, Yumiko; Numakawa, Tadahiro; Matsumoto, Tomoya; Yagasaki, Yuki; Kumamaru, Emi; Kunugi, Hiroshi; Taguchi, Takahisa; Niki, Etsuo

    2006-05-01

    The role of vitamin E in the CNS has not been fully elucidated. In the present study, we found that pre-treatment with vitamin E analogs including alphaT (alpha-tocopherol), alphaT3 (alpha -tocotrienol), gammaT, and gammaT3 for 24 h prevented the cultured cortical neurons from cell death in oxidative stress stimulated by H2O2, while Trolox, a cell-permeable analog of alphaT, did not. The preventive effect of alphaT was dependent on de novo protein synthesis. Furthermore, we found that alphaT exposure induced the activation of both the MAP kinase (MAPK) and PI3 kinase (PI3K) pathways and that the alphaT-dependent survival effect was blocked by the inhibitors, U0126 (an MAPK pathway inhibitor) or LY294002 (a PI3K pathway inhibitor). Interestingly, the up-regulation of Bcl-2 (survival promoting molecule) was induced by alphaT application. The up-regulation of Bcl-2 did not occur in the presence of U0126 or LY294002, suggesting that alphaT-up-regulated Bcl-2 is mediated by these kinase pathways. These observations suggest that vitamin E analogs play an essential role in neuronal maintenance and survival in the CNS.

  12. WNT16B is a new marker of cellular senescence that regulates p53 activity and the phosphoinositide 3-kinase/AKT pathway.

    PubMed

    Binet, Romuald; Ythier, Damien; Robles, Ana I; Collado, Manuel; Larrieu, Delphine; Fonti, Claire; Brambilla, Elisabeth; Brambilla, Christian; Serrano, Manuel; Harris, Curtis C; Pedeux, Rémy

    2009-12-15

    Senescence is a tumor suppression mechanism that is induced by several stimuli, including oncogenic signaling and telomere shortening, and controlled by the p53/p21(WAF1) signaling pathway. Recently, a critical role for secreted factors has emerged, suggesting that extracellular signals are necessary for the onset and maintenance of senescence. Conversely, factors secreted by senescent cells may promote tumor growth. By using expression profiling techniques, we searched for secreted factors that were overexpressed in fibroblasts undergoing replicative senescence. We identified WNT16B, a member of the WNT family of secreted proteins. We found that WNT16B is overexpressed in cells undergoing stress-induced premature senescence and oncogene-induced senescence in both MRC5 cell line and the in vivo murine model of K-Ras(V12)-induced senescence. By small interfering RNA experiments, we observed that both p53 and WNT16B are necessary for the onset of replicative senescence. WNT16B expression is required for the full transcriptional activation of p21(WAF1). Moreover, WNT16B regulates activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway. Overall, we identified WNT16B as a new marker of senescence that regulates p53 activity and the PI3K/AKT pathway and is necessary for the onset of replicative senescence.

  13. Structure-Based Design of an Organoruthenium Phosphatidyl-inositol-3-Kinase Inhibitor Reveals a Switch Governing Lipid Kinase Potency and Selectivity

    SciTech Connect

    Xie,P.; Williams, D.; Atilla-Gokcumen, G.; Milk, L.; Xiao, M.; Smalley, K.; Herlyn, M.; Meggers, E.; Marmorstein, R.

    2008-01-01

    Mutations that constitutively activate the phosphatidyl-inositol-3-kinase (PI3K) signaling pathway, including alterations in PI3K, PTEN, and AKT, are found in a variety of human cancers, implicating the PI3K lipid kinase as an attractive target for the development of therapeutic agents to treat cancer and other related diseases. In this study, we report on the combination of a novel organometallic kinase inhibitor scaffold with structure-based design to develop a PI3K inhibitor, called E5E2, with an IC50 potency in the mid-low-nanomolar range and selectivity against a panel of protein kinases. We also show that E5E2 inhibits phospho-AKT in human melanoma cells and leads to growth inhibition. Consistent with a role for the PI3K pathway in tumor cell invasion, E5E2 treatment also inhibits the migration of melanoma cells in a 3D spheroid assay. The structure of the PI3K?/E5E2 complex reveals the molecular features that give rise to this potency and selectivity toward lipid kinases with implications for the design of a subsequent generation of PI3K-isoform-specific organometallic inhibitors.

  14. Overexpression of protein-tyrosine phosphatase-1B in adipocytes inhibits insulin-stimulated phosphoinositide 3-kinase activity without altering glucose transport or Akt/Protein kinase B activation.

    PubMed

    Venable, C L; Frevert, E U; Kim, Y B; Fischer, B M; Kamatkar, S; Neel, B G; Kahn, B B

    2000-06-16

    Previous studies suggested that protein-tyrosine phosphatase 1B (PTP1B) antagonizes insulin action by catalyzing dephosphorylation of the insulin receptor (IR) and/or other key proteins in the insulin signaling pathway. In adipose tissue and muscle of obese humans and rodents, PTP1B expression is increased, which led to the hypothesis that PTP1B plays a role in the pathogenesis of insulin resistance. Consistent with this, mice in which the PTP1B gene was disrupted exhibit increased insulin sensitivity. To test whether increased expression of PTP1B in an insulin-sensitive cell type could contribute to insulin resistance, we overexpressed wild-type PTP1B in 3T3L1 adipocytes using adenovirus-mediated gene delivery. PTP1B expression was increased approximately 3-5-fold above endogenous levels at 16 h, approximately 14-fold at 40 h, and approximately 20-fold at 72 h post-transduction. Total protein-tyrosine phosphatase activity was increased by 50% at 16 h, 3-4-fold at 40 h, and 5-6-fold at 72 h post-transduction. Compared with control cells, cells expressing high levels of PTP1B showed a 50-60% decrease in maximally insulin-stimulated tyrosyl phosphorylation of IR and insulin receptor substrate-1 (IRS-1) and phosphoinositide 3-kinase (PI3K) activity associated with IRS-1 or with phosphotyrosine. Akt phosphorylation and activity were unchanged. Phosphorylation of p42 and p44 MAP kinase (MAPK) was reduced approximately 32%. Overexpression of PTP1B had no effect on basal, submaximally or maximally (100 nm) insulin-stimulated glucose transport or on the EC(50) for transport. Our results suggest that: 1) insulin stimulation of glucose transport in adipocytes requires

  15. 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

  16. Genes

    MedlinePlus

    ... Search Search MedlinePlus GO GO About MedlinePlus Site Map FAQs Customer Support Health Topics Drugs & Supplements Videos & Tools Español You Are Here: Home → Medical Encyclopedia → Genes URL of this page: //medlineplus.gov/ency/article/ ...

  17. 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

  18. 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.

  19. Differential regulation of insulin receptor substrates-1 and -2 (IRS-1 and IRS-2) and phosphatidylinositol 3-kinase isoforms in liver and muscle of the obese diabetic (ob/ob) mouse.

    PubMed Central

    Kerouz, N J; Hörsch, D; Pons, S; Kahn, C R

    1997-01-01

    Intracellular insulin signaling involves a series of alternative and complementary pathways created by the multiple substrates of the insulin receptor (IRS) and the various isoforms of SH2 domain signaling molecules that can interact with these substrates. In this study, we have evaluated the roles of IRS-1 and IRS-2 in signaling to the phosphatidylinositol (PI) 3-kinase pathway in the ob/ob mouse, a model of the insulin resistance of obesity and non-insulin-dependent diabetes mellitus. We find that the levels of expression of both IRS-1 and IRS-2 are decreased approximately 50% in muscle, whereas in liver the decrease is significantly greater for IRS-2 (72%) than for IRS-1 (29%). This results in differential decreases in IRS-1 and IRS-2 phosphorylation, docking of the p85alpha regulatory subunit of PI 3-kinase, and activation of this enzyme in these two insulin target tissues. In ob/ob liver there is also a change in expression of the alternatively spliced isoforms of the regulatory subunits for PI 3-kinase that was detected at the protein and mRNA level. This resulted in a 45% decrease in the p85alpha form of PI 3-kinase, a ninefold increase in the AS53/p55alpha, and a twofold increase in p50alpha isoforms. Thus, there are multiple alterations in the early steps of insulin signaling in the ob/ob mouse, with differential regulation of IRS-1 and IRS-2, various PI 3-kinase regulatory isoforms, and a lack of compensation for the decrease in insulin signaling by any of the known alternative pathways at these levels. PMID:9399964

  20. The phosphoinositide 3-kinase inhibitor PI-103 downregulates choline kinase alpha leading to phosphocholine and total choline decrease detected by magnetic resonance spectroscopy.

    PubMed

    Al-Saffar, Nada M S; Jackson, L Elizabeth; Raynaud, Florence I; Clarke, Paul A; Ramírez de Molina, Ana; Lacal, Juan C; Workman, Paul; Leach, Martin O

    2010-07-01

    The phosphoinositide 3-kinase (PI3K) pathway is a major target for cancer drug development. PI-103 is an isoform-selective class I PI3K and mammalian target of rapamycin inhibitor. The aims of this work were as follows: first, to use magnetic resonance spectroscopy (MRS) to identify and develop a robust pharmacodynamic (PD) biomarker for target inhibition and potentially tumor response following PI3K inhibition; second, to evaluate mechanisms underlying the MRS-detected changes. Treatment of human PTEN null PC3 prostate and PIK3CA mutant HCT116 colon carcinoma cells with PI-103 resulted in a concentration- and time-dependent decrease in phosphocholine (PC) and total choline (tCho) levels (P < 0.05) detected by phosphorus ((31)P)- and proton ((1)H)-MRS. In contrast, the cytotoxic microtubule inhibitor docetaxel increased glycerophosphocholine and tCho levels in PC3 cells. PI-103-induced MRS changes were associated with alterations in the protein expression levels of regulatory enzymes involved in lipid metabolism, including choline kinase alpha (ChoK(alpha)), fatty acid synthase (FAS), and phosphorylated ATP-citrate lyase (pACL). However, a strong correlation (r(2) = 0.9, P = 0.009) was found only between PC concentrations and ChoK(alpha) expression but not with FAS or pACL. This study identified inhibition of ChoK(alpha) as a major cause of the observed change in PC levels following PI-103 treatment. We also showed the capacity of (1)H-MRS, a clinically well-established technique with higher sensitivity and wider applicability compared with (31)P-MRS, to assess response to PI-103. Our results show that monitoring the effects of PI3K inhibitors by MRS may provide a noninvasive PD biomarker for PI3K inhibition and potentially of tumor response during early-stage clinical trials with PI3K inhibitors.

  1. Role of phosphatidylinositol-3 kinase-gamma in the actions of glucagon-like peptide-2 on the murine small intestine.

    PubMed

    Anini, Younes; Izzo, Angelo; Oudit, Gavin Y; Backx, Peter H; Brubaker, Patricia L

    2007-06-01

    Glucagon-like peptide-2 (GLP-2) enhances intestinal growth and function through a cAMP-linked G protein-coupled receptor (GPCR) expressed in the mucosal layer and enteric nervous system. Because the type 1B gamma-isoform of phosphatidylinositol 3-kinase (PI3-K) is activated by GPCRs, we determined whether this enzyme plays a role in the intestinal actions of GLP-2 by using PI3-Kgamma knockout (KO) mice. Wild-type (WT), heterozygous, and KO mice were treated with vehicle or 1 microg Gly2-GLP-2 (a long-acting analog) twice daily for 10 days and analyzed for changes in intestinal growth, motility, and cAMP production. Basal small intestinal wet weight was increased in KO mice in association with enhanced crypt-villus height and crypt cell proliferation (P < 0.05-0.01). However, the GLP-2-induced changes in these parameters were not different between KO and WT animals. GLP-2 treatment also enhanced the number of mucous cells in the intestinal epithelium, but this effect was lost in the PI3-Kgamma KO mice. Both basal and GLP-2-induced suppression of intestinal transit were normal in KO mice. In contrast, the ability of GLP-2 to stimulate cAMP levels in isolated muscle strips was abrogated by loss of PI3-Kgamma, despite the expression of GLP-2 receptor mRNA transcripts in this tissue. Together, the results of this study demonstrate a role for PI3-Kgamma in basal but not GLP-2-induced small intestinal mucosal growth. However, PI3-Kgamma is important for the enhancement of mucous cell number by GLP-2 and in the ability of the GLP-2 receptor to couple to cAMP in the enteric nervous system.

  2. Inhibition of phosphatidylinositol 3-kinase promotes tumor cell resistance to chemotherapeutic agents via a mechanism involving delay in cell cycle progression

    SciTech Connect

    McDonald, Gail T.; Sullivan, Richard; Pare, Genevieve C.; Graham, Charles H.

    2010-11-15

    Approaches to overcome chemoresistance in cancer cells have involved targeting specific signaling pathways such as the phosphatidylinositol 3-kinase (PI3K) pathway, a stress response pathway known to be involved in the regulation of cell survival, apoptosis and growth. The present study determined the effect of PI3K inhibition on the clonogenic survival of human cancer cells following exposure to various chemotherapeutic agents. Treatment with the PI3K inhibitors LY294002 or Compound 15e resulted in increased survival of MDA-MB-231 breast carcinoma cells after exposure to doxorubicin, etoposide, 5-fluorouracil, and vincristine. Increased survival following PI3K inhibition was also observed in DU-145 prostate, HCT-116 colon and A-549 lung carcinoma cell lines exposed to doxorubicin. Increased cell survival mediated by LY294002 was correlated with a decrease in cell proliferation, which was linked to an increase in the proportion of cells in the G{sub 1} phase of the cell cycle. Inhibition of PI3K signaling also resulted in higher levels of the cyclin-dependent kinase inhibitors p21{sup Waf1/Cip1} and p27{sup Kip1}; and knockdown of p27{sup kip1} with siRNA attenuated resistance to doxorubicin in cells treated with LY294002. Incubation in the presence of LY294002 after exposure to doxorubicin resulted in decreased cell survival. These findings provide evidence that PI3K inhibition leads to chemoresistance in human cancer cells by causing a delay in cell cycle; however, the timing of PI3K inhibition (either before or after exposure to anti-cancer agents) may be a critical determinant of chemosensitivity.

  3. Overexpression of phospholipase D prevents actinomycin D-induced apoptosis through potentiation of phosphoinositide 3-kinase signalling pathways in Chinese-hamster ovary cells.

    PubMed Central

    Yamada, Momoko; Banno, Yoshiko; Takuwa, Yoh; Koda, Masahiro; Hara, Akira; Nozawa, Yoshinori

    2004-01-01

    To examine the roles of PLD (phospholipase D) in the regulation of the apoptotic process, PLD1 and PLD2 were stably overexpressed in S1P3-CHO cells [CHO (Chinese-hamster ovary) cells expressing the S1P (sphingosine 1-phosphate) receptor S1P3]. Treatment of S1P3-CHO cells with ActD (actinomycin D) induced apoptosis, as shown by the occurrence of nuclear fragmentation and the caspase-dependent proteolytic cleavage of PARP [poly(ADP-ribose) polymerase] and protein kinase Cd. Overexpression of either PLD1 or PLD2 protected S1P3-CHO cells from ActD-induced apoptosis, as demonstrated by an increased number of viable cells and inhibition of PARP and protein kinase Cd cleavage. However, in the early phase of apoptosis, ActD induced an increase in PLD activity and activation of key factors in the cell-survival signalling pathways, such as PI3K (phosphoinositide 3-kinase), Akt, p70S6K (p70 S6 kinase) and ERK (extracellular-signal-regulated kinase). Furthermore, the ActD-induced activation of these survival signalling enzymes was potentiated by overexpression of either PLD1 or PLD2. The PI3K inhibitor LY294002 inhibited the ActD-induced activation of Akt and p70S6K, and completely abolished the effects of PLD1 or PLD2, whereas inhibition of ERK activity by the MEK inhibitor U0126 had a milder effect. The ActD-induced activation of p70S6K and ERKs was blocked by 1-butanol, but not by t-butanol; similar to S1P, exogenous PLD suppressed the ActD-induced events in the apoptosis signalling pathways. These results show that, in S1P3-CHO cells, increased expression of PLDs prevents ActD-induced apoptosis by enhanced activation of the PI3K signalling pathways. PMID:14640974

  4. PI3 kinase/Akt activation mediates estrogen and IGF-1 nigral DA neuronal neuroprotection against a unilateral rat model of Parkinson's disease.

    PubMed

    Quesada, Arnulfo; Lee, Becky Y; Micevych, Paul E

    2008-04-01

    Recently, using the medial forebrain bundle (MFB) 6-hydroxydopmaine (6-OHDA) lesion rat model of Parkinson's disease (PD), we have demonstrated that blockade of central IGF-1 receptors (IGF-1R) attenuated estrogen neuroprotection of substantia nigra pars compacta (SNpc) DA neurons, but exacerbated 6-OHDA lesions in IGF-1 only treated rats (Quesada and Micevych [2004]: J Neurosci Res 75:107-116). This suggested that the IGF-1 system is a central mechanism through which estrogen acts to protect the nigrostriatal DA system. Moreover, these results also suggest that IGF-1R-induced intracellular signaling pathways are involved in the estrogen mechanism that promotes neuronal survival. In vitro, two convergent intracellular signaling pathways used by estrogen and IGF-1, the mitogen-activated protein kinase (MAPK/ERK), and phosphatidyl-inositol-3-kinase/Akt (PI3K/Akt), have been demonstrated to be neuroprotective. Continuous central infusions of MAPK/ERK and PI3K/Akt inhibitors were used to test the hypothesis that one or both of these signal transduction pathways mediates estrogen and/or IGF-1 neuroprotection of SNpc DA neurons after a unilateral administration of 6-OHDA into the MFB of rats. Motor behavior tests and tyrosine hydroxylase immunoreactivity revealed that the inhibitor of the PI3K/Akt pathway (LY294002) blocked the survival effects of both estrogen and IGF-1, while an inhibitor of the MAPK/ERK signaling (PD98059) was ineffective. Western blot analyses showed that estrogen and IGF-1 treatments increased PI3K/Akt activation in the SN; however, MAPK/ERK activation was decreased in the SN. Indeed, continuous infusions of inhibitors blocked phosphorylation of PI3K/Akt and MAPK/ERK. These findings indicate that estrogen and IGF-1-mediated SNpc DA neuronal protection is dependent on PI3K/Akt signaling, but not on the MAPK/ERK pathway.

  5. Protective effects of statins on L-DOPA neurotoxicity due to the activation of phosphatidylinositol 3-kinase and free radical scavenging in PC12 cell culture.

    PubMed

    Koh, Seong-Ho; Park, Hyun-Hee; Choi, Na-Young; Lee, Kyu-Yong; Kim, Sangjae; Lee, Young Joo; Kim, Hee-Tae

    2011-01-25

    Neurotoxic effects have been suggested for l-3,4-dihydroxyphenylalanine (L-DOPA), while neuroprotective effects have been proposed for statins. We investigated whether certain statins (simvastatin or pitavastatin) could inhibit L-DOPA neurotoxicity. Neuronally-differentiated PC12 (nPC12) cells were treated with L-DOPA and/or statins for 24h, and their viabilities were measured using a cell counting kit, trypan blue staining, and 4',6-diamidino-2-phenylindole (DAPI) staining. Free radical and specific intracellular signaling protein levels were measured with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and Western blotting, respectively. High concentrations of l-DOPA reduced nPC12 cell viability, but combined treatment with statins restored viability. Treatment with 200 μM L-DOPA increased free radical and hydroxyl radical levels, but combined treatment with 5 μM statins decreased these levels. Survival-related signaling proteins were decreased in nPC12 cells treated with 200 μM L-DOPA, but combined treatment with 5μM statins significantly increased the levels of these proteins. Treatment with 200 μM L-DOPA significantly increased death-related signaling proteins, while combined treatment with 5 μM statins reduced the levels of these proteins. Pretreatment with LY294002, a phosphatidylinositol 3 kinase (PI3K) inhibitor, before combined treatment with statins and L-DOPA almost completely blocked the protective effects of statins. These results indicate that statins reduce L-DOPA neurotoxicity by lowering oxidative stress and by enhancing survival signals and inhibiting death signals via activation of the PI3K pathway.

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

    PubMed

    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

  7. 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

  8. 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

  9. 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

  10. Dexmedetomidine increases the activity of excitatory amino acid transporter type 3 expressed in Xenopus oocytes: the involvement of protein kinase C and phosphatidylinositol 3-kinase.

    PubMed

    Do, Sang-Hwan; Park, Seong-Joo; Shin, Hyun-Jung; Paik, Hye-Sun; Zuo, Zhiyi; Yoon, Hea-Jo; Ryu, Jung-Hee

    2014-09-05

    Dexmedetomidine, an α2 adrenergic agonist, has neuroprotective and anticonvulsant properties in addition to its sedative and anxiolytic effects. We hypothesized that dexmedetomidine would increase the activity of excitatory amino acid transporter type 3 (EAAT3) and that this effect would involve protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K), two protein kinases known to regulate EAAT3 activity. EAAT3 was expressed in Xenopus oocytes by injecting its mRNA. Two-electrode voltage clamping was used to record membrane currents before, during, and after application of 30 μM l-glutamate in the presence of 0.1-30 nM dexmedetomidine. Dexmedetomidine-treated oocytes were also exposed to a PKC activator (phorbol-12-myristate-13-acetate [PMA]), PKC inhibitors (chelerythrine, staurosporine, and calphostin C), and PI3K inhibitors (wortmannin and LY294002) before current measurement. Dexmedetomidine application resulted in a concentration-dependent increase in the EAAT3 activity in response to l-glutamate. The kinetic study showed that dexmedetomidine significantly increased the Vmax without changing Km. Treatment of oocytes with PMA significantly increased transporter currents compared with controls, but treatment with dexmedetomidine plus PMA did not further increase the response compared with PMA or dexmedetomidine alone. In addition, pre-treatment of oocytes with PKC inhibitors and PI3K inhibitors significantly abolished the dexmedetomidine-enhanced EAAT3 activity. These results suggest that dexmedetomidine increases the activity of EAAT3 expressed in Xenopus oocytes. PKC and PI3K seem to mediate this effect. These findings may explain the neuroprotective and anticonvulsant effects of dexmedetomidine.

  11. Ceramide limits phosphatidylinositol-3-kinase C2β-controlled cell motility in ovarian cancer: potential of ceramide as a metastasis-suppressor lipid

    PubMed Central

    Kitatani, Kazuyuki; Toshinori, Usui; Sriraman, Shravan Kumar; Toyoshima, Masafumi; Ishibashi, Masumi; Shigeta, Shogo; Nagase, Satoru; Sakamoto, Masahiro; Ogiso, Hideo; Okazaki, Toshiro; Hannun, Yusuf A.; Torchilin, Vladimir P.; Yaegashi, Nobuo

    2015-01-01

    Targeting cell motility, which is required for dissemination and metastasis, has therapeutic potential for ovarian cancer metastasis, and regulatory mechanisms of cell motility need to be uncovered for developing novel therapeutics. Invasive ovarian cancer cells spontaneously formed protrusions, such as lamellipodia, which are required for generating locomotive force in cell motility. siRNA screening identified class II phosphatidylinositol 3-kinase C2β (PI3KC2β) as the predominant isoform of PI3K involved in lamellipodia formation of ovarian cancer cells. The bioactive sphingolipid ceramide has emerged as an antitumorigenic lipid, and treatment with short-chain C6-ceramide decreased the number of ovarian cancer cells with PI3KC2β-driven lamellipodia. Pharmacological analysis demonstrated that long-chain ceramide regenerated from C6-ceramide through the salvage/recycling pathway, at least in part, mediated the action of C6-ceramide. Mechanistically, ceramide was revealed to interact with the PIK-catalytic domain of PI3KC2β and affect its compartmentalization, thereby suppressing PI3KC2β activation and its driven cell motility. Ceramide treatment also suppressed cell motility promoted by epithelial growth factor, which is a prometastatic factor. To examine the role of ceramide in ovarian cancer metastasis, ceramide liposomes were employed and confirmed to suppress cell motility in vitro. Ceramide liposomes had an inhibitory effect on peritoneal metastasis in a murine xenograft model of human ovarian cancer. Metastasis of PI3KC2β knocked-down cells was insensitive to treatment with ceramide liposomes, suggesting specific involvement of ceramide interaction with PI3KC2β in metastasis suppression. Our study identified ceramide as a bioactive lipid that limits PI3KC2β-governed cell motility, and ceramide is proposed to serve as a metastasis-suppressor lipid in ovarian cancer. These findings could be translated into developing ceramide-based therapy for

  12. 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.

  13. Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

    PubMed Central

    Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko

    2016-01-01

    Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing. PMID:27541856

  14. Ellagic acid prevents rat colon carcinogenesis induced by 1, 2 dimethyl hydrazine through inhibition of AKT-phosphoinositide-3 kinase pathway.

    PubMed

    Umesalma, Syed; Sudhandiran, Ganapasam

    2011-06-25

    Colon cancer is the third most malignant neoplasm in the world and chemoprevention through dietary intervention is an emerging option to reduce its mortality. Ellagic acid (EA) a major component of berries possesses attractive biological deeds. This study is aimed to investigate the effect of ellagic acid in fostering apoptosis in 1,2-dimethyl hydrazine (DMH) mediated experimental colon carcinogenesis model. Wistar male rats were segregated into four groups: group I-control rats, group II-rats received ellagic acid (60 mg/kg body weight p.o. every day), rats in group III-induced with DMH (20 mg/kg body weight, s.c.) for 15 weeks, DMH-induced group IV rats were initiated with ellagic acid treatment. The present study is designed to explore the significance of phosphoinositide-3-kinase (PI3K)/Akt molecular pathway as well as ellagic acid's chemopreventive effect in colon cancer. DMH-induced rats exhibited elevated expressions of PI3K and Akt as confirmed by immunofluorescence, immunoblot and confocal microscopic analysis. Mechanistically, ellagic acid was found to prevent PI3K/Akt activation that in turn, results in modulation of its downstream Bcl-2 family proteins. Bax expression and caspase-3 activation was noted after ellagic acid supplementation leading to elevation of cytochrome c (cyt c) levels and finally cell death. These observations were supported by the DNA fragmentation results, which showed the occurrence of apoptosis. This study reveals the involvement of PI3K-Akt signaling through which ellagic acid induces apoptosis and subsequently suppresses colon cancer during DMH-induced rat colon carcinogenesis. In conclusion, our findings demonstrate that ellagic acid begets apoptosis in DMH-induced colon carcinoma.

  15. Phosphoinositide 3-kinase alpha-dependent regulation of branching morphogenesis in murine embryonic lung: evidence for a role in determining morphogenic properties of FGF7.

    PubMed

    Carter, Edward; Miron-Buchacra, Gabriela; Goldoni, Silvia; Danahay, Henry; Westwick, John; Watson, Malcolm L; Tosh, David; Ward, Stephen G

    2014-01-01

    Branching morphogenesis is a critical step in the development of many epithelial organs. The phosphoinositide-3-kinase (PI3K) pathway has been identified as a central component of this process but the precise role has not been fully established. Herein we sought to determine the role of PI3K in murine lung branching using a series of pharmacological inhibitors directed at this pathway. The pan-class I PI3K inhibitor ZSTK474 greatly enhanced the branching potential of whole murine lung explants as measured by an increase in the number of terminal branches compared with controls over 48 hours. This enhancement of branching was also observed following inhibition of the downstream signalling components of PI3K, Akt and mTOR. Isoform selective inhibitors of PI3K identified that the alpha isoform of PI3K is a key driver in branching morphogenesis. To determine if the effect of PI3K inhibition on branching was specific to the lung epithelium or secondary to an effect on the mesenchyme we assessed the impact of PI3K inhibition in cultures of mesenchyme-free lung epithelium. Isolated lung epithelium cultured with FGF7 formed large cyst-like structures, whereas co-culture with FGF7 and ZSTK474 induced the formation of defined branches with an intact lumen. Together these data suggest a novel role for PI3K in the branching program of the murine embryonic lung contradictory to that reported in other branching organs. Our observations also point towards PI3K acting as a morphogenic switch for FGF7 signalling.

  16. Ellagitannin-rich cloudberry inhibits hepatocyte growth factor induced cell migration and phosphatidylinositol 3-kinase/AKT activation in colon carcinoma cells and tumors in Min mice

    PubMed Central

    Pajari, Anne-Maria; Päivärinta, Essi; Paavolainen, Lassi; Vaara, Elina; Koivumäki, Tuuli; Garg, Ritu; Heiman-Lindh, Anu; Mutanen, Marja; Marjomäki, Varpu; Ridley, Anne J.

    2016-01-01

    Berries have been found to inhibit colon carcinogenesis in animal models, and thus represent a potential source of compounds for prevention and treatment of colorectal cancer. The mechanistic basis for their effects is not well understood. We used human colon carcinoma cells and Min mice to investigate the effects of ellagitannin-rich cloudberry (Rubus chamaemorus) extract on cancer cell migration and underlying cell signaling. Intrinsic and hepatocyte growth factor (HGF) -induced cell motility in human HT29 and HCA7 colon carcinoma cells was assessed carrying out cell scattering and scratch wound healing assays using time-lapse microscopy. Activation of Met, AKT, and ERK in cell lines and tumors of cloudberry-fed Min mice were determined using immunoprecipitation, Western blot and immunohistochemical analyses. Cloudberry extract significantly inhibited particularly HGF-induced cancer cell migration in both cell lines. Cloudberry extract inhibited the Met receptor tyrosine phosphorylation by HGF and strongly suppressed HGF-induced AKT and ERK activation in both HT29 and HCA7 cells. Consistently, cloudberry feeding (10% w/w freeze-dried berries in diet for 10 weeks) reduced the level of active AKT and prevented phosphoMet localization at the edges in tumors of Min mice. These results indicate that cloudberry reduces tumor growth and cancer cell motility by inhibiting Met signaling and consequent activation of phosphatidylinositol 3-kinase/AKT in vitro and in tumors in vivo. As the Met receptor is recognized to be a major target in cancer treatment, our results suggest that dietary phytochemicals may have therapeutic value in reducing cancer progression and metastasis. PMID:27270323

  17. 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

    Zhou, Long Dian; Xiong, Xu; Long, Xin Hua; Liu, Zhi Li; Huang, Shan Hu; Zhang, Wei

    2014-11-01

    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.

  18. Nitric oxide decreases subventricular zone stem cell proliferation by inhibition of epidermal growth factor receptor and phosphoinositide-3-kinase/Akt pathway.

    PubMed

    Torroglosa, Ana; Murillo-Carretero, Maribel; Romero-Grimaldi, Carmen; Matarredona, Esperanza R; Campos-Caro, Antonio; Estrada, Carmen

    2007-01-01

    Nitric oxide (NO) inhibits proliferation of subventricular zone (SVZ) neural precursor cells in adult mice in vivo under physiological conditions. The mechanisms underlying this NO effect have now been investigated using SVZ-derived neural stem cells, which generate neurospheres in vitro when stimulated by epidermal growth factor (EGF). In these cultures, NO donors decreased the number of newly formed neurospheres as well as their size, which indicates that NO was acting on the neurosphere-forming neural stem cells and the daughter neural progenitors. The effect of NO was cytostatic, not proapoptotic, and did not involve cGMP synthesis. Neurosphere cells expressed the neuronal and endothelial isoforms of NO synthase (NOS) and produced NO in culture. Inhibition of NOS activity by N(omega)-nitro-L-arginine methylester (L-NAME) promoted neurosphere formation and growth, thus revealing an autocrine/paracrine action of NO on the neural precursor cells. Both exogenous and endogenous NO impaired the EGF-induced activation of the EGF receptor (EGFR) tyrosine kinase and prevented the EGF-induced Akt phosphorylation in neurosphere cells. Inhibition of the phosphoinositide-3-kinase (PI3-K)/Akt pathway by LY294002 significantly reduced the number of newly formed neurospheres, which indicates that this is an essential pathway for neural stem cell self-renewal. Chronic administration of l-NAME to adult mice enhanced phospho-Akt staining in the SVZ and reduced nuclear p27(Kip1) in the SVZ and olfactory bulb. The inhibition of EGFR and PI3-K pathway by NO explains, at least in part, its antimitotic effect on neurosphere cells and may be a mechanism involved in the physiological role of NO as a negative regulator of SVZ neurogenesis in adult mice.

  19. Dystrophin glycoprotein complex-associated Gbetagamma subunits activate phosphatidylinositol-3-kinase/Akt signaling in skeletal muscle in a laminin-dependent manner.

    PubMed

    Xiong, Yongmin; Zhou, Yanwen; Jarrett, Harry W

    2009-05-01

    Previously, we showed that laminin-binding to the dystrophin glycoprotein complex (DGC) of skeletal muscle causes a heterotrimeric G-protein (Galphabetagamma) to bind, changing the activation state of the Gsalpha subunit. Others have shown that laminin-binding to the DGC also leads to Akt activation. Gbetagamma, released when Gsalpha is activated, is known to bind phosphatidylinositol-3-kinase (PI3K), which activates Akt in other cells. Here, we investigate whether muscle Akt activation results from Gbetagamma, using immunoprecipitation and immunoblotting, and purified Gbetagamma. In the presence of laminin, PI3K-binding to the DGC increases and Akt becomes phosphorylated and activated (pAkt), and glycogen synthase kinase is phosphorylated. Antibodies, which specifically block laminin-binding to alpha-dystroglycan, prevent PI3K-binding to the DGC. Purified bovine brain Gbetagamma also caused PI3K and Akt activation. These results show that DGC-Gbetagamma is binding PI3K and activating pAkt in a laminin-dependent manner. Mdx mice, which have greatly diminished amounts of DGC proteins, display elevated pAkt signaling and increased expression of integrin beta1 compared to normal muscle. This integrin binds laminin, Gbetagamma, and PI3K. Collectively, these suggest that PI3K is an important target for the Gbetagamma, which normally binds to DGC syntrophin, and activates PI3K/Akt signaling. Disruption of the DGC in mdx mouse is causing dis-regulation of the laminin-DGC-Gbetagamma-PI3K-Akt signaling and is likely to be important to the pathogenesis of muscular dystrophy. Upregulating integrin beta1 expression and activating the PI3K/Akt pathway in muscular dystrophy may partially compensate for the loss of the DGC. The results suggest new therapeutic approaches to muscle disease.

  20. 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

  1. 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

  2. MRI reveals the in vivo cellular and vascular response to BEZ235 in ovarian cancer xenografts with different PI3-kinase pathway activity

    PubMed Central

    Cebulla, J; Huuse, E M; Pettersen, K; van der Veen, A; Kim, E; Andersen, S; Prestvik, W S; Bofin, A M; Pathak, A P; Bjørkøy, G; Bathen, T F; Moestue, S A

    2015-01-01

    Background: The phosphoinositide-3 kinase (PI3K) pathway is an attractive therapeutic target. However, difficulty in predicting therapeutic response limits the clinical implementation of PI3K inhibitors. This study evaluates the utility of clinically relevant magnetic resonance imaging (MRI) biomarkers for noninvasively assessing the in vivo response to the dual PI3K/mTOR inhibitor BEZ235 in two ovarian cancer models with differential PI3K pathway activity. Methods: The PI3K signalling activity of TOV-21G and TOV-112D human ovarian cancer cells was investigated in vitro. Cellular and vascular response of the xenografts to BEZ235 treatment (65 mg kg−1, 3 days) was assessed in vivo using diffusion-weighted (DW) and dynamic contrast-enhanced (DCE)-MRI. Micro-computed tomography was performed to investigate changes in vascular morphology. Results: The TOV-21G cells showed higher PI3K signalling activity than TOV-112D cells in vitro and in vivo. Treated TOV-21G xenografts decreased in volume and DW-MRI revealed an increased water diffusivity that was not found in TOV-112D xenografts. Treatment-induced improvement in vascular functionality was detected with DCE-MRI in both models. Changes in vascular morphology were not found. Conclusions: Our results suggest that DW- and DCE-MRI can detect cellular and vascular response to PI3K/mTOR inhibition in vivo. However, only DW-MRI could discriminate between a strong and weak response to BEZ235. PMID:25535727

  3. 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

  4. Class III phosphoinositide 3-kinase--Beclin1 complex mediates the amino acid-dependent regulation of autophagy in C2C12 myotubes.

    PubMed Central

    Tassa, Amina; Roux, Marie Paule; Attaix, Didier; Bechet, Daniel M

    2003-01-01

    Increased proteolysis contributes to muscle atrophy that prevails in many diseases. Elucidating the signalling pathways responsible for this activation is of obvious clinical importance. Autophagy is a ubiquitous degradation process, induced by amino acid starvation, that delivers cytoplasmic components to lysosomes. Starvation markedly stimulates autophagy in myotubes, and the present studies investigate the mechanisms of this regulation. In C(2)C(12) myotubes incubated with serum growth factors, amino acid starvation stimulated autophagic proteolysis independently of p38 and p42/p44 mitogen-activated protein kinases, but in a PI3K (phosphoinositide 3-kinase)-dependent manner. Starvation, however, did not alter activities of class I and class II PI3Ks, and was not sufficient to affect major signalling proteins downstream from class I PI3K (glycogen synthase kinase, Akt/protein kinase B and protein S6). In contrast, starvation increased class III PI3K activity in whole-myotube extracts. In fact, this increase was most pronounced for a population of class III PI3K that coimmunoprecipitated with Beclin1/Apg6 protein, a major determinant in the initiation of autophagy. Stimulation of proteolysis was reproduced by feeding myotubes with synthetic dipalmitoyl-PtdIns3 P, the class III PI3K product. Conversely, protein transfection of anti-class III PI3K inhibitory antibody into starved myotubes inverted the induction of proteolysis. Therefore, independently of class I PI3K/Akt, protein S6 and mitogen-activated protein kinase pathways, amino acid starvation stimulates proteolysis in myotubes by regulating class III PI3K-Beclin1 autophagic complexes. PMID:12967324

  5. Herpesvirus gB-induced fusion between the virion envelope and outer nuclear membrane during virus egress is regulated by the viral US3 kinase.

    PubMed

    Wisner, Todd W; Wright, Catherine C; Kato, Akihisa; Kawaguchi, Yasushi; Mou, Fan; Baines, Joel D; Roller, Richard J; Johnson, David C

    2009-04-01

    Herpesvirus capsids collect along the inner surface of the nuclear envelope and bud into the perinuclear space. Enveloped virions then fuse with the outer nuclear membrane (NM). We previously showed that herpes simplex virus (HSV) glycoproteins gB and gH act in a redundant fashion to promote fusion between the virion envelope and the outer NM. HSV mutants lacking both gB and gH accumulate enveloped virions in herniations, vesicles that bulge into the nucleoplasm. Earlier studies had shown that HSV mutants lacking the viral serine/threonine kinase US3 also accumulate herniations. Here, we demonstrate that HSV gB is phosphorylated in a US3-dependent manner in HSV-infected cells, especially in a crude nuclear fraction. Moreover, US3 directly phosphorylated the gB cytoplasmic (CT) domain in in vitro assays. Deletion of gB in the context of a US3-null virus did not add substantially to defects in nuclear egress. The majority of the US3-dependent phosphorylation of gB involved the CT domain and amino acid T887, a residue present in a motif similar to that recognized by US3 in other proteins. HSV recombinants lacking gH and expressing either gB substitution mutation T887A or a gB truncated at residue 886 displayed substantial defects in nuclear egress. We concluded that phosphorylation of the gB CT domain is important for gB-mediated fusion with the outer NM. This suggested a model in which the US3 kinase is incorporated into the tegument layer (between the capsid and envelope) in HSV virions present in the perinuclear space. By this packaging, US3 might be brought close to the gB CT tail, leading to phosphorylation and triggering fusion between the virion envelope and the outer NM.

  6. Phosphopeptide binding to the N-terminal SH2 domain of the p85 alpha subunit of PI 3'-kinase: a heteronuclear NMR study.

    PubMed Central

    Hensmann, M.; Booker, G. W.; Panayotou, G.; Boyd, J.; Linacre, J.; Waterfield, M.; Campbell, I. D.

    1994-01-01

    The N-terminal src-homology 2 domain of the p85 alpha subunit of phosphatidylinositol 3' kinase (SH2-N) binds specifically to phosphotyrosine-containing sequences. Notably, it recognizes phosphorylated Tyr 751 within the kinase insert of the cytoplasmic domain of the activated beta PDGF receptor. A titration of a synthetic 12-residue phosphopeptide (ESVDY*VPMLDMK) into a solution of the SH2-N domain was monitored using heteronuclear 2D and 3D NMR spectroscopy. 2D-(15N-1H) heteronuclear single-quantum correlation (HSQC) experiments were performed at each point of the titration to follow changes in both 15N and 1H chemical shifts in NH groups. When mapped onto the solution structure of the SH2-N domain, these changes indicate a peptide-binding surface on the protein. Line shape analysis of 1D profiles of individual (15N-1H)-HSQC peaks at each point of the titration suggests a kinetic exchange model involving at least 2 steps. To characterize changes in the internal dynamics of the domain, the magnitude of the (15N-1H) heteronuclear NOE for the backbone amide of each residue was determined for the SH2-N domain with and without bound peptide. These data indicate that, on a nanosecond timescale, there is no significant change in the mobility of either loops or regions of secondary structure. A mode of peptide binding that involves little conformational change except in the residues directly involved in the 2 binding pockets of the p85 alpha SH2-N domain is suggested by this study. PMID:7522724

  7. Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase.

    PubMed

    Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko

    2016-11-01

    Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.

  8. Transduction of Growth or Mitogenic Signals into Translational Activation of TOP mRNAs Is Fully Reliant on the Phosphatidylinositol 3-Kinase-Mediated Pathway but Requires neither S6K1 nor rpS6 Phosphorylation

    PubMed Central

    Stolovich, Miri; Tang, Hua; Hornstein, Eran; Levy, Galit; Cohen, Ruth; Bae, Sun Sik; Birnbaum, Morris J.; Meyuhas, Oded

    2002-01-01

    Translation of terminal oligopyrimidine tract (TOP) mRNAs, which encode multiple components of the protein synthesis machinery, is known to be controlled by mitogenic stimuli. We now show that the ability of cells to progress through the cell cycle is not a prerequisite for this mode of regulation. TOP mRNAs can be translationally activated when PC12 or embryonic stem (ES) cells are induced to grow (increase their size) by nerve growth factor and retinoic acid, respectively, while remaining mitotically arrested. However, both growth and mitogenic signals converge via the phosphatidylinositol 3-kinase (PI3-kinase)-mediated pathway and are transduced to efficiently translate TOP mRNAs. Translational activation of TOP mRNAs can be abolished by LY294002, a PI3-kinase inhibitor, or by overexpression of PTEN as well as by dominant-negative mutants of PI3-kinase or its effectors, PDK1 and protein kinase Bα (PKBα). Likewise, overexpression of constitutively active PI3-kinase or PKBα can relieve the translational repression of TOP mRNAs in quiescent cells. Both mitogenic and growth signals lead to phosphorylation of ribosomal protein S6 (rpS6), which precedes the translational activation of TOP mRNAs. Nevertheless, neither rpS6 phosphorylation nor its kinase, S6K1, is essential for the translational response of these mRNAs. Thus, TOP mRNAs can be translationally activated by growth or mitogenic stimuli of ES cells, whose rpS6 is constitutively unphosphorylated due to the disruption of both alleles of S6K1. Similarly, complete inhibition of mammalian target of rapamycin (mTOR) and its effector S6K by rapamycin in various cell lines has only a mild repressive effect on the translation of TOP mRNAs. It therefore appears that translation of TOP mRNAs is primarily regulated by growth and mitogenic cues through the PI3-kinase pathway, with a minor role, if any, for the mTOR pathway. PMID:12417714

  9. Dehydroepiandrosterone Stimulates Phosphorylation of FoxO1 in Vascular Endothelial Cells via Phosphatidylinositol 3-Kinase- and Protein Kinase A-dependent Signaling Pathways to Regulate ET-1 Synthesis and Secretion*

    PubMed Central

    Chen, Hui; Lin, Alice Seraphina; Li, Yunhua; Reiter, Chad E. N.; Ver, Maria R.; Quon, Michael J.

    2008-01-01

    Dehydroepiandrosterone (DHEA) is an endogenous adrenal steroid hormone with controversial actions in humans. We previously reported that DHEA has opposing actions in endothelial cells to stimulate phosphatidylinositol (PI) 3-kinase/Akt/endothelial nitric-oxide synthase leading to increased production of nitric oxide while simultaneously stimulating MAPK-dependent secretion of the vasoconstrictor ET-1. In the present study we hypothesized that DHEA may stimulate PI 3-kinase-dependent phosphorylation of FoxO1 in endothelial cells to help regulate endothelial function. In bovine or human aortic endothelial cells (BAEC and HAEC), treatment with DHEA (100 nm) acutely enhanced phosphorylation of FoxO1. DHEA-stimulated phosphorylation of FoxO1 was inhibited by pretreatment of cells with wortmannin (PI 3-kinase inhibitor) or H89 (protein kinase A (PKA) inhibitor) but not ICI182780 (estrogen receptor blocker), or PD98059 (MEK (MAPK/extracellular signal-regulated kinase kinase) inhibitor). Small interfering RNA knockdown of PKA inhibited DHEA-stimulated phosphorylation of FoxO1. DHEA promoted nuclear exclusion of FoxO1 that was blocked by pretreatment of cells with wortmannin, H89, or by small interfering RNA knockdown of PKA. DHEA treatment of endothelial cells increased PKA activity and intracellular cAMP concentrations. Transfection of BAEC with a constitutively nuclear FoxO1 mutant transactivated a co-transfected ET-1 promoter luciferase reporter. Treatment of BAEC with DHEA inhibited transactivation of the ET-1 promoter reporter in cells overexpressing FoxO1. ET-1 promoter activity and secretion in response to DHEA treatment was augmented by PI 3-kinase blockade and inhibited by MAPK blockade. We conclude that DHEA stimulates phosphorylation of FoxO1 via PI 3-kinase- and PKA-dependent pathways in endothelial cells that negatively regulates ET-1 promoter activity and secretion. Balance between PI 3-kinase-dependent inhibition and MAPK-dependent stimulation of ET-1

  10. 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…

  11. Regulation of sphingosine 1-phosphate-induced endothelial cytoskeletal rearrangement and barrier enhancement by S1P1 receptor, PI3 kinase, Tiam1/Rac1, and alpha-actinin.

    PubMed

    Singleton, Patrick A; Dudek, Steven M; Chiang, Eddie T; Garcia, Joe G N

    2005-10-01

    Endothelial cell (EC) barrier dysfunction results in increased vascular permeability observed in inflammation, tumor angiogenesis, and atherosclerosis. The platelet-derived phospholipid sphingosine-1-phosphate (S1P) decreases EC permeability in vitro and in vivo and thus has obvious therapeutic potential. We examined S1P-mediated human pulmonary artery EC signaling and barrier regulation in caveolin-enriched microdomains (CEM). Immunoblotting from S1P-treated EC revealed S1P-mediated rapid recruitment (1 microM, 5 min) to CEMs of the S1P receptors S1P1 and S1P3, p110 PI3 kinase alpha and beta catalytic subunits, the Rac1 GEF, Tiam1, and alpha-actinin isoforms 1 and 4. Immunoprecipitated p110 PI3 kinase catalytic subunits from S1P-treated EC exhibited PIP3 production in CEMs. Immunoprecipitation of S1P receptors from CEM fractions revealed complexes containing Tiam1 and S1P1. PI3 kinase inhibition (LY294002) attenuated S1P-induced Tiam1 association with S1P1, Tiam1/Rac1 activation, alpha-actinin-1/4 recruitment, and EC barrier enhancement. Silencing of either S1P1 or Tiam1 expression resulted in the loss of S1P-mediated Rac1 activation and alpha-actinin-1/4 recruitment to CEM. Finally, silencing S1P1, Tiam1, or both alpha-actinin isoforms 1/4 inhibits S1P-induced cortical F-actin rearrangement and S1P-mediated barrier enhancement. Taken together, these results suggest that S1P-induced recruitment of S1P1 to CEM fractions promotes PI3 kinase-mediated Tiam1/Rac1 activation required for alpha-actinin-1/4-regulated cortical actin rearrangement and EC barrier enhancement.

  12. 17β-estradiol impedes Bax-involved mitochondrial apoptosis of retinal nerve cells induced by oxidative damage via the phosphatidylinositol 3-kinase/Akt signal pathway.

    PubMed

    Li, Hongbo; Wang, Baoying; Zhu, Chunhui; Feng, Yan; Wang, Shaolan; Shahzad, Muhammad; Hu, Chenghu; Mo, Mingshu; Du, Fangying; Yu, Xiaorui

    2013-07-01

    Oxidative stress leading to retinal nerve cells (RNCs) apoptosis is a major cause of neurodegenerative disorders of the retina. 17β-Estradiol (E2) has been suggested to be a neuroprotective agent in the central nervous system; however, at present, the underlying mechanisms are not well understood, and the related research on the RNCs is less reported. Here, in order to investigate the protective role and mechanism of E2 against oxidative stress-induced damage on RNCs, the transmission electron microscopy and annexin V-FITC/propidium iodide assay were applied to detect the RNCs apoptosis. Western blot and real-time PCR were used to determine the expression of the critical molecules in Bcl-2 and caspase family associated with apoptosis. The transmission electron microscopy results showed that H(2)O(2) could induce typical features of apoptosis in RNCs, including formation of the apoptosome. E2 could, however, suppress the H(2)O(2)-induced morphological changes of apoptosis. Intriguingly, we observed E2-mediated phagocytic scavenging of apoptosome. In response to H(2)O(2)-induced apoptosis, Bax, acting as one of the pivotal pro-apoptotic members of Bcl-2 family, increased significantly, which directly resulted in an increased ratio of Bax to anti-apoptotic protein Bcl-2 (Bax/Bcl-2). Additionally, caspases 9 and 3, which are the critical molecules of the mitochondrial apoptosis pathway, were activated by H(2)O(2). In contrast, E2 exerted anti-apoptotic effects by reducing the expression of Bax to decrease the ratio of Bax/Bcl-2 and impeded the caspases 9/3 activation. Moreover, LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, could sharply block the effect of E2 in reducing the percentage of apoptotic cells resistance to H(2)O(2). And the attenuation of Bax, the reduced activities of caspases 9/3 and the impeded release of mitochondrial cytochrome c mediated by E2 resistance to H(2)O(2) damage were significantly retrieved by LY294002 administration. Taken

  13. Phosphatidylinositide 3-kinase (PI3K) and PI3K-related kinase (PIKK) activity contributes to radioresistance in thyroid carcinomas

    PubMed Central

    Burrows, Natalie; Williams, Joseph; Telfer, Brian A; Resch, Julia; Valentine, Helen R; Fitzmaurice, Richard J; Eustace, Amanda; Irlam, Joely; Rowling, Emily J; Hoang-Vu, Cuong; West, Catharine M; Brabant, Georg; Williams, Kaye J

    2016-01-01

    Anaplastic (ATC) and certain follicular thyroid-carcinomas (FTCs) are radioresistant. The Phosphatidylinositide 3-kinase (PI3K) pathway is commonly hyperactivated in thyroid-carcinomas. PI3K can modify the PI3K-related kinases (PIKKs) in response to radiation: How PIKKs interact with PI3K and contribute to radioresistance in thyroid-carcinomas is unknown. Further uncertainties exist in how these interactions function under the radioresistant hypoxic microenvironment. Under normoxia/anoxia, ATC (8505c) and FTC (FTC-133) cells were irradiated, with PI3K-inhibition (via GDC-0941 and PTEN-reconstitution into PTEN-null FTC-133s) and effects on PIKK-activation, DNA-damage, clonogenic-survival and cell cycle, assessed. FTC-xenografts were treated with 5 × 2 Gy, ± 50 mg/kg GDC-0941 (twice-daily; orally) for 14 days and PIKK-activation and tumour-growth assessed. PIKK-expression was additionally assessed in 12 human papillary thyroid-carcinomas, 13 FTCs and 12 ATCs. GDC-0941 inhibited radiation-induced activation of Ataxia-telangiectasia mutated (ATM), ATM-and Rad3-related (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Inhibition of ATM and DNA-PKcs was PI3K-dependent, since activation was reduced in PTEN-reconstituted FTC-133s. Inhibition of PIKK-activation was greater under anoxia: Consequently, whilst DNA-damage was increased and prolonged under both normoxia and anoxia, PI3K-inhibition only reduced clonogenic-survival under anoxia. GDC-0941 abrogated radiation-induced cell cycle arrest, an effect most likely linked to the marked inhibition of ATR-activation. Importantly, GDC-0941 inhibited radiation-induced PIKK-activation in FTC-xenografts leading to a significant increase in time taken for tumours to triple in size: 26.5 ± 5 days (radiation-alone) versus 31.5 ± 5 days (dual-treatment). PIKKs were highly expressed across human thyroid-carcinoma classifications, with ATM scoring consistently lower. Interestingly, some loss of ATM and DNA

  14. 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.

  15. Inhibitory Role of α6β4-Associated Erbb-2 and Phosphoinositide 3-Kinase in Keratinocyte Haptotactic Migration Dependent on α3β1 Integrin

    PubMed Central

    Hintermann, Edith; Bilban, Martin; Sharabi, Andrew; Quaranta, Vito

    2001-01-01

    Keratinocytes and other epithelial cells express two receptors for the basement membrane (BM) extracellular matrix component laminin-5 (Ln-5), integrins α3β1 and α6β4. While α3β1 mediates adhesion, spreading, and migration (Kreidberg, J.A. 2000. Curr. Opin. Cell Biol. 12:548–553), α6β4 is involved in BM anchorage via hemidesmosomes (Borradori, L., and A. Sonnenberg. 1999. J. Invest. Dermatol. 112:411–418). We investigated a possible regulatory interplay between α3β1 and α6β4 in cell motility using HaCaT keratinocytes as a model. We found that α6β4 antibodies inhibit α3β1-mediated migration on Ln-5, but only when migration is haptotactic (i.e., spontaneous or stimulated by α3β1 activation), and not when chemotactic (i.e., triggered by epidermal growth factor receptor). Inhibition of migration by α6β4 depends upon phosphoinositide 3-kinase (PI3-K) since it is abolished by PI3-K blockers and by dominant-negative PI3-K, and constitutively active PI3-K prevents haptotaxis. In HaCaT cells incubated with anti–α6β4 antibodies, activation of PI3-K is mediated by α6β4-associated erbB-2, as indicated by erbB-2 autophosphorylation and erbB-2/p85 PI3-K coprecipitation. Furthermore, dominant-negative erbB-2 abolishes inhibition of haptotaxis by anti–α6β4 antibodies. These results support a model whereby (a) haptotactic cell migration on Ln-5 is regulated by concerted action of α3β1 and α6β4 integrins, (b) α6β4-associated erbB-2 and PI3-K negatively affect haptotaxis, and (c) chemotaxis on Ln-5 is not affected by α6β4 antibodies and may require PI3-K activity. This model could be of general relevance to motility of epithelial cells in contact with BM. PMID:11331299

  16. Participation of phosphatidyl inositol 3-kinase/protein kinase B and ERK1/2 pathways in interleukin-1beta stimulation of lactate production in Sertoli cells.

    PubMed

    Riera, María Fernanda; Galardo, María Noel; Pellizzari, Eliana Herminia; Meroni, Silvina Beatriz; Cigorraga, Selva Beatriz

    2007-04-01

    Interleukin-1beta (IL1beta ) belongs to a set of intratesticular regulators that provide the fine-tuning of cellular processes implicated in the maintenance of spermatogenesis. The aim of the present study was to analyze the signaling pathways that may participate in IL1beta regulation of Sertoli cell function. Sertoli cell cultures from 20-day-old rat were used. Stimulation of the cultures with IL1beta showed increments in phosphorylated protein kinase B (PKB), P70S6K, and ERK1/2 levels. A phosphatidyl inositol 3-kinase (PI3K) inhibitor (wortmannin (W)), a mammalian target of rapamycin inhibitor (rapamycin (R)), and a MEK inhibitor (PD98059 (PD)) were utilized to evaluate the participation of PI3K/PKB, P70S6K, and ERK1/2 pathways in the regulation of lactate production by IL1beta . PD and W, but not R, decreased IL1beta-stimulated lactate production. The participation of these pathways in the regulation of glucose uptake and lactate dehydrogenase (LDH) A mRNA levels by IL1beta was also analyzed. It was observed that W decreased IL1beta-stimulated glucose uptake, whereas PD and R did not modify it. On the other hand, PD decreased the stimulation of LDH A mRNA levels by IL1beta , whereas W and R did not modify it. In summary, results presented herein demonstrate that IL1beta stimulates PI3K/PKB-, P70S6K-, and ERK1/2-dependent pathways in rat Sertoli cells. Moreover, these results show that while IL1beta utilizes the PI3K/PKB pathway to regulate glucose transport, it utilizes the ERK1/2 pathway to regulate LDH A mRNA levels. This study reveals that IL1beta utilizes different signal transduction pathways to modify the biochemical steps that are important to regulate lactate production in rat Sertoli cells.

  17. Selective inhibitors of phosphoinositide 3-kinase delta: modulators of B-cell function with potential for treating autoimmune inflammatory diseases and B-cell malignancies

    PubMed Central

    Puri, Kamal D.; Gold, Michael R.

    2012-01-01

    The delta isoform of the p110 catalytic subunit (p110δ) of phosphoinositide 3-kinase is expressed primarily in hematopoietic cells and plays an essential role in B-cell development and function. Studies employing mice lacking a functional p110δ protein, as well as the use of highly-selective chemical inhibitors of p110δ, have revealed that signaling via p110δ-containing PI3K complexes (PI3Kδ) is critical for B-cell survival, migration, and activation, functioning downstream of key receptors on B cells including the B-cell antigen receptor, chemokine receptors, pro-survival receptors such as BAFF-R and the IL-4 receptor, and co-stimulatory receptors such as CD40 and Toll-like receptors (TLRs). Similarly, this PI3K isoform plays a key role in the survival, proliferation, and dissemination of B-cell lymphomas. Herein we summarize studies showing that these processes can be inhibited in vitro and in vivo by small molecule inhibitors of p110δ enzymatic activity, and that these p110δ inhibitors have shown efficacy in clinical trials for the treatment of several types of B-cell malignancies including chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). PI3Kδ also plays a critical role in the activation, proliferation, and tissue homing of self-reactive B cells that contribute to autoimmune diseases, in particular innate-like B-cell populations such as marginal zone (MZ) B cells and B-1 cells that have been strongly linked to autoimmunity. We discuss the potential utility of p110δ inhibitors, either alone or in combination with B-cell depletion, for treating autoimmune diseases such as lupus, rheumatoid arthritis, and type 1 diabetes. Because PI3Kδ plays a major role in both B-cell-mediated autoimmune inflammation and B-cell malignancies, PI3Kδ inhibitors may represent a promising therapeutic approach for treating these diseases. PMID:22936933

  18. Combined blockade of ADP receptors and PI3-kinase p110β fully prevents platelet and leukocyte activation during hypothermic extracorporeal circulation.

    PubMed

    Krajewski, Stefanie; Kurz, Julia; Geisler, Tobias; Peter, Karlheinz; Wendel, Hans Peter; Straub, Andreas

    2012-01-01

    Extracorporeal circulation (ECC) and hypothermia are used to maintain stable circulatory parameters and improve the ischemia tolerance of patients in cardiac surgery. However, ECC and hypothermia induce activation mechanisms in platelets and leukocytes, which are mediated by the platelet agonist ADP and the phosphoinositide-3-kinase (PI3K) p110β. Under clinical conditions these processes are associated with life-threatening complications including thromboembolism and inflammation. This study analyzes effects of ADP receptor P(2)Y(12) and P(2)Y(1) blockade and PI3K p110β inhibition on platelets and granulocytes during hypothermic ECC. Human blood was treated with the P(2)Y(12) antagonist 2-MeSAMP, the P(2)Y(1) antagonist MRS2179, the PI3K p110β inhibitor TGX-221, combinations thereof, or PBS and propylene glycol (controls). Under static in vitro conditions a concentration-dependent effect regarding the inhibition of ADP-induced platelet activation was found using 2-MeSAMP or TGX-221. Further inhibition of ADP-mediated effects was achieved with MRS2179. Next, blood was circulated in an ex vivo ECC model at 28°C for 30 minutes and various platelet and granulocyte markers were investigated using flow cytometry, ELISA and platelet count analysis. GPIIb/IIIa activation induced by hypothermic ECC was inhibited using TGX-221 alone or in combination with P(2)Y blockers (p<0.05), while no effect of hypothermic ECC or antiplatelet agents on GPIIb/IIIa and GPIbα expression and von Willebrand factor binding was observed. Sole P(2)Y and PI3K blockade or a combination thereof inhibited P-selectin expression on platelets and platelet-derived microparticles during hypothermic ECC (p<0.05). P(2)Y blockade alone or combined with TGX-221 prevented ECC-induced platelet-granulocyte aggregate formation (p<0.05). Platelet adhesion to the ECC surface, platelet loss and Mac-1 expression on granulocytes were inhibited by combined P(2)Y and PI3K blockade (p<0.05). Combined blockade of P

  19. Role of the phosphatidylinositol 3-kinase/Akt and mTOR/P70S6-kinase pathways in the proliferation and apoptosis in multiple myeloma.

    PubMed

    Pene, Frédéric; Claessens, Yann-Erick; Muller, Odile; Viguié, Franck; Mayeux, Patrick; Dreyfus, François; Lacombe, Catherine; Bouscary, Didier

    2002-09-26

    Multiple myeloma (MM) is a plasma cell malignancy preliminary localized in the bone marrow and characterized by its capacity to disseminate. IL-6 and IGF-1 have been shown to mediate proliferative and anti-apoptotic signals in plasmocytes. However, in primary plasma-cell leukemia (PCL) and in end-stage aggressive extramedullar disease, the cytokine requirement for both effects may be not mandatory. This suggests that constitutive activation of signaling pathways occurs. One of the signaling pathways whose deregulation may play an oncogenic role in MM is the phosphatidylinositol 3-kinase (PI 3-K) pathway. In human growth factor-independent MM cell lines OPM2 and RPMI8226, we show that the PI 3-K inhibitors LY294002 and Wortmannin strongly inhibited cell proliferation, whereas inhibition of the mammalian Target Of Rapamycin (mTOR)/P70-S6-kinase (P70(S6K)) pathway with rapamycin or of the Mitogen-Activated Protein Kinase (MAPK) pathway with PD98059 had minimal effect on proliferation. In both cell lines, constitutive activation of the PI 3-K/Akt/FKHRL-1, mTOR/P70(S6K) and MAPK pathways was detected. LY294002 inhibited phosphorylation of Akt, FKHRL-1 and P70(S6K) but had no effect on ERK1/2 phosphorylation, indicating that the PI 3-K and MAPK pathways are independent. IGF-1 but not IL-6 increased phosphorylation of Akt, FKHRL-1 and P70(S6K). Purified plasmocytes from four patients with MM and two patients with primary PCL were studied. In three of them including the two patients with PCL, constitutive phosphorylation of Akt, FKHRL-1 and P70(S6K) was present, inhibited by LY294002 and enhanced by IGF-1. In these patients with constitutive Akt activation, normal PTEN expression was detected. PI 3-K inhibition induced caspase-dependent apoptosis as confirmed by inhibition with the large spectrum caspase inhibitor Z-VAD-FMK and cleavage of pro-caspase-3. Both cell lines spontaneously expressed Skp2 and cyclin D1 proteins at high levels but no p27(Kip1) protein. In the

  20. Differential Phosphatidylinositol-3-Kinase-Akt-mTOR Activation by Semliki Forest and Chikungunya Viruses Is Dependent on nsP3 and Connected to Replication Complex Internalization

    PubMed Central

    Biasiotto, Roberta; Eng, Kai; Neuvonen, Maarit; Götte, Benjamin; Rheinemann, Lara; Mutso, Margit; Utt, Age; Varghese, Finny; Balistreri, Giuseppe; Merits, Andres; Ahola, Tero; McInerney, Gerald M.

    2015-01-01

    ABSTRACT Many viruses affect or exploit the phosphatidylinositol-3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway, a crucial prosurvival signaling cascade. We report that this pathway was strongly activated in cells upon infection with the Old World alphavirus Semliki Forest virus (SFV), even under conditions of complete nutrient starvation. We mapped this activation to the hyperphosphorylated/acidic domain in the C-terminal tail of SFV nonstructural protein nsP3. Viruses with a deletion of this domain (SFV-Δ50) but not of other regions in nsP3 displayed a clearly delayed and reduced capacity of Akt stimulation. Ectopic expression of the nsP3 of SFV wild type (nsP3-wt), but not nsP3-Δ50, equipped with a membrane anchor was sufficient to activate Akt. We linked PI3K-Akt-mTOR stimulation to the intracellular dynamics of viral replication complexes, which are formed at the plasma membrane and subsequently internalized in a process blocked by the PI3K inhibitor wortmannin. Replication complex internalization was observed upon infection of cells with SFV-wt and SFV mutants with deletions in nsP3 but not with SFV-Δ50, where replication complexes were typically accumulated at the cell periphery. In cells infected with the closely related chikungunya virus (CHIKV), the PI3K-Akt-mTOR pathway was only moderately activated. Replication complexes of CHIKV were predominantly located at the cell periphery. Exchanging the hypervariable C-terminal tail of nsP3 between SFV and CHIKV induced the phenotype of strong PI3K-Akt-mTOR activation and replication complex internalization in CHIKV. In conclusion, infection with SFV but not CHIKV boosts PI3K-Akt-mTOR through the hyperphosphorylated/acidic domain of nsP3 to drive replication complex internalization. IMPORTANCE SFV and CHIKV are very similar in terms of molecular and cell biology, e.g., regarding replication and molecular interactions, but are strikingly different regarding pathology: CHIKV is a relevant human

  1. 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.

  2. Role of Protein Kinase C, PI3-kinase and Tyrosine Kinase in Activation of MAP Kinase by Glucose and Agonists of G-protein Coupled Receptors in INS-1 Cells

    PubMed Central

    Böcker, Dietmar

    2001-01-01

    MAP (mitogen-activated protein) kinase (also called Erk 1/2) plays a crucial role in cell proliferation and differentiation. Its impact on secretory events is less well established. The interplay of protein kinase C (PKC), PI3-kinase nd cellular tyrosine kinase with MAP kinase activity using inhibitors and compounds such as glucose, phorbol 12-myristate 13-acetate (PMA) and agonists of G-protein coupled receptors like gastrin releasing peptide (GRP), oxytocin (OT) and glucose-dependent insulinotropic peptide (GIP) was investigated in INS-1 cells, an insulin secreting cell line. MAP kinase activity was determined by using a peptide derived from the EGF receptor as a MAP kinase substrate and [ P 32 ]ATP. Glucose as well as GRP, OT and GIP exhibited a time-dependent increase in MAP kinase activity with a maximum at time point 2.5 min. All further experiments were performed using 2.5 min incubations. The flavone PD 098059 is known to bind to the inactive forms of MEK1 (MAPK/ERK-Kinase) thus preventing activation by upstream activators. 20 μM PD 098059 ( IC 50 =51 μM) inhibited MAP kinase stimulated by either glucose, GRP, OT, GIP or PMA. Inhibiton (“downregulation”) of PKC by a long term (22h) pretreatment with 1 μM PMA did not influence MAP kinase activity when augmented by either of the above mentioned compound. To investigate whether PI3-kinase and cellular tyrosine kinase are involved in G-protein mediated effects on MAP kinase, inhibitors were used: 100 nM wortmannin (PI3-kinase inhibitor) reduced the effects of GRP, OT and GIP but not that of PMA; 100 μM genistein (tyrosine kinase inhibitor) inhibited the stimulatory effect of either above mentioned compound on MAP kinase activation. Inhibition of MAP kinase by 20 μM PD 098059 did not influence insulin secretion modulated by either compound (glucose, GRP, OT or GIP). [ H 3 ]Thymidine incorporation, however, was severely inhibited by PD 098059. Thus MAP kinase is important for INS-1 cell proliferation but

  3. The Development of Novel Small Molecule Inhibitors of the Phosphoinositide- 3-Kinase Pathway through High-Throughput Cell-Based Screens

    DTIC Science & Technology

    2006-02-01

    1997, the tumor suppressor gene PTEN was cloned from the 10q23 region, a region frequently targeted by loss of heterozygosity (LOH) in advanced...compromised as a result of lymphoproliferation and tumors of intestines, mammary, thyroid, endome - trial and adrenal glands. Thus, it has been difficult to

  4. Mutation of Tyr697, a GRB2-binding site, and Tyr721, a PI 3-kinase binding site, abrogates signal transduction by the murine CSF-1 receptor expressed in Rat-2 fibroblasts.

    PubMed Central

    van der Geer, P; Hunter, T

    1993-01-01

    The receptor for the myeloid cell growth factor colony stimulating factor 1 (CSF-1) is a protein tyrosine kinase that is closely related to the PDGF receptor. Ligand binding results in kinase activation and autophosphorylation. Three autophosphorylation sites, Tyr697, Tyr706 and Tyr721, have been mapped to the kinase insert domain. Deletion of the entire kinase insert domain completely abrogates signal transduction by the CSF-1 receptor expressed in Rat-2 fibroblasts. To investigate the function of individual phosphorylation sites present in the CSF-1 receptor kinase insert domain, a number of phosphorylation site mutants were expressed in Rat-2 fibroblasts. Mutation of either Tyr697 or Tyr721 compromised signal transduction by the CSF-1 receptor. A mutant receptor, in which both Tyr697 and Tyr721 were replaced by phenylalanine, has lost all ability to induce changes in morphology or to increase cell growth rate in response to CSF-1. Tyr721 has been identified recently as the binding site for PI 3-kinase. Here we report that GRB2 associates with the CSF-1 receptor upon ligand binding. The phosphorylation on tyrosine of SHC and several other GRB2-associated proteins increased upon stimulation with CSF-1. Tyr697 was identified as a binding site for GRB2. We suggest that PI 3-kinase, GRB2 and some of the GRB2-associated proteins could play an important role in signal transduction by the CSF-1 receptor. Images PMID:8262059

  5. The Development of Novel Small Molecule Inhibitors of the Phosphoinositide- 3-Kinase Pathway through High-Throughput Cell-Based Screens

    DTIC Science & Technology

    2007-02-01

    pipettemodified Eagle’s medium (DMEM) containing 10% Fetal Clone (Clonetech) (ThermoLabsystems) in DMEM. Media were aspirated from infected cellsand 100 g/ml...according to the manufacturer’s protocol Fixed cells were then stained with M5 anti-FLAG antibody (Sigma) followed(Boehinger Mannheim). Stable clones ...Sellers and Sawyers, 2002). In 1997, the tumor suppressor gene PTEN was cloned from the 10q23 region, a region frequently targeted by loss of

  6. Novel Role for p110β PI 3-Kinase in Male Fertility through Regulation of Androgen Receptor Activity in Sertoli Cells

    PubMed Central

    Guillermet-Guibert, Julie; Smith, Lee B.; Halet, Guillaume; Whitehead, Maria A.; Pearce, Wayne; Rebourcet, Diane; León, Kelly; Crépieux, Pascale; Nock, Gemma; Strömstedt, Maria; Enerback, Malin; Chelala, Claude; Graupera, Mariona; Carroll, John; Cosulich, Sabina; Saunders, Philippa T. K.; Huhtaniemi, Ilpo; Vanhaesebroeck, Bart

    2015-01-01

    The organismal roles of the ubiquitously expressed class I PI3K isoform p110β remain largely unknown. Using a new kinase-dead knockin mouse model that mimics constitutive pharmacological inactivation of p110β, we document that full inactivation of p110β leads to embryonic lethality in a substantial fraction of mice. Interestingly, the homozygous p110β kinase-dead mice that survive into adulthood (maximum ~26% on a mixed genetic background) have no apparent phenotypes, other than subfertility in females and complete infertility in males. Systemic inhibition of p110β results in a highly specific blockade in the maturation of spermatogonia to spermatocytes. p110β was previously suggested to signal downstream of the c-kit tyrosine kinase receptor in germ cells to regulate their proliferation and survival. We now report that p110β also plays a germ cell-extrinsic role in the Sertoli cells (SCs) that support the developing sperm, with p110β inactivation dampening expression of the SC-specific Androgen Receptor (AR) target gene Rhox5, a homeobox gene critical for spermatogenesis. All extragonadal androgen-dependent functions remain unaffected by global p110β inactivation. In line with a crucial role for p110β in SCs, selective inactivation of p110β in these cells results in male infertility. Our study is the first documentation of the involvement of a signalling enzyme, PI3K, in the regulation of AR activity during spermatogenesis. This developmental pathway may become active in prostate cancer where p110β and AR have previously been reported to functionally interact. PMID:26132308

  7. The PI3 kinase, p38 SAP kinase, and NF-kappaB signal transduction pathways are involved in the survival and maturation of lipopolysaccharide-stimulated human monocyte-derived dendritic cells.

    PubMed

    Ardeshna, K M; Pizzey, A R; Devereux, S; Khwaja, A

    2000-08-01

    As a dendritic cell (DC) matures, it becomes more potent as an antigen-presenting cell. This functional change is accompanied by a change in DC immunophenotype. The signal transduction events underlying this process are poorly characterized. In this study, we have investigated the signal transduction pathways involved in the lipopolysaccharide (LPS)-induced maturation of human monocyte-derived DCs (MoDCs) in vitro. We show that exposure of immature MoDCs to LPS activates the p38 stress-activated protein kinase (p38SAPK), extracellular signal-regulated protein kinase (ERK), phosphoinositide 3-OH kinase (PI3 kinase)/Akt, and nuclear factor (NF)-kappaB pathways. Studies using inhibitors demonstrate that PI3 kinase/Akt but not the other pathways are important in maintaining survival of LPS-stimulated MoDCs. Inhibiting p38SAPK prevented activation of the transcription factors ATF-2 and CREB and significantly reduced the LPS-induced up-regulation of CD80, CD83, and CD86, but did not have any significant effect on the LPS-induced changes in macropinocytosis or HLA-DR, CD40, and CD1a expression. Inhibiting the NF-kappaB pathway significantly reduced the LPS-induced up-regulation of HLA-DR as well as CD80, CD83, and CD86. Inhibiting the p38SAPK and NF-kappaB pathways simultaneously had variable effects depending on the cell surface marker studied. It thus appears that different aspects of LPS-induced MoDC maturation are regulated by different and sometimes overlapping pathways.

  8. Tannin 1-alpha-O-galloylpunicalagin induces the calcium-dependent activation of endothelial nitric-oxide synthase via the phosphatidylinositol 3-kinase/Akt pathway in endothelial cells.

    PubMed

    Chen, Lih-Geeng; Liu, Yen-Chin; Hsieh, Chia-Wen; Liao, Being-Chyuan; Wung, Being-Sun

    2008-10-01

    Many polyphenols have been found to increase endothelial nitric oxide (NO) production. In our present study, we investigated the effects of 1-alpha-O-galloylpunicalagin upon endothelial nitric oxide synthase (eNOS) activity in endothelial cells (ECs). Both 1-alpha-O-galloylpunicalagin and punicalagin induced NO production in a dose-dependent manner in ECs. Despite having similar chemical structures, punicalagin induced lower levels of NO production than 1-alpha-O-galloylpunicalagin. After 1-alpha-O-galloylpunicalagin addition, a rise in the intracellular Ca(2+) concentration preceded NO production. The Ca(2+) ionophore A23187 stimulated eNOS phosphorylation and augmented NO production. Pretreatment with Ca(2+) chelators inhibited 1-alpha-O-galloylpunicalagin-induced eNOS phosphorylation and NO production. Treatment with 1-alpha-O-galloylpunicalagin did not alter the eNOS protein levels but, unlike punicalagin, induced a sustained activation of eNOS Ser(1179) phosphorylation. 1-alpha-O-galloylpunicalagin was also found to activate ERK1/2, JNK and Akt in ECs. Moreover, simultaneous treatment of these cells with specific phosphatidylinositol-3-kinase inhibitors significantly inhibited the observed increases in eNOS activity and phosphorylation levels. In contrast, the inhibition of (ERK)1/2, JNK and p38 had no influence on eNOS Ser(1179) phosphorylation. Our present results thus indicate that the 1-alpha-O-galloylpunicalagin-induced calcium-dependent activation of eNOS is primarily mediated via a phosphatidylinositol 3-kinase/Akt-dependent increase in eNOS activity, and occurs independently of the eNOS protein content.

  9. Follicle-stimulating hormone-induced aromatase in immature rat Sertoli cells requires an active phosphatidylinositol 3-kinase pathway and is inhibited via the mitogen-activated protein kinase signaling pathway.

    PubMed

    McDonald, Claudia A; Millena, Ana C; Reddy, Sheila; Finlay, Sheila; Vizcarra, Jorge; Khan, Shafiq A; Davis, John S

    2006-03-01

    Postnatal development and function of testicular Sertoli cells are regulated primarily by FSH. During this early period of development, estrogens play a role in proliferation of somatic cells, which contributes significantly to testicular development. Growth factors like epidermal growth factor (EGF) are produced in the testis and play a role in regulation of estradiol production and male fertility. Although these divergent factors modulate gonadal function, little is known about their mechanism of action in Sertoli cells. The present study investigates the intracellular events that take place down-stream of FSH and EGF receptors in Sertoli cells isolated from immature (10-d-old) rats, and examines which intracellular signals may be involved in their effects on aromatase activity and estradiol production in immature rat Sertoli cells. Primary cultures of rat Sertoli cells were treated with FSH in combination with EGF and signaling pathway-specific inhibitors. Levels of estradiol production, aromatase mRNA (Cyp19a1), and aromatase protein (CYP19A1) were determined. Western blot analysis was performed to determine the effects of FSH and EGF on levels of activated (phosphorylated) AKT1 and p42 ERK2 and p44 ERK1, also named MAPK1 and MAPK3, respectively. The stimulatory actions of FSH on aromatase mRNA, aromatase protein, and estradiol production were blocked by inhibition of the phosphatidylinositol 3-kinase/AKT1 signaling pathway. In contrast, inhibition of ERK signaling augmented the stimulatory effects of FSH on estradiol production, aromatase mRNA, and protein levels. Furthermore, EGF inhibited the expression of aromatase mRNA and protein in response to FSH, and these inhibitory effects of EGF were critically dependent on the activation of the ERK signaling pathway. We conclude that an active phosphatidylinositol 3-kinase /AKT signaling pathway is required for the stimulatory actions of FSH, whereas an active ERK/MAPK pathway inhibits estradiol production and

  10. Active β-catenin is regulated by the PTEN/PI3 kinase pathway: a role for protein phosphatase PP2A

    PubMed Central

    Persad, Amit; Venkateswaran, Geetha; Hao, Li; Garcia, Maria E.; Yoon, Jenny; Sidhu, Jaskiran; Persad, Sujata

    2016-01-01

    Dysregulation of Wnt/β-catenin signaling has been associated with the development and progression of many cancers. The stability and subcellular localization of β-catenin, a dual functional protein that plays a role in intracellular adhesion and in regulating gene expression, is tightly regulated. However, little is known about the transcriptionally active form of β-catenin, Active Beta Catenin (ABC), that is unphosphorylated at serine 37 (Ser37) and threonine 41 (Thr41). Elucidating the mechanism by which β-catenin is activated to generate ABC is vital to the development of therapeutic strategies to block β-catenin signaling for cancer treatment. Using melanoma, breast and prostate cancer cell lines, we show that while cellular β-catenin levels are regulated by the Wnt pathway, cellular ABC levels are mainly regulated by the PI3K pathway and are dependent on the phosphatase activity of the protein phosphatase PP2A. Furthermore, we demonstrate that although the PI3K/PTEN pathway does not regulate total β-catenin protein levels within the cell, it plays a role in regulating the subcellular localization of β-catenin. Our results support a novel functional interaction/cross-talk between the PTEN/PI3K and Wnt pathways in the regulation of the subcellular/nuclear levels of ABC, which is crucially important for the protein's activity as a transcription factor and its biological effects in health and disease. PMID:28191283

  11. Characterization of mutations of the phosphoinositide-3-kinase regulatory subunit, PIK3R2, in perisylvian polymicrogyria: a next generation sequencing study

    PubMed Central

    Mirzaa, Ghayda; Conti, Valerio; Timms, Andrew E.; Smyser, Christopher D.; Ahmed, Sarah; Carter, Melissa; Barnett, Sarah; Hufnagel, Robert B.; Goldstein, Amy; Narumi-Kishimoto, Yoko; Olds, Carissa; Collins, Sarah; Johnston, Kathreen; Deleuze, Jean-François; Nitschké, Patrick; Friend, Kathryn; Harris, Catharine; Goetsch, Allison; Martin, Beth; Boyle, Evan August; Parrini, Elena; Mei, Davide; Tattini, Lorenzo; Slavotinek, Anne; Blair, Ed; Barnett, Christopher; Shendure, Jay; Chelly, Jamel; Dobyns, William B.; Guerrini, Renzo

    2015-01-01

    SUMMARY Background Bilateral perisylvian polymicrogyria (BPP), the most common form of regional polymicrogyria, causes the congenital bilateral perisylvian syndrome, featuring oromotor dysfunction, cognitive impairment and epilepsy. BPP is etiologically heterogeneous, but only a few genetic causes have been reported. The aim of this study was to identify additional genetic etiologies of BPP and delineate their frequency in this patient population. Methods We performed child-parent (trio)-based whole exome sequencing (WES) on eight children with BPP. Following the identification of mosaic PIK3R2 mutations in two of these eight children, we performed targeted screening of PIK3R2 in a cohort of 118 children with BPP who were ascertained from 1980 until 2015 using two methods. First, we performed targeted sequencing of the entire PIK3R2 gene by single molecule molecular inversion probes (smMIPs) on 38 patients with BPP with normal-large head size. Second, we performed amplicon sequencing of the recurrent PIK3R2 mutation (p.Gly373Arg) on 80 children with various types of polymicrogyria including BPP. One additional patient underwent clinical WES independently, and was included in this study given the phenotypic similarity to our cohort. All patients included in this study were children (< 18 years of age) with polymicrogyria enrolled in our research program. Findings Using WES, we identified a mosaic mutation (p.Gly373Arg) in the regulatory subunit of the PI3K-AKT-MTOR pathway, PIK3R2, in two children with BPP. Of the 38 patients with BPP and normal-large head size who underwent targeted next generation sequencing by smMIPs, we identified constitutional and mosaic PIK3R2 mutations in 17 additional children. In parallel, one patient was found to have the recurrent PIK3R2 mutation by clinical WES. Seven patients had BPP alone, and 13 had BPP in association with features of the megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome (MPPH). Nineteen patients had

  12. Loss of function mutations in PTPN6 promote STAT3 deregulation via JAK3 kinase in diffuse large B-cell lymphoma.

    PubMed

    Demosthenous, Christos; Han, Jing Jing; Hu, Guangzhen; Stenson, Mary; Gupta, Mamta

    2015-12-29

    PTPN6 (SHP1) is a tyrosine phosphatase that negatively controls the activity of multiple signaling pathways including STAT signaling, however role of mutated PTPN6 is not much known. Here we investigated whether PTPN6 might also be a potential target for diffuse large B cell lymphoma (DLBCL) and performed Sanger sequencing of the PTPN6 gene. We have identified missense mutations within PTPN6 (N225K and A550V) in 5% (2/38) of DLBCL tumors. Site directed mutagenesis was performed to mutate wild type (WT) PTPN6 and stable cell lines were generated by lentiviral transduction of PTPN6(WT), PTPN6(N225K) and PTPN6(A550V) constructs, and effects of WT or mutated PTPN6 on STAT3 signaling were analyzed. WT PTPN6 dephosphorylated STAT3, but had no effect on STAT1, STAT5 or STAT6 phosphorylation. Both PTPN6 mutants were unable to inhibit constitutive, as well as cytokines induced STAT3 activation. Both PTPN6 mutants also demonstrated reduced tyrosine phosphatase activity and exhibited enhanced STAT3 transactivation activity. Intriguingly, a lack of direct binding between STAT3 and WT or mutated PTPN6 was observed. However, compared to WT PTPN6, cells expressing PTPN6 mutants exhibited increased binding between JAK3 and PTPN6 suggesting a more dynamic interaction of PTPN6 with upstream regulators of STAT3. Consistent with this notion, both the mutants demonstrated increased resistance to JAK3 inhibitor, WHIP-154 relative to WT PTPN6. Overall, this is the first study, which demonstrates that N225K and A550V PTPN6 mutations cause loss-of-function leading to JAK3 mediated deregulation of STAT3 pathway and uncovers a mechanism that tumor cells can use to control PTPN6 substrate specificity.

  13. Resveratrol modulates interleukin-1β-induced phosphatidylinositol 3-kinase and nuclear factor κB signaling pathways in human tenocytes.

    PubMed

    Busch, Franziska; Mobasheri, Ali; Shayan, Parviz; Lueders, Cora; Stahlmann, Ralf; Shakibaei, Mehdi

    2012-11-02

    Resveratrol, an activator of histone deacetylase Sirt-1, has been proposed to have beneficial health effects due to its antioxidant and anti-inflammatory properties. However, the mechanisms underlying the anti-inflammatory effects of resveratrol and the intracellular signaling pathways involved are poorly understood. An in vitro model of human tenocytes was used to examine the mechanism of resveratrol action on IL-1β-mediated inflammatory signaling. Resveratrol suppressed IL-1β-induced activation of NF-κB and PI3K in a dose- and time-dependent manner. Treatment with resveratrol enhanced the production of matrix components collagen types I and III, tenomodulin, and tenogenic transcription factor scleraxis, whereas it inhibited gene products involved in inflammation and apoptosis. IL-1β-induced NF-κB and PI3K activation was inhibited by resveratrol or the inhibitors of PI3K (wortmannin), c-Src (PP1), and Akt (SH-5) through inhibition of IκB kinase, IκBα phosphorylation, and inhibition of nuclear translocation of NF-κB, suggesting that PI3K signaling pathway may be one of the signaling pathways inhibited by resveratrol to abrogate NF-κB activation. Inhibition of PI3K by wortmannin attenuated IL-1β-induced Akt and p65 acetylation, suggesting that p65 is a downstream component of PI3K/Akt in these responses. The modulatory effects of resveratrol on IL-1β-induced activation of NF-κB and PI3K were found to be mediated at least in part by the association between Sirt-1 and scleraxis and deacetylation of NF-κB and PI3K. Overall, these results demonstrate that activated Sirt-1 plays an essential role in the anti-inflammatory effects of resveratrol and this may be mediated at least in part through inhibition/deacetylation of PI3K and NF-κB.

  14. Resveratrol Modulates Interleukin-1β-induced Phosphatidylinositol 3-Kinase and Nuclear Factor κB Signaling Pathways in Human Tenocytes

    PubMed Central

    Busch, Franziska; Mobasheri, Ali; Shayan, Parviz; Lueders, Cora; Stahlmann, Ralf; Shakibaei, Mehdi

    2012-01-01

    Resveratrol, an activator of histone deacetylase Sirt-1, has been proposed to have beneficial health effects due to its antioxidant and anti-inflammatory properties. However, the mechanisms underlying the anti-inflammatory effects of resveratrol and the intracellular signaling pathways involved are poorly understood. An in vitro model of human tenocytes was used to examine the mechanism of resveratrol action on IL-1β-mediated inflammatory signaling. Resveratrol suppressed IL-1β-induced activation of NF-κB and PI3K in a dose- and time-dependent manner. Treatment with resveratrol enhanced the production of matrix components collagen types I and III, tenomodulin, and tenogenic transcription factor scleraxis, whereas it inhibited gene products involved in inflammation and apoptosis. IL-1β-induced NF-κB and PI3K activation was inhibited by resveratrol or the inhibitors of PI3K (wortmannin), c-Src (PP1), and Akt (SH-5) through inhibition of IκB kinase, IκBα phosphorylation, and inhibition of nuclear translocation of NF-κB, suggesting that PI3K signaling pathway may be one of the signaling pathways inhibited by resveratrol to abrogate NF-κB activation. Inhibition of PI3K by wortmannin attenuated IL-1β-induced Akt and p65 acetylation, suggesting that p65 is a downstream component of PI3K/Akt in these responses. The modulatory effects of resveratrol on IL-1β-induced activation of NF-κB and PI3K were found to be mediated at least in part by the association between Sirt-1 and scleraxis and deacetylation of NF-κB and PI3K. Overall, these results demonstrate that activated Sirt-1 plays an essential role in the anti-inflammatory effects of resveratrol and this may be mediated at least in part through inhibition/deacetylation of PI3K and NF-κB. PMID:22936809

  15. Non-Invasive Imaging of Phosphoinositide-3-Kinase-Catalytic-Subunit-Alpha (PIK3CA) Promoter Modulation in Small Animal Models

    PubMed Central

    Gaikwad, Snehal M.; Gunjal, Lata; Junutula, Anitha R.; Astanehe, Arezoo; Gambhir, Sanjiv Sam; Ray, Pritha

    2013-01-01

    Activation of the PI3K/Akt pathway, a critical step for survival in cancer cells is often associated with decreased sensitivity to several chemotherapeutic drugs. PIK3CA gene amplification is observed in 16–24% of epithelial ovarian cancer (EOC) patients in conjunction with p53 mutations. A 900 bp long PIK3CA promoter is shown to be negatively regulated by p53 in ovarian surface epithelial cells but the consequence of chemotherapeutic drug treatments on this promoter in ovarian cancer cells is largely unknown. We aim to study the modulation of this promoter by cisplatin using an improved fusion reporter in ovarian cancer cells and tumor xenografts by non-invasive imaging approach. A PIK3CA sensor was developed using a bi-fusion reporter from a newly constructed library of bi- and tri-fusion vectors comprising of two mutant far red fluorescent proteins (mcherry/mch and tdTomato/tdt), a mutant firefly luciferase (fluc2), and a PET reporter protein (ttk). In vivo imaging of mice implanted with 293T cells transiently expressing these bi- and tri-fusion reporters along with respective controls revealed comparable activity of each reporter in the fusion background and fluc2-tdt as the most sensitive one. Repression of the PIK3CA sensor by drugs was inversely proportional to cellular p53 level in a germline (PA1) and in an EOC (A2780) cell line but not in a p53 deficient EOC (SKOV3) cell line. Bioluminescence imaging of tumor xenografts stably expressing the PIK3CA sensor in PA1 and A2780 cells exhibited attenuating activity without any change in SKOV3 tumors expressing the PIK3CA sensor after cisplatin treatment. Sequential mutation at p53 binding sites showed gradual increase in promoter activity and decreased effects of the drugs. These newly developed PIK3CA-fluc2-tdt and the mutant reporter sensors thus would be extremely useful for screening new drugs and for functional assessment of PIK3CA expression from intact cells to living subjects. PMID:23393606

  16. Class III PI 3-kinase is the main source of PtdIns3P substrate and membrane recruitment signal for PIKfyve constitutive function in podocyte endomembrane homeostasis.

    PubMed

    Ikonomov, Ognian C; Sbrissa, Diego; Venkatareddy, Madhusudan; Tisdale, Ellen; Garg, Puneet; Shisheva, Assia

    2015-05-01

    The evolutionarily conserved PIKfyve, which synthesizes PtdIns5P from PtdIns, and PtdIns(3,5)P2 from PtdIns3P, requires PtdIns3P as both an enzyme substrate and a membrane recruitment signal. Whereas the PtdIns3P source is undetermined, class III PI3K (Vps34), the only evolutionarily conserved of the eight mammalian PI3Ks, is presumed as a main candidate. A hallmark of PIKfyve deficiency is formation of multiple translucent cytoplasmic vacuoles seen by light microscopy in cells cultured in complete media. Such an aberrant phenotype is often observed in cells from conditional Vps34 knockout (KO) mice. To clarify the mechanism of Vps34 KO-triggered vacuolation and the PtdIns3P source for PIKfyve functionality, here we have characterized a podocyte cell type derived from Vps34fl/fl mice, which, upon Cre-mediated gene KO, robustly formed cytoplasmic vacuoles resembling those in PikfyveKO MEFs. Vps34wt, expressed in Vps34KO podocytes restored the normal morphology, but only if the endogenous PIKfyve activity was intact. Conversely, expressed PIKfyvewt rescued completely the vacuolation only in PikfyveKO MEFs but not in Vps34KO podocytes. Analyses of phosphoinositide profiles by HPLC and localization patterns by a PtdIns3P biosensor revealed that Vps34 is the main supplier of localized PtdIns3P not only for PIKfyve activity but also for membrane recruitment. Concordantly, Vps34KO podocytes had severely reduced steady-state levels of both PtdIns(3,5)P2 and PtdIns5P, along with PtdIns3P. We further revealed a plausible physiologically-relevant Vps34-independent PtdIns3P supply for PIKfyve, operating through activated class I PI3Ks. Our data provide the first evidence that the vacuolation phenotype in Vps34KO podocytes is due to PIKfyve dysfunction and that Vps34 is a main PtdIns3P source for constitutive PIKfyve functionality.

  17. 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 Central

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

    2016-01-01

    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

  18. Regulation of NGF-driven neurite outgrowth by Ins(1,4,5)P3 kinase is specifically associated with the two isoenzymes Itpka and Itpkb in a model of PC12 cells.

    PubMed

    Koenig, Sandra; Moreau, Colette; Dupont, Geneviève; Scoumanne, Ariane; Erneux, Christophe

    2015-07-01

    Four inositol phosphate kinases catalyze phosphorylation of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3 ] to inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4 ]: these enzymes comprise three isoenzymes of inositol 1,4,5-trisphosphate 3-kinase (Itpk), referred to as Itpka, Itpkb and Itpkc, and the inositol polyphosphate multikinase (IPMK). The four enzymes that act on Ins(1,4,5)P3 are all expressed in rat pheochromocytoma PC12 cells, a model that is used to study neurite outgrowth induced by nerve growth factor (NGF). We compared the effect of over-expression of the four GFP-tagged kinases on NGF-induced neurite outgrowth. Our data show that over-expression of the Itpka and Itpkb isoforms inhibits NGF-induced neurite outgrowth, but over-expression of Itpkc and IPMK does not. Surprisingly, over-expression of the N-terminal F-actin binding domain of Itpka, which lacks catalytic activity, was as effective at inhibiting neurite outgrowth as the full-length enzyme. Neurite length was also significantly decreased in cells over-expressing Itpka and Itpkb but not Itpkc or IPMK. This result did not depend on the over-expression level of any of the kinases. PC12 cells over-expressing GFP-tagged kinase-dead mutants Itpka/b have shorter neurites than GFP control cells. The decrease in neurite length was never as pronounced as observed with wild-type GFP-tagged Itpka/b. Finally, the percentage of neurite-bearing cells was increased in cells over-expressing the membranous type I Ins(1,4,5)P3 5-phosphatase. We conclude that Itpka and Itpkb inhibit neurite outgrowth through both F-actin binding and localized Ins(1,4,5)P3 3-kinase activity. Itpkc and IPMK do not influence neurite outgrowth or neurite length in this model.

  19. Restoration of SHIP activity in a human leukemia cell line downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

    PubMed

    Horn, S; Endl, E; Fehse, B; Weck, M M; Mayr, G W; Jücker, M

    2004-11-01

    The inositol 5-phosphatase SHIP (SHIP-1) is a negative regulator of signal transduction in hematopoietic cells and targeted disruption of SHIP in mice leads to a myeloproliferative disorder. We analyzed the effects of SHIP on the human leukemia cell line Jurkat in which expression of endogenous SHIP protein is not detectable. Restoration of SHIP expression in Jurkat cells with an inducible expression system caused a 69% reduction of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and a 65% reduction of Akt kinase activity, which was associated with reduced phosphorylation of glycogen synthase kinase 3beta (GSK-3beta) (Ser-9) without changing the phosphorylation of Bad (Ser-136), FKHR (Ser-256) or MAPK (Thr-202/Tyr-204). SHIP-expressing Jurkat cells showed an increased transit time through the G1 phase of the cell cycle, but SHIP did not cause a complete cell cycle arrest or apoptosis. Extension of the G1 phase was associated with an increased stability of the cell cycle inhibitor p27(Kip1) and reduced phosphorylation of the retinoblastoma protein Rb at serine residue 780. Our data indicate that restoration of SHIP activity in a human leukemia cell line, which has lost expression of endogenous SHIP, downregulates constitutively activated phosphatidylinositol 3-kinase/Akt/GSK-3beta signaling and leads to an increased transit time through the G1 phase of the cell cycle.

  20. The phosphatidylinositol 3-kinases (PI3K) inhibitor GS-1101 synergistically potentiates histone deacetylase inhibitor-induced proliferation inhibition and apoptosis through the inactivation of PI3K and extracellular signal-regulated kinase pathways.

    PubMed

    Bodo, Juraj; Zhao, Xiaoxian; Sharma, Arishya; Hill, Brian T; Portell, Craig A; Lannutti, Brian J; Almasan, Alexandru; Hsi, Eric D

    2013-10-01

    Previously, we showed that inhibition of the protein kinase C β (PKCβ)/AKT pathway augments engagement of the histone deacetylase inhibitor (HDI)-induced apoptosis in lymphoma cells. In the present study, we investigated the cytotoxicity and mechanisms of cell death induced by the delta isoform-specific phosphatidylinositide 3-kinase (PI3K) inhibitor, GS-1101, in combination with the HDI, panobinostat (LBH589) and suberoylanilide hydroxamic acid (SAHA). Lymphoma cell lines, primary non-Hodgkin Lymphoma (NHL) and chronic lymphocytic leukaemia (CLL) cells were simultaneously treated with the HDI, LBH589 and GS-1101. An interaction of the LBH589/GS-1101 combination was formally examined by using various concentrations of LBH589 and GS-1101. Combined treatment resulted in a synergistic inhibition of proliferation and showed synergistic effect on apoptotic induction in all tested cell lines and primary NHL and CLL cells. This study indicates that interference with PI3K signalling dramatically increases HDI-mediated apoptosis in malignant haematopoietic cells, possibly through both AKT-dependent or AKT- independent mechanisms. Moreover, the increase in HDI-related apoptosis observed in PI3K inhibitor-treated cells appears to be related to the disruption of the extracellular signal-regulated kinase (ERK) signalling pathway. This study provides a strong rational for testing the combination of PI3K inhibitors and HDI in the clinic.

  1. 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.

  2. Activation of sonic hedgehog signaling enhances cell migration and invasion by induction of matrix metalloproteinase-2 and -9 via the phosphoinositide-3 kinase/AKT signaling pathway in glioblastoma.

    PubMed

    Chang, Liang; Zhao, Dan; Liu, Hui-Bin; Wang, Qiu-Shi; Zhang, Ping; Li, Chen-Long; Du, Wen-Zhong; Wang, Hong-Jun; Liu, Xing; Zhang, Zhi-Ren; Jiang, Chuan-Lu

    2015-11-01

    Aberrant hedgehog signaling contributes to the development of various malignancies, including glioblastoma (GBM). However, the potential mechanism of hedgehog signaling in GBM migration and invasion has remained to be elucidated. The present study showed that enhanced hedgehog signaling by recombinant human sonic hedgehog N‑terminal peptide (rhSHH) promoted the adhesion, invasion and migration of GBM cells, accompanied by increases in mRNA and protein levels of matrix metalloproteinase‑2 (MMP‑2) and MMP‑9. However, inhibition of hedgehog signaling with cyclopamine suppressed the adhesion, invasion and migration of GBM cells, accompanied by decreases in mRNA and protein levels of MMP‑2 and ‑9. Furthermore, it was found that MMP‑2- and MMP‑9-neutralizing antibodies or GAM6001 reversed the inductive effects of rhSHH on cell migration and invasion. In addition, enhanced hedgehog signaling by rhSHH increased AKT phosphorylation, whereas blockade of hedgehog signaling decreased AKT phosphorylations. Further experiments showed that LY294002, an inhibitor of phosphoinositide-3 kinase (PI3K), decreased rhSHH‑induced upregulation of MMP‑2 and ‑9. Finally, the protein expression of glioblastoma-associated oncogene 1 was positively correlated with levels of phosphorylated AKT as well as protein expressions of MMP‑2 and ‑9 in GBM tissue samples. In conclusion, the present study indicated that the hedgehog pathway regulates GBM-cell migration and invasion by increasing MMP-2 and MMP-9 production via the PI3K/AKT pathway.

  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. 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.

  5. PI 3-kinase-dependent phosphorylation of Plk1–Ser99 promotes association with 14-3-3γ and is required for metaphase–anaphase transition

    PubMed Central

    Kasahara, Kousuke; Goto, Hidemasa; Izawa, Ichiro; Kiyono, Tohru; Watanabe, Nobumoto; Elowe, Sabine; Nigg, Erich A; Inagaki, Masaki

    2013-01-01

    Polo-like kinase 1 (Plk1) controls multiple aspects of mitosis and is activated through its phosphorylation at Thr210. Here we identify Ser99 on Plk1 as a novel mitosis-specific phosphorylation site, which operates independently of Plk1–Thr210 phosphorylation. Plk1–Ser99 phosphorylation creates a docking site for 14-3-3γ, and this interaction stimulates the catalytic activity of Plk1. Knockdown of 14-3-3γ or replacement of wild-type (WT) Plk1 by a Ser99-phospho-blocking mutant leads to a prometaphase/metaphase-like arrest due to the activation of the spindle assembly checkpoint. Inhibition of phosphatidylinositol 3-kinase (PI3K) and Akt significantly reduces the level of Plk1–Ser99 phosphorylation and delays metaphase to anaphase transition. Plk1–Ser99 phosphorylation requires not only Akt activity but also protein(s) associated with Plk1 in a mitosis-specific manner. Therefore, mitotic Plk1 activity is regulated not only by Plk1–Thr210 phosphorylation, but also by Plk1 binding to 14-3-3γ following Plk1–Ser99 phosphorylation downstream of the PI3K–Akt signalling pathway. This novel Plk1 activation pathway controls proper progression from metaphase to anaphase. PMID:23695676

  6. Phosphatidylinositol 3-kinase activity and asymmetrical accumulation of F-actin are necessary for establishment of cell polarity in the early development of monospores from the marine red alga Porphyra yezoensis.

    PubMed

    Li, Lin; Saga, Naotsune; Mikami, Koji

    2008-01-01

    The polarized distribution of F-actin is important in providing the driving force for directional migration in mammalian leukocytes and Dictyostelium cells, in which compartmentation of phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol phosphatase is critical for the establishment of cell polarity. Since monospores from the red alga Porphyra yezoensis are a real example of migrating plant cells, the involvement of the cytoskeleton and PI3K was investigated during their early development. Our results indicate that the asymmetrical localization of F-actin at the leading edge is fixed by the establishment of the anterior-posterior axis in migrating monospores, which is PI3K-dependent and protein synthesis-independent. After migration, monospores adhere to the substratum and then become upright, developing into multicellular thalli via the establishment of the apical-basal axis. In this process, F-actin usually accumulates at the bottom of the basal cell and development after migration requires new protein synthesis. These findings suggest that the establishment of anterior-posterior and apical-basal axes are differentially regulated during the early development of monospores. Our results also indicate that PI3K-dependent F-actin asymmetry is evolutionally conserved in relation to the establishment of cell polarity in migrating eukaryotic cells.

  7. Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes.

    PubMed Central

    Panayotou, G; Bax, B; Gout, I; Federwisch, M; Wroblowski, B; Dhand, R; Fry, M J; Blundell, T L; Wollmer, A; Waterfield, M D

    1992-01-01

    Circular dichroism and fluorescence spectroscopy were used to investigate the structure of the p85 alpha subunit of the PI 3-kinase, a closely related p85 beta protein, and a recombinant SH2 domain-containing fragment of p85 alpha. Significant spectral changes, indicative of a conformational change, were observed on formation of a complex with a 17 residue peptide containing a phosphorylated tyrosine residue. The sequence of this peptide is identical to the sequence surrounding Tyr751 in the kinase-insert region of the platelet-derived growth factor beta-receptor (beta PDGFR). The rotational correlation times measured by fluorescence anisotropy decay indicated that phosphopeptide binding changed the shape of the SH2 domain-containing fragment. The CD and fluorescence spectroscopy data support the secondary structure prediction based on sequence analysis and provide evidence for flexible linker regions between the various domains of the p85 proteins. The significance of these results for SH2 domain-containing proteins is discussed. Images PMID:1330535

  8. Phosphatidylinositol 3-kinase signaling determines kidney size.

    PubMed

    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

    2015-06-01

    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 (Pten(ptKO)) 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 p21(Cip1/WAF) and p27(Kip1). Administration of rapamycin to Pten(ptKO) mice diminished hypertrophy. Proximal tubule-specific deletion of Egfr in Pten(ptKO) mice also attenuated class I PI3K/mTORC2/AKT signaling and reduced the size of enlarged kidneys. In Pten(ptKO) 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 Pten(ptKO) 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.

  9. Inhibitors of glycogen synthase 3 kinase

    DOEpatents

    Schultz, Peter; Ring, David B.; Harrison, Stephen D.; Bray, Andrew M.

    2006-05-30

    Compounds of formula 1: ##STR00001## wherein R.sub.1 is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0 3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro; R.sub.2 is hydroxy, amino, or lower alkoxy; R.sub.3 is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amino-acyl; R.sub.4 is H or lower alkyl; and pharmaceutically acceptable salts and esters thereof; are effective inhibitors of GSK3.

  10. Inhibitors of glycogen synthase 3 kinase

    DOEpatents

    Schultz, Peter; Ring, David B.; Harrison, Stephen D.; Bray, Andrew M.

    2000-01-01

    Compounds of formula 1: ##STR1## wherein R.sub.1 is alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or heteroaralkyl, substituted with 0-3 substituents selected from lower alkyl, halo, hydroxy, lower alkoxy, amino, lower alkyl-amino, and nitro; R.sub.2 is hydroxy, amino, or lower alkoxy; R.sub.3 is H, lower alkyl, lower acyl, lower alkoxy-acyl, or amnino-acyl; R.sub.4 is H or lower alkyl; and pharmaceutically acceptable salts and esters thereof; are effective inhibitors of GSK3.

  11. Regulation of Gene33 expression by insulin requires MEK-ERK activation.

    PubMed

    Keeton, Adam B; Xu, Jie; Franklin, J Lee; Messina, Joseph L

    2004-09-17

    Gene33 and its human homologue, mitogen inducible gene-6/receptor-associated late transducer (mig-6, RALT), is a 53-kDa soluble protein that was identified as a hepatic gene regulated by glucocorticoids and insulin. Its mRNA is expressed in numerous tissues in addition to the liver. Mitogen inducibility of Gene33 mRNA has been described in several experimental systems. Recent reports have suggested a role for Gene33 in inhibition of proliferation induced by factors that bind to members of the ErbB family of receptors. In the present work, we examine the regulation of Gene33 protein by insulin in hepatoma cells of rat (H4IIE) and human (HepG2/Hep3B) origin. Inhibition of MEK1 significantly inhibited extracellularly regulated kinase (ERK)1/2 activation and insulin-regulated Gene33 transcription and protein levels in H4IIE cells. Inhibition of phosphatidylinositol 3-kinase (PI3-K) activity alone did not significantly alter transcription of Gene33. In Hep3B and HepG2 cells, insulin did not significantly induce either ERK1/2 activation or Gene33 expression. This work suggests that the MEK-ERK, but not the phosphatidylinositol 3-kinase (PI3-K), pathway plays a direct role in insulin regulation of Gene33 transcription and protein expression.

  12. Characterization of VPS34-IN1, a selective inhibitor of Vps34, reveals that the phosphatidylinositol 3-phosphate-binding SGK3 protein kinase is a downstream target of class III phosphoinositide 3-kinase.

    PubMed

    Bago, Ruzica; Malik, Nazma; Munson, Michael J; Prescott, Alan R; Davies, Paul; Sommer, Eeva; Shpiro, Natalia; Ward, Richard; Cross, Darren; Ganley, Ian G; Alessi, Dario R

    2014-11-01

    The Vps34 (vacuolar protein sorting 34) class III PI3K (phosphoinositide 3-kinase) phosphorylates PtdIns (phosphatidylinositol) at endosomal membranes to generate PtdIns(3)P that regulates membrane trafficking processes via its ability to recruit a subset of proteins possessing PtdIns(3)P-binding PX (phox homology) and FYVE domains. In the present study, we describe a highly selective and potent inhibitor of Vps34, termed VPS34-IN1, that inhibits Vps34 with 25 nM IC50 in vitro, but does not significantly inhibit the activity of 340 protein kinases or 25 lipid kinases tested that include all isoforms of class I as well as class II PI3Ks. Administration of VPS34-IN1 to cells induces a rapid dose-dependent dispersal of a specific PtdIns(3)P-binding probe from endosome membranes, within 1 min, without affecting the ability of class I PI3K to regulate Akt. Moreover, we explored whether SGK3 (serum- and glucocorticoid-regulated kinase-3), the only protein kinase known to interact specifically with PtdIns(3)P via its N-terminal PX domain, might be controlled by Vps34. Mutations disrupting PtdIns(3)P binding ablated SGK3 kinase activity by suppressing phosphorylation of the T-loop [PDK1 (phosphoinositide-dependent kinase 1) site] and hydrophobic motif (mammalian target of rapamycin site) residues. VPS34-IN1 induced a rapid ~50-60% loss of SGK3 phosphorylation within 1 min. VPS34-IN1 did not inhibit activity of the SGK2 isoform that does not possess a PtdIns(3)P-binding PX domain. Furthermore, class I PI3K inhibitors (GDC-0941 and BKM120) that do not inhibit Vps34 suppressed SGK3 activity by ~40%. Combining VPS34-IN1 and GDC-0941 reduced SGK3 activity ~80-90%. These data suggest SGK3 phosphorylation and hence activity is controlled by two pools of PtdIns(3)P. The first is produced through phosphorylation of PtdIns by Vps34 at the endosome. The second is due to the conversion of class I PI3K product, PtdIns(3,4,5)P3 into PtdIns(3)P, via the sequential actions of the Ptd

  13. Blockade of the phosphatidylinositol-3-kinase-Akt signaling pathway enhances the induction of apoptosis by microtubule-destabilizing agents in tumor cells in which the pathway is constitutively activated.

    PubMed

    Fujiwara, Yusuke; Hosokawa, Yoshihisa; Watanabe, Kazushi; Tanimura, Susumu; Ozaki, Kei-ichi; Kohno, Michiaki

    2007-03-01

    Constitutive activation of the phosphatidylinositol-3-kinase (PI3K)-Akt signaling pathway is associated with the neoplastic phenotype in many human tumor cell types. Given the antiapoptotic role of this pathway, we examined whether its specific blockade might sensitize human tumor cells to the induction of apoptosis by various anticancer drugs. Although specific blockade of the PI3K-Akt pathway alone with inhibitors such as LY294002 did not induce cell death, it resulted in marked and selective enhancement of the induction of apoptosis by microtubule-destabilizing agents such as vincristine. This effect was apparent only in tumor cells in which the PI3K-Akt pathway is constitutively activated. Blockade of the PI3K-Akt pathway induced the activation of glycogen synthase kinase-3beta, which phosphorylates microtubule-associated proteins such as tau and thereby reduces their ability to bind and stabilize microtubules. The consequent destabilization of microtubules induced by the inhibition of PI3K-Akt signaling appeared to increase their sensitivity to low concentrations of microtubule-destabilizing agents that alone do not lead to the disruption of cytoplasmic microtubules in tumor cells. Such a synergistic effect on microtubule integrity was not apparent for stable microtubules in the neurites of neuronal cells. These results suggest that the administration of a combination of a PI3K-Akt pathway inhibitor and a microtubule-destabilizing agent is a potential chemotherapeutic strategy for the treatment of tumor cells in which this signaling pathway is constitutively activated.

  14. Activation of phosphoinositide 3-kinase/PKB pathway by CB(1) and CB(2) cannabinoid receptors expressed in prostate PC-3 cells. Involvement in Raf-1 stimulation and NGF induction.

    PubMed

    Sánchez, María G; Ruiz-Llorente, Lidia; Sánchez, Ana M; Díaz-Laviada, Inés

    2003-09-01

    Cannabinoids exert a variety of physiological and pharmacological responses in humans through interaction with specific cannabinoid receptors. Cannabinoid receptors described to date belong to the seven-transmembrane-domain receptor superfamily and are coupled through the inhibitory G(i) protein to adenylyl cyclase inhibition. However, downstream signal transduction mechanisms triggered by cannabinoids are poorly understood. We examined here the involvement of the phosphoinositide 3-kinase (PI3K)/PKB pathway in the mechanism of action of cannabinoids in human prostate epithelial PC-3 cells. Cannabinoid receptors CB(1) and CB(2) are expressed in these cells, as shown by RT-PCR, Western blot and immunofluorescence techniques. Treatment of PC-3 cells with either Delta(9)-tetrahydrocannabinol (THC), the major psychoactive ingredient of marijuana, or R-(+)-methanandamide (MET), an analogue of the endogenous cannabinoid anandamide, increased phosphorylation of PKB in Thr308 and Ser473. The stimulation of PKB induced by cannabinoids was blocked by the two cannabinoid receptor antagonists, SR 141716 and SR 144528, and by the PI3K inhibitor LY 294002. These results indicate that activation of cannabinoid receptors in PC-3 cells stimulate the PI3K/PKB pathway. We further investigated the involvement of Raf-1/Erk activation in the mechanism of action of cannabinoid receptors. THC and MET induced translocation of Raf-1 to the membrane and phosphorylation of p44/42 Erk kinase, which was reversed by cannabinoid receptor antagonists and PI3K inhibitor. These results point to a sequential connection between cannabinoid receptors/PI3K/PKB pathway and Raf-1/Erk in prostate PC-3 cells. We also show that this pathway is involved in the mechanism of NGF induction exerted by cannabinoids in PC-3 cells.

  15. Roles of tyrosine kinase-, 1-phosphatidylinositol 3-kinase-, and mitogen-activated protein kinase-signaling pathways in ethanol-induced contractions of rat aortic smooth muscle: possible relation to alcohol-induced hypertension.

    PubMed

    Yang, Zhi-wei; Wang, Jun; Zheng, Tao; Altura, Bella T; Altura, Burton M

    2002-08-01

    Insights into the relations between and among ethanol-induced contractions in rat aorta, tyrosine kinases (including src family of cytoplasmic tyrosine kinases), 1-phosphatidylinositol 3-kinases (PI-3Ks), mitogen-activated protein kinases (MAPKs), and regulation of intracellular free Ca(2+) ([Ca(2+)](i)) were investigated in the present study. Ethanol-induced concentration-dependent contractions in isolated rat aortic rings were attenuated greatly by pretreatment of the arteries with low concentrations of an antagonist of protein tyrosine kinases (genistein), an src homology domain 2 (SH2) inhibitor peptide, a highly specific antagonist of p38 MAPK (SB-203580), a potent, selective antagonist of two specific MAPK kinases-MEK1/MEK2 (U0126)-and a selective antagonist of mitogen-activated protein kinase kinase (MAPKK) (PD-98059), as well as by treatment with wortmannin or LY-294002 (both are selective antagonists of PI-3Ks). Inhibitory concentration 50 (IC(50)) levels obtained for these seven antagonists were consistent with reported inhibition constant (Ki) values for these tyrosine kinase, MAPK, and MAPKK antagonists. Ethanol-induced transient and sustained increases in [Ca(2+)](i) in primary single smooth muscle cells from rat aorta were markedly attenuated in the presence of genistein, an SH2 domain inhibitor peptide, SB-203580, U0126, PD-98059, wortmannin, and LY-294002. A variety of specific antagonists of known endogenously formed vasoconstrictors did not inhibit or attenuate either the ethanol-induced contractions or the elevations of [Ca(2+)](i). Results of the present study support the suggestion that activation of tyrosine kinases (including the src family of cytoplasmic tyrosine kinases), PI-3Ks, and MAPK seems to play an important role in ethanol-induced contractions and the elevation of [Ca(2+)](i) in smooth muscle cells from rat aorta. These signaling pathways thus may be important in hypertension in human beings associated with chronic alcohol

  16. Enhanced expression and purification of inositol 1,4,5-trisphosphate 3-kinase A through use of the pCold1-GST vector and a C-terminal hexahistidine tag in Escherichia coli.

    PubMed

    Lee, Dongmin; Han, Seungrie; Woo, Seungkyun; Lee, Hyun Woo; Sun, Woong; Kim, Hyun

    2014-05-01

    Inositol 1,4,5-trisphosphate 3-kinase A (IP3K-A, alternative name: ITPKA) is a neuron-specific enzyme that converts 1,4,5-trisphosphate (IP3) into inositol 1,3,4,5-tetrakisphosphate (IP4) through its kinase domain. In addition, transient overexpression of IP3K-A induces morphological changes in dendritic spines of excitatory synapses in a kinase-independent manner, apparently by modulating the organization of the neuronal cytoskeleton. Although the procurement of a purified recombinant IP3K-A protein would be indispensable for the biochemical elucidation of its physiological roles, production of recombinant IP3K-A has proven technically challenging in conventional Escherichia coli expression systems. These difficulties stem from low enzyme solubility, as well as poor protein quality caused by the tendency of IP3K-A to split into partial fragments. In present study, we newly introduced cold-shock expression vector (pCold1) together with a C-terminal hexahistidine tag (C-HIS) to enhance the expression levels of recombinant IP3K-A in E. coli. Importantly, when compared with other commonly-employed bacterial expression systems, the pCold1 system improved the yield and the purity of full-length IP3K-A due to the exclusion of truncated enzyme forms, and also enhanced the solubility of the enzyme. Furthermore, the functional integrity of purified IP3K-A was confirmed in both kinase activity assay and microtubule binding assay. Recombinant IP3K-A acquired via this modified protocol will be expected to facilitate the exploration of the enzyme's biochemical profile, both structurally and functionally.

  17. 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

  18. A randomized, phase 2 trial of docetaxel with or without PX-866, an irreversible oral phosphatidylinositol 3-kinase inhibitor, in patients with relapsed or metastatic head and neck squamous cell cancer

    PubMed Central

    Jimeno, Antonio; Bauman, Julie E.; Weissman, Charles; Adkins, Douglas; Schnadig, Ian; Beauregard, Patrice; Bowles, Daniel W.; Spira, Alexander; Levy, Benjamin; Seetharamu, Nagashree; Hausman, Diana; Walker, Luke; Rudin, Charles M.; Shirai, Keisuke

    2016-01-01

    SUMMARY Introduction The phosphotidylinositol-3 kinase (PI3K)/serine–threonine kinase (AKT)/mammalian target of rapamycin (mTOR) signaling pathway is frequently altered in head and neck squamous cell cancer (HNSCC). PX-866 is an oral, irreversible, pan-isoform inhibitor of PI3K. Preclinical models revealed synergy with docetaxel and a phase 1 trial demonstrated tolerability of this combination. This randomized phase 2 study evaluated PX-866 combined with docetaxel in patients with advanced, refractory HNSCC. Methods Patients with locally advanced, recurrent or metastatic HNSCC who had received at least one and no more than two prior systemic treatment regimens were randomized (1:1) to a combination of docetaxel (75 mg/m2 IV every 21 days) with or without PX-866 (8 mg PO daily; Arms A and B, respectively). The primary endpoint was progression free survival (PFS). Secondary endpoints included objective response rate (RR), overall survival (OS), toxicity, and correlation of biomarker analyses with efficacy outcomes. Results 85 patients were enrolled. There was a non-significant improvement in response rate in the combination arm (14% vs. 5%; P = 0.13). Median PFS was 92 days in Arm A and 82 days in Arm B (P = 0.42). There was no difference in OS between the two arms (263 vs. 195 days; P = 0.62). Grade 3 or higher adverse events were infrequent, but more common in the combination arm with respect to diarrhea (17% vs. 2%), nausea (7% vs. 0%), and febrile neutropenia (21% vs. 5%); grade 3 or higher anemia was more frequent in arm B (7% vs. 27%). PIK3CA mutations or PTEN loss were infrequently observed. Conclusion The addition of PX-866 to docetaxel did not improve PFS, RR, or OS in patients with advanced, refractory HNSCC without molecular pre-selection. PMID:25593016

  19. 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

    Chuang, Wan-Ling; Su, Chin-Cheng; Lin, Ping-Yi; Lin, Chi-Chen; Chen, Yao-Li

    2015-08-01

    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.

  20. 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

  1. A Phase Ib Study of BEZ235, a Dual Inhibitor of Phosphatidylinositol 3-Kinase (PI3K) and Mammalian Target of Rapamycin (mTOR), in Patients With Advanced Renal Cell Carcinoma

    PubMed Central

    Molina, Ana M.; Lakhman, Yulia; Patil, Sujata; Woo, Kaitlin; DeLuca, John; Lee, Chung-Han; Hsieh, James J.; Feldman, Darren R.; Motzer, Robert J.; H. Voss, Martin

    2016-01-01

    Lessons Learned Our results highlight additional toxicities of dual PI3K/mTOR inhibition in the clinical setting that were unforeseen from preclinical models. Because of toxicity and lack of efficacy, BEZ235 should not be further developed in the current formulation for patients with renal cell carcinoma. Background. Allosteric inhibitors of the mammalian target of rapamycin complex 1 (mTORC1) are approved for advanced renal cell carcinoma (RCC). Preclinical models have suggested that dual inhibition of phosphatidylinositol 3-kinase (PI3K) and mTOR kinase may establish superior anticancer effect. We aimed to establish safety for BEZ235, a potent inhibitor of both PI3K and mTOR, in advanced RCC. Methods. Patients with advanced RCC who had previously failed standard therapy received escalating doses of BEZ235 in sachet formulation twice daily until progression or unacceptable toxicity. Primary endpoints were to identify the maximally tolerated dose (MTD) and to determine the recommended dose for the phase II study. Results. The study was terminated early because of high incidence of dose-limiting toxicities (DLTs) across all dose levels tested. Ten patients were treated with BEZ235—six with clear cell and four with non-clear cell subtypes. Five of these patients suffered DLTs: 2 of 2 patients in the original 400 mg b.i.d. cohort, 1 of 6 in the 200 mg b.i.d. cohort, and 2 of 2 in the 300 mg b.i.d. cohort. DLTs included fatigue, rash, nausea and vomiting, diarrhea, mucositis, anorexia, and dysgeusia. Five patients were evaluable for response: Two had stable disease as best response, and three had progressive disease. Conclusion. BEZ235 twice daily resulted in significant toxicity without objective responses; further development of this compound will not be pursued in this disease. PMID:27286790

  2. Progesterone increases brain-derived neuroptrophic factor expression and protects against glutamate toxicity in a mitogen-activated protein kinase- and phosphoinositide-3 kinase-dependent manner in cerebral cortical explants.

    PubMed

    Kaur, Paramjit; Jodhka, Parmeet K; Underwood, Wendy A; Bowles, Courtney A; de Fiebre, Nancyellen C; de Fiebre, Christopher M; Singh, Meharvan

    2007-08-15

    The higher prevalence and risk for Alzheimer's disease in women relative to men has been partially attributed to the precipitous decline in gonadal hormone levels that occurs in women following the menopause. Although considerable attention has been focused on the consequence of estrogen loss, and thus estrogen's neuroprotective potential, it is important to recognize that the menopause results in a precipitous decline in progesterone levels as well. In fact, progesterone is neuroprotective, although the precise mechanisms involved remain unclear. Based on our previous observation that progesterone elicits the phosphorylation of ERK and Akt, key effectors of the neuroprotective mitogen-activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI3-K) pathways, respectively, we determined whether activation of either of these pathways was necessary for progesterone-induced protection. With organotypic explants (slice culture) of the cerebral cortex, we found that progesterone protected against glutamate-induced toxicity. Furthermore, these protective effects were inhibited by either the MEK1/2 inhibitor UO126 or the PI3-K inhibitor LY294002, supporting the requirement for both the MAPK and PI3-K pathways in progesterone-induced protection. In addition, at a concentration and duration of treatment consistent with our neuroprotection data, progesterone also increased the expression of brain-derived neurotrophic factor (BDNF), at the level of both protein and mRNA. This induction of BDNF may be relevant to the protective effects of progesterone, in that inhibition of Trk signaling, with K252a, inhibited the protective effects of progesterone. Collectively, these data suggest that progesterone is protective via multiple and potentially related mechanisms. (c) 2007 Wiley-Liss, Inc.

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

    PubMed

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

    2010-11-24

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

  4. Guanosine is neuroprotective against oxygen/glucose deprivation in hippocampal slices via large conductance Ca²+-activated K+ channels, phosphatidilinositol-3 kinase/protein kinase B pathway activation and glutamate uptake.

    PubMed

    Dal-Cim, T; Martins, W C; Santos, A R S; Tasca, C I

    2011-06-02

    Guanine derivatives (GD) have been implicated in many relevant brain extracellular roles, such as modulation of glutamate transmission and neuronal protection against excitotoxic damage. GD are spontaneously released to the extracellular space from cultured astrocytes and during oxygen/glucose deprivation (OGD). The aim of this study has been to evaluate the potassium channels and phosphatidilinositol-3 kinase (PI3K) pathway involvement in the mechanisms related to the neuroprotective role of guanosine in rat hippocampal slices subjected to OGD. The addition of guanosine (100 μM) to hippocampal slices subjected to 15 min of OGD and followed by 2 h of re-oxygenation is neuroprotective. The presence of K+ channel blockers, glibenclamide (20 μM) or apamin (300 nM), revealed that neuroprotective effect of guanosine was not dependent on ATP-sensitive K+ channels or small conductance Ca²+-activated K+ channels. The presence of charybdotoxin (100 nM), a large conductance Ca²+-activated K+ channel (BK) blocker, inhibited the neuroprotective effect of guanosine. Hippocampal slices subjected to OGD and re-oxygenation showed a significant reduction of glutamate uptake. Addition of guanosine in the re-oxygenation period has blocked the reduction of glutamate uptake. This guanosine effect was inhibited when hippocampal slices were pre-incubated with charybdotoxin or wortmanin (a PI3K inhibitor, 1 μM) in the re-oxygenation period. Guanosine promoted an increase in Akt protein phosphorylation. However, the presence of charybdotoxin blocked such effect. In conclusion, the neuroprotective effect of guanosine involves augmentation of glutamate uptake, which is modulated by BK channels and the activation of PI3K pathway. Moreover, neuroprotection caused by guanosine depends on the increased expression of phospho-Akt protein.

  5. Inhibition of net HepG2 cell apolipoprotein B secretion by the citrus flavonoid naringenin involves activation of phosphatidylinositol 3-kinase, independent of insulin receptor substrate-1 phosphorylation.

    PubMed

    Borradaile, Nica M; de Dreu, Linda E; Huff, Murray W

    2003-10-01

    The flavonoid naringenin improves hyperlipidemia and hyperglycemia in streptozotocin-treated rats. In HepG2 human hepatoma cells, naringenin inhibits apolipoprotein B (apoB) secretion primarily by inhibiting microsomal triglyceride transfer protein and enhances LDL receptor (LDLr)-mediated apoB-containing lipoprotein uptake. Phosphatidylinositol 3-kinase (PI3K) activation by insulin increases sterol regulatory element-binding protein (SREBP)-1 and LDLr expression and inhibits apoB secretion in hepatocytes. Thus, we determined whether naringenin activates this pathway. Insulin and naringenin induced PI3K-dependent increases in cytosolic and nuclear SREBP-1 and LDLr expression. Similar PI3K-mediated increases in SREBP-1 were observed in McA-RH7777 rat hepatoma cells, which express predominantly SREBP-1c. Reductions in HepG2 cell media apoB with naringenin were partially attenuated by wortmannin, whereas the effect of insulin was completely blocked. Both treatments reduced apoB100 secretion in wild-type and LDLr(-/-) mouse hepatocytes to the same extent. Insulin and naringenin increased HepG2 cell PI3K activity and decreased insulin receptor substrate (IRS)-2 levels. In sharp contrast to insulin, naringenin did not induce tyrosine phosphorylation of IRS-1. We conclude that naringenin increases LDLr expression in HepG2 cells via PI3K-mediated upregulation of SREBP-1, independent of IRS-1 phosphorylation. Although this pathway may not regulate apoB secretion in primary hepatocytes, PI3K activation by this novel mechanism may explain the insulin-like effects of naringenin in vivo.

  6. Estrogen Receptor β Signaling through Phosphatase and Tensin Homolog/Phosphoinositide 3-Kinase/Akt/Glycogen Synthase Kinase 3 Down-Regulates Blood-Brain Barrier Breast Cancer Resistance Protein

    PubMed Central

    Hartz, A. M. S.; Madole, E. K.; Miller, D. S.

    2010-01-01

    Breast cancer resistance protein (BCRP) is an ATP-driven efflux pump at the blood-brain barrier that limits central nervous system pharmacotherapy. Our previous studies showed rapid loss of BCRP transport activity in rat brain capillaries exposed to low concentrations of 17-β-estradiol (E2); this occurred without acute change in BCRP protein expression. Here, we describe a pathway through which sustained, extended exposure to E2 signals down-regulation of BCRP at the blood-brain barrier. Six-hour exposure of isolated rat and mouse brain capillaries to E2 reduced BCRP transport activity and BCRP monomer and dimer expression. Experiments with brain capillaries from estrogen receptor (ER)α and ERβ knockout mice and with ER agonists and antagonists showed that E2 signaled through ERβ to down-regulate BCRP expression. In rat brain capillaries, E2 increased unphosphorylated, active phosphatase and tensin homolog (PTEN); decreased phosphorylated, active Akt; and increased phosphorylated, active glycogen synthase kinase (GSK)3. Consistent with this, inhibition of phosphoinositide 3-kinase (PI3K) or Akt decreased BCRP activity and protein expression, and inhibition of PTEN or GSK3 reversed the E2 effect on BCRP. Lactacystin, a proteasome inhibitor, abolished E2-mediated BCRP down-regulation, suggesting internalization followed by transporter degradation. Dosing mice with E2 reduced BCRP activity in brain capillaries within 1 h; this reduction persisted for 24 h. BCRP protein expression in brain capillaries was unchanged 1 h after E2 dosing but was substantially reduced 6 and 24 h after dosing. Thus, E2 signals through ERβ, PTEN/PI3K/Akt/GSK3 to stimulate proteasomal degradation of BCRP. These in vitro and in vivo findings imply that E2-mediated down-regulation of blood-brain barrier BCRP has the potential to increase brain uptake of chemotherapeutics that are BCRP substrates. PMID:20460386

  7. First-in-human Phase I study of Pictilisib (GDC-0941), a potent pan-class I phosphatidylinositol-3-kinase (PI3K) inhibitor, in patients with advanced solid tumors

    PubMed Central

    Baird, Richard; Kristeleit, Rebecca; Shah, Krunal; Moreno, Victor; Clarke, Paul A.; Raynaud, Florence I.; Levy, Gallia; Ware, Joseph A; Mazina, Kathryn; Lin, Ray; Wu, Jenny; Fredrickson, Jill; Spoerke, Jill M; Lackner, Mark R; Yan, Yibing; Friedman, Lori S.; Kaye, Stan B.; Derynck, Mika K.; Workman, Paul; de Bono, Johann S.

    2014-01-01

    Purpose This first-in-human dose-escalation trial evaluated the safety, tolerability, maximal tolerated dose (MTD), dose limiting toxicities (DLTs), pharmacokinetics, pharmacodynamics and preliminary clinical activity of pictilisib (GDC-0941), an oral, potent and selective inhibitor of the Class I phosphatidylinositol-3-kinases (PI3K). Patients and Methods Sixty patients with solid tumors received pictilisib at 14 dose levels from 15 to 450mg once-daily, initially on days 1-21 every 28 days and later, utilizing continuous dosing for selected dose levels. Pharmacodynamic studies incorporated 18F-FDG-PET, and assessment of phosphorylated AKT and S6 ribosomal protein in platelet-rich plasma and tumor tissue. Results Pictilisib was well-tolerated. The most common toxicities were grade 1-2 nausea, rash and fatigue while the DLT was grade 3 maculopapular rash (450mg, 2 of 3 patients; 330mg, 1 of 7 patients). The pharmacokinetic profile was dose-proportional and supported once-daily dosing. Levels of phosphorylated serine-473 AKT were suppressed >90% in platelet rich plasma at 3 hours post-dose at the MTD and in tumor at pictilisib doses associated with AUC >20uM.hr. Significant increase in plasma insulin and glucose levels, and >25% decrease in 18F-FDG uptake by PET in 7 of 32 evaluable patients confirmed target modulation. A patient with V600E BRAF mutant melanoma and another with platinum-refractory epithelial ovarian cancer exhibiting PTEN loss and PIK3CA amplification demonstrated partial response by RECIST and GCIG-CA125 criteria, respectively. Conclusion Pictilisib was safely administered with a dose-proportional pharmacokinetic profile, on-target pharmacodynamic activity at dose levels ≥100mg and signs of antitumor activity. The recommended Phase II dose was continuous dosing at 330mg once-daily. PMID:25370471

  8. Integrin αvβ3 mediates the synergetic regulation of core-binding factor α1 transcriptional activity by gravity and insulin-like growth factor-1 through phosphoinositide 3-kinase signaling.

    PubMed

    Dai, Zhongquan; Guo, Feima; Wu, Feng; Xu, Hongjie; Yang, Chao; Li, Jinqiao; Liang, Peilong; Zhang, Hongyu; Qu, Lina; Tan, Yingjun; Wan, Yumin; Li, Yinghui

    2014-12-01

    Mechanical stimulation and biological factors coordinately regulate bone development and regeneration; however, the underlying mechanisms are poorly understood. Microgravity induces bone loss, which may be partly related to the development of resistance to local cytokines, including insulin-like growth factor 1 (IGF-1). Here, we report the involvement of integrin αvβ3 in microgravity-associated bone loss. An established OSE-3T3 cell model was stably transfected with a 6OSE2 (Osteoblast-Specific Element 2)-luciferase reporter and cultured under simulated microgravity (SMG) and hypergravity (HG) conditions in the presence or absence of IGF-1, the disintegrin echistatin, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, or combinations of these agents. Activity of core-binding factor α1 (Cbfa1), an essential transcription factor for osteoblastic differentiation and osteogenesis, was reflected by luciferase activity. Different gravity conditions affected the induction of IGF-1 and subsequent effects on Cbfa1 transcription activity. SMG and HG influenced the expression and activity of integrin αvβ3 and phosphorylation level of p85. LY294002 inhibited the effects of HG or IGF-1 on Cbfa1 activity, indicating that HG and IGF-1 could increase Cbfa1 activity via PI3K signaling. Inhibition of integrin αvβ3 by echistatin attenuated the induction of IGF-1 and thus its effect on Cbfa1 activity under normal and HG conditions. Co-immunoprecipitation demonstrated that integrin β3 interacted with insulin receptor substrate 1, and that this interaction was decreased under SMG and increased under HG conditions. These results suggest that integrin αvβ3 mediates the synergetic regulation of Cbfa1 transcription activity by gravity and IGF-1 via PI3K signaling.

  9. c-Met Overexpression Contributes to the Acquired Apoptotic Resistance of Nonadherent Ovarian Cancer Cells through a Cross Talk Mediated by Phosphatidylinositol 3-Kinase and Extracellular Signal-Regulated Kinase 1/212

    PubMed Central

    Tang, Maggie K S; Zhou, Hong Y; Yam, Judy W P; Wong, Alice S T

    2010-01-01

    Ovarian cancer is the most lethal gynecologic cancer mainly because of widespread peritoneal dissemination and malignant ascites. Key to this is the capacity of tumor cells to escape suspension-induced apoptosis (anoikis), which also underlies their resistance to chemotherapy. Here, we used a nonadherent cell culture model to investigate the molecular mechanisms of apoptotic resistance of ovarian cancer cells that may mimic the chemoresistance found in solid tumors. We found that ovarian cancer cells acquired a remarkable resistance to anoikis and apoptosis induced by exposure to clinically relevant doses of two front-line chemotherapeutic drugs cisplatin and paclitaxel when grown in three-dimensional than monolayer cultures. Inhibition of the hepatocyte growth factor (HGF) receptor c-Met, which is frequently overexpressed in ovarian cancer, by a specific inhibitor or small interfering RNA blocked the acquired anoikis resistance and restored chemosensitivity in three-dimensional not in two-dimensional cultures. These effects were found to be dependent on both phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) 1/2 signaling pathways. Inhibitors of PI3K/Akt abrogated ERK1/2 activation and its associated anoikis resistance in response to HGF, suggesting a signaling relay between these two pathways. Furthermore, we identified a central role of Ras as a mechanism of this cross talk. Interestingly, Ras did not lie upstream of PI3K/Akt, whereas PI3K/Akt signaling to ERK1/2 involved Ras. These findings shed new light on the apoptotic resistance mechanism of nonadherent ovarian cancer ascites cells and may have important clinical implications. PMID:20126471

  10. Triptolide, a diterpenoid triepoxide, induces antitumor proliferation via activation of c-Jun NH{sub 2}-terminal kinase 1 by decreasing phosphatidylinositol 3-kinase activity in human tumor cells

    SciTech Connect

    Miyata, Yoshiki; Sato, Takashi . E-mail: satotak@ps.toyaku.ac.jp; Ito, Akira

    2005-11-04

    Triptolide, a diterpenoid triepoxide extracted from the Chinese herb Tripterygium wilfordii Hook f., exerts antitumorigenic actions against several tumor cells, but the intracellular target signal molecule(s) for this antitumorigenesis activity of triptolide remains to be identified. In the present study, we demonstrated that triptolide, in a dose-dependent manner, inhibited the proliferation of human fibrosarcoma HT-1080, human squamous carcinoma SAS, and human uterine cervical carcinoma SKG-II cells. In addition, triptolide was found to decrease phosphatidylinositol 3-kinase (PI3K) activity. A PI3K inhibitor, LY-294002, mimicked the triptolide-induced antiproliferative activity in HT-1080, SAS, and SKG-II cells. There was no change in the activity of Akt or protein kinase C (PKC), both of which are downstream effectors in the PI3K pathway. Furthermore, the phosphorylation of Ras, Raf, and mitogen-activated protein/extracellular signal-regulated kinase 1/2 was not modified in HT-1080 cells treated with triptolide. However, the phosphorylation of c-Jun NH{sub 2}-terminal kinase 1 (JNK1) was found to increase in both triptolide- and LY-294002-treated cells. Furthermore, the triptolide-induced inhibition of HT-1080 cell proliferation was not observed by JNK1 siRNA-treatment. These results provide novel evidence that PI3K is a crucial target molecule in the antitumorigenic action of triptolide. They further suggest a possible triptolide-induced inhibitory signal for tumor cell proliferation that is initiated by the decrease in PI3K activity, which in turn leads to the augmentation of JNK1 phosphorylation via the Akt and/or PKC-independent pathway(s). Moreover, it is likely that the activation of JNK1 is required for the triptolide-induced inhibition of tumor proliferation.

  11. L-leucine availability regulates phosphatidylinositol 3-kinase, p70 S6 kinase and glycogen synthase kinase-3 activity in L6 muscle cells: evidence for the involvement of the mammalian target of rapamycin (mTOR) pathway in the L-leucine-induced up-regulation of system A amino acid transport.

    PubMed Central

    Peyrollier, K; Hajduch, E; Blair, A S; Hyde, R; Hundal, H S

    2000-01-01

    Amino acid availability is known to regulate diverse cell processes including the activation of p70 S6 kinase, initiation factors involved in mRNA translation, gene expression and cellular amino acid uptake. Essential amino acids, in particular the branched-chain amino acids (e.g. leucine), have been shown to be the dominant players in mediating these effects, although the precise nature by which they regulate these processes remain poorly understood. In this study we have investigated the mechanisms involved in the leucine-induced modulation of p70 S6 kinase and addressed whether this kinase participates in the up-regulation of the System A amino acid transporter in L6 muscle cells. Incubation of muscle cells that had been amino acid-deprived for 1 h with L-leucine (2 mM) led to a rapid (>2-fold) activation of p70 S6 kinase, which was suppressed by both wortmannin and rapamycin. Consistent with this finding, addition of leucine caused a rapid ( approximately 5-fold) but transient stimulation of phosphatidylinositol 3-kinase (PI3K). PI3K activation was inhibited by wortmannin and was not dependent upon insulin receptor substrate-1 activation. Unlike stimulation by insulin, activation of neither protein kinase B nor p42/p44 mitogen-activated protein kinase accompanied the leucine-induced stimulation of PI3K. However, the leucine-induced activation of PI3K and p70 S6 kinase did result in the concomitant inactivation of glycogen synthase kinase-3 (GSK-3). Leucine enhanced System A transport by approximately 50%. We have shown previously that this stimulation is protein-synthesis-dependent and in the current study we show that it was blocked by both wortmannin and rapamycin. Our findings indicate that PI3K and the mammalian target of rapamycin are components of a nutrient signalling pathway that regulates the activation of p70 S6 kinase and induction of System A in L6 cells. The activation of this pathway by leucine is also responsible for the inactivation of GSK-3

  12. Genes and Gene Therapy

    MedlinePlus

    ... correctly, a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... or prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  13. First-in-human phase I study of copanlisib (BAY 80-6946), an intravenous pan-class I phosphatidylinositol 3-kinase inhibitor, in patients with advanced solid tumors and non-Hodgkin's lymphomas

    PubMed Central

    Patnaik, A.; Appleman, L. J.; Tolcher, A. W.; Papadopoulos, K. P.; Beeram, M.; Rasco, D. W.; Weiss, G. J.; Sachdev, J. C.; Chadha, M.; Fulk, M.; Ejadi, S.; Mountz, J. M.; Lotze, M. T.; Toledo, F. G. S.; Chu, E.; Jeffers, M.; Peña, C.; Xia, C.; Reif, S.; Genvresse, I.; Ramanathan, R. K.

    2016-01-01

    Background To evaluate the safety, tolerability, pharmacokinetics, and maximum tolerated dose (MTD) of copanlisib, a phosphatidylinositol 3-kinase inhibitor, in patients with advanced solid tumors or non-Hodgkin's lymphoma (NHL). Patients and methods Phase I dose-escalation study including patients with advanced solid tumors or NHL, and a cohort of patients with type 2 diabetes mellitus. Patients received three weekly intravenous infusions of copanlisib per 28-day cycle over the dose range 0.1–1.2 mg/kg. Plasma copanlisib levels were analyzed for pharmacokinetics. Biomarker analysis included PIK3CA, KRAS, BRAF, and PTEN mutational status and PTEN immunohistochemistry. Whole-body [18F]-fluorodeoxyglucose positron emission tomography (18FDG-PET) was carried out at baseline and following the first dose to assess early pharmacodynamic effects. Plasma glucose and insulin levels were evaluated serially. Results Fifty-seven patients received treatment. The MTD was 0.8 mg/kg copanlisib. The most frequent treatment-related adverse events were nausea and transient hyperglycemia. Copanlisib exposure was dose-proportional with no accumulation; peak exposure positively correlated with transient hyperglycemia post-infusion. Sixteen of 20 patients treated at the MTD had reduced 18FDG-PET uptake; 7 (33%) had a reduction >25%. One patient achieved a complete response (CR; endometrial carcinoma exhibiting both PIK3CA and PTEN mutations and complete PTEN loss) and two had a partial response (PR; both metastatic breast cancer). Among the nine NHL patients, all six with follicular lymphoma (FL) responded (one CR and five PRs) and one patient with diffuse large B-cell lymphoma had a PR by investigator assessment; two patients with FL who achieved CR (per post hoc independent radiologic review) were on treatment >3 years. Conclusion Copanlisib, dosed intermittently on days 1, 8, and 15 of a 28-day cycle, was well tolerated and the MTD was determined to be 0.8 mg/kg. Copanlisib

  14. Different inhibition of Gβγ-stimulated class IB phosphoinositide 3-kinase (PI3K) variants by a monoclonal antibody. Specific function of p101 as a Gβγ-dependent regulator of PI3Kγ enzymatic activity.

    PubMed

    Shymanets, Aliaksei; Prajwal; Vadas, Oscar; Czupalla, Cornelia; LoPiccolo, Jaclyn; Brenowitz, Michael; Ghigo, Alessandra; Hirsch, Emilio; Krause, Eberhard; Wetzker, Reinhard; Williams, Roger L; Harteneck, Christian; Nürnberg, Bernd

    2015-07-01

    Class IB phosphoinositide 3-kinases γ (PI3Kγ) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled receptors (GPCRs). PI3Kγ variants have one catalytic p110γ subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110γ and p101-p110γ allowing integration into distinct signalling pathways. Pharmacological tools enabling distinct modulation of the two variants are missing. The ability of an anti-p110γ monoclonal antibody [mAb(A)p110γ] to block PI3Kγ enzymatic activity attracted us to characterize this tool in detail using purified proteins. In order to get insight into the antibody-p110γ interface, hydrogen-deuterium exchange coupled to MS (HDX-MS) measurements were performed demonstrating binding of the monoclonal antibody to the C2 domain in p110γ, which was accompanied by conformational changes in the helical domain harbouring the Gβγ-binding site. We then studied the modulation of phospholipid vesicles association of PI3Kγ by the antibody. p87-p110γ showed a significantly reduced Gβγ-mediated phospholipid recruitment as compared with p101-p110γ. Concomitantly, in the presence of mAb(A)p110γ, Gβγ did not bind to p87-p110γ. These data correlated with the ability of the antibody to block Gβγ-stimulated lipid kinase activity of p87-p110γ 30-fold more potently than p101-p110γ. Our data argue for differential regulatory functions of the non-catalytic subunits and a specific Gβγ-dependent regulation of p101 in PI3Kγ activation. In this scenario, we consider the antibody as a valuable tool to dissect the distinct roles of the two PI3Kγ variants downstream of GPCRs.

  15. Phosphatidylinositol 3-Kinase-Associated Protein (PI3KAP)/XB130 Crosslinks Actin Filaments through Its Actin Binding and Multimerization Properties In Vitro and Enhances Endocytosis in HEK293 Cells

    PubMed Central

    Yamanaka, Daisuke; Akama, Takeshi; Chida, Kazuhiro; Minami, Shiro; Ito, Koichi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2016-01-01

    Actin-crosslinking proteins control actin filament networks and bundles and contribute to various cellular functions including regulation of cell migration, cell morphology, and endocytosis. Phosphatidylinositol 3-kinase-associated protein (PI3KAP)/XB130 has been reported to be localized to actin filaments (F-actin) and required for cell migration in thyroid carcinoma cells. Here, we show a role for PI3KAP/XB130 as an actin-crosslinking protein. First, we found that the carboxyl terminal region of PI3KAP/XB130 containing amino acid residues 830–840 was required and sufficient for localization to F-actin in NIH3T3 cells, and this region is directly bound to F-actin in vitro. Moreover, actin-crosslinking assay revealed that recombinant PI3KAP/XB130 crosslinked F-actin. In general, actin-crosslinking proteins often multimerize to assemble multiple actin-binding sites. We then investigated whether PI3KAP/XB130 could form a multimer. Blue native-PAGE analysis showed that recombinant PI3KAP/XB130 was detected at 250–1200 kDa although the molecular mass was approximately 125 kDa, suggesting that PI3KAP/XB130 formed multimers. Furthermore, we found that the amino terminal 40 amino acids were required for this multimerization by co-immunoprecipitation assay in HEK293T cells. Deletion mutants of PI3KAP/XB130 lacking the actin-binding region or the multimerizing region did not crosslink actin filaments, indicating that actin binding and multimerization of PI3KAP/XB130 were necessary to crosslink F-actin. Finally, we examined roles of PI3KAP/XB130 on endocytosis, an actin-related biological process. Overexpression of PI3KAP/XB130 enhanced dextran uptake in HEK 293 cells. However, most of the cells transfected with the deletion mutant lacking the actin-binding region incorporated dextran to a similar extent as control cells. Taken together, these results demonstrate that PI3KAP/XB130 crosslinks F-actin through both its actin-binding region and multimerizing region and

  16. Differential Modulation of Brainstem Phosphatidylinositol 3-Kinase/Akt and Extracellular Signal-Regulated Kinase 1/2 Signaling Underlies WIN55,212-2 Centrally Mediated Pressor Response in Conscious Rats

    PubMed Central

    Ibrahim, Badr Mostafa

    2012-01-01

    Our recent study demonstrated that central cannabinoid receptor 1 (CB1R) activation caused dose-related pressor response in conscious rats, and reported studies implicated the brainstem phosphatidylinositol 3-kinase (PI3K)/Akt-extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in blood pressure control. Therefore, in this study, we tested the hypothesis that the modulation of brainstem PI3K/Akt-ERK1/2 signaling plays a critical role in the central CB1R-mediated pressor response. In conscious freely moving rats, the pressor response elicited by intracisternal (i.c.) (R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate salt (WIN55,212-2) (15 μg) was associated with significant increases in ERK1/2 phosphorylation in the rostral ventrolateral medulla (RVLM) and the nucleus tractus solitarius (NTS). In contrast, Akt phosphorylation was significantly reduced in the same neuronal pools. Pretreatment with the selective CB1R antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) (30 μg i.c.) attenuated the neurochemical responses elicited by central CB1R activation. Furthermore, pretreatment with the ERK/mitogen-activated protein kinase kinase inhibitor 2′-amino-3′-methoxyflavone (PD98059) (5 μg i.c.) abrogated WIN55,212-2-evoked increases in blood pressure and neuronal ERK1/2 phosphorylation but not the reduction in Akt phosphorylation. On the other hand, prior PI3K inhibition with wortmannin (0.4 μg i.c.) exacerbated the WIN55,212-2 (7.5 and 15 μg i.c.) dose-related increases in blood pressure and ERK1/2 phosphorylation in the RVLM. The present neurochemical and integrative studies yield new insight into the critical role of two brainstem kinases, PI3K and ERK1/2, in the pressor response elicited by central CB1R activation in conscious rats. PMID:21946192

  17. Differential usage of signal transduction pathways defines two types of serum response factor target gene.

    PubMed

    Gineitis, D; Treisman, R

    2001-07-06

    Activation of the transcription factor serum response factor (SRF) is dependent on Rho-controlled changes in actin dynamics. We used pathway-specific inhibitors to compare the roles of actin dynamics, extracellular signal-regulated kinase (ERK) signaling, and phosphatidylinositol 3-kinase in signaling either to SRF itself or to four cellular SRF target genes. Serum, lysophosphatidic acid, platelet-derived growth factor, and phorbol 12-myristate 13-acetate (PMA) each activated transcription of a stably integrated SRF reporter gene dependent on functional RhoA GTPase. Inhibition of mitogen-activated protein kinase-ERK kinase (MEK) signalling reduced activation of the SRF reporter by all stimuli by about 50%, except for PMA, which was effectively blocked. Inhibition of phosphatidylinositol 3-kinase slightly reduced reporter activation by serum and lysophosphatidic acid but substantially inhibited activation by platelet-derived growth factor and PMA. Reporter induction by all stimuli was absolutely dependent on actin dynamics. Regulation of the SRF (srf) and vinculin (vcl) genes was similar to that of the SRF reporter gene; activation by all stimuli was Rho-dependent and required actin dynamics but was largely independent of MEK activity. In contrast, activation of fos and egr1 occurred independently of RhoA and actin polymerization but was almost completely dependent on MEK activation. These results show that at least two classes of SRF target genes can be distinguished on the basis of their relative sensitivity to RhoA-actin and MEK-ERK signaling pathways.

  18. Genetic basis and gene therapy trials for thyroid cancer.

    PubMed

    Al-Humadi, Hussam; Zarros, Apostolos; Al-Saigh, Rafal; Liapi, Charis

    2010-01-01

    Gene therapy is regarded as one of the most promising novel therapeutic approaches for hopeless cases of thyroid cancer and those not responding to traditional treatment. In the last two decades, many studies have focused on the genetic factors behind the origin and the development of thyroid cancer, in order to investigate and shed more light on the molecular pathways implicated in different differentiated or undifferentiated types of thyroid tumors. We, herein, review the current data on the main genes that have been proven to (or thought to) be implicated in thyroid cancer etiology, and which are involved in several well-known signaling pathways (such as the mitogen-activated protein kinase and phosphatidylinositol-3-kinase/Akt pathways). Moreover, we review the results of the efforts made through multiple gene therapy trials, via several gene therapy approaches/strategies, on different thyroid carcinomas. Our review leads to the conclusion that future research efforts should seriously consider gene therapy for the treatment of thyroid cancer, and, thus, should: (a) shed more light on the molecular basis of thyroid cancer tumorigenesis, (b) focus on the development of novel gene therapy approaches that can achieve the required antitumoral efficacy with minimum normal tissue toxicity, as well as (c) perform more gene therapy clinical trials, in order to acquire more data on the efficacy of the examined approaches and to record the provoked adverse effects.

  19. Predicting the structures of complexes between phosphoinositide 3-kinase (PI3K) and romidepsin-related compounds for the drug design of PI3K/histone deacetylase dual inhibitors using computational docking and the ligand-based drug design approach.

    PubMed

    Oda, Akifumi; Saijo, Ken; Ishioka, Chikashi; Narita, Koichi; Katoh, Tadashi; Watanabe, Yurie; Fukuyoshi, Shuichi; Takahashi, Ohgi

    2014-11-01

    Predictions of the three-dimensional (3D) structures of the complexes between phosphoinositide 3-kinase (PI3K) and two inhibitors were conducted using computational docking and the ligand-based drug design approach. The obtained structures were refined by structural optimizations and molecular dynamics (MD) simulations. The ligands were located deep inside the ligand binding pocket of the p110α subunit of PI3K, and the hydrogen bond formations and hydrophobic effects of the surrounding amino acids were predicted. Although rough structures were obtained for the PI3K-inhibitor complexes before the MD simulations, the refinement of the structures by these simulations clarified the hydrogen bonding patterns of the complexes.

  20. Studying Genes

    MedlinePlus

    ... Area What are genes? Genes are sections of DNA that contain instructions for making the molecules—many ... material in an organism. This includes genes and DNA elements that control the activity of genes. Does ...