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Sample records for 3-kinase pathway-dependent keratinocyte

  1. Phosphoinositide 3-kinase signaling to Akt promotes keratinocyte differentiation versus death.

    PubMed

    Calautti, Enzo; Li, Jian; Saoncella, Stefania; Brissette, Janice L; Goetinck, Paul F

    2005-09-23

    Signaling pathways regulating the differentiation program of epidermal cells overlap widely with those activated during apoptosis. How differentiating cells remain protected from premature death, however, is still poorly defined. We show here that the phosphoinositide 3-kinase (PI3K)/Akt pathway is activated at early stages of mouse keratinocyte differentiation both in culture and in the intact epidermis in vivo. Expression of active Akt in keratinocytes promotes growth arrest and differentiation, whereas pharmacological blockade of PI3K inhibits the expression of "late" differentiation markers and leads to death of cells that would otherwise differentiate. Mechanistically, the activation of the PI3K/Akt pathway in keratinocyte differentiation depends on the activity of the epidermal growth factor receptor and Src families of tyrosine kinases and the engagement of E-cadherin-mediated adhesion. During this process, PI3K associates increasingly with cadherin-catenin protein complexes bearing tyrosine phosphorylated YXXM motifs. Thus, the PI3K signaling pathway regulates the choice between epidermal cell differentiation and death at the cross-talk between tyrosine kinases and cadherin-associated catenins.

  2. 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.

  3. 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

  4. 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.

  5. Targeting the phosphoinositide 3-kinase pathway in hematologic malignancies

    PubMed Central

    Jabbour, Elias; Ottmann, Oliver G.; Deininger, Michael; Hochhaus, Andreas

    2014-01-01

    The phosphoinositide 3-kinase pathway represents an important anticancer target because it has been implicated in cancer cell growth, survival, and motility. Recent studies show that PI3K may also play a role in the development of resistance to currently available therapies. In a broad range of cancers, various components of the phosphoinositide 3-kinase signaling axis are genetically modified, and the pathway can be activated through many different mechanisms. The frequency of genetic alterations in the phosphoinositide 3-kinase pathway, coupled with the impact in oncogenesis and disease progression, make this signaling axis an attractive target in anticancer therapy. A better understanding of the critical function of the phosphoinositide 3-kinase pathway in leukemias and lymphomas has led to the clinical evaluation of novel rationally designed inhibitors in this setting. Three main categories of phosphoinositide 3-kinase inhibitors have been developed so far: agents that target phosphoinositide 3-kinase and mammalian target of rapamycin (dual inhibitors), pan-phosphoinositide 3-kinase inhibitors that target all class I isoforms, and isoform-specific inhibitors that selectively target the α, -β, -γ, or -δ isoforms. Emerging data highlight the promise of phosphoinositide 3-kinase inhibitors in combination with other therapies for the treatment of patients with hematologic malignancies. Further evaluation of phosphoinositide 3-kinase inhibitors in first-line or subsequent regimens may improve clinical outcomes. This article reviews the role of phosphoinositide 3-kinase signaling in hematologic malignancies and the potential clinical utility of inhibitors that target this pathway. PMID:24425689

  6. The TRAF-interacting protein (TRIP) is a regulator of keratinocyte proliferation.

    PubMed

    Almeida, Stéphanie; Ryser, Stephan; Obarzanek-Fojt, Magdalena; Hohl, Daniel; Huber, Marcel

    2011-02-01

    The TRAF-interacting protein (TRIP/TRAIP) is a RING-type E3 ubiquitin ligase inhibiting tumor necrosis factor-α (TNF-α)-mediated NF-κB activation. TRIP ablation results in early embryonic lethality in mice. To investigate TRIP function in epidermis, we examined its expression and the effect of TRIP knockdown (KD) in keratinocytes. TRIP mRNA expression was strongly downregulated in primary human keratinocytes undergoing differentiation triggered by high cell density or high calcium. Short-term phorbol-12-myristate-13-acetate (TPA) treatment or inhibition of phosphatidylinositol-3 kinase signaling in proliferative keratinocytes suppressed TRIP transcription. Inhibition by TPA was protein kinase C dependent. Keratinocytes undergoing KD of TRIP expression by lentiviral short-hairpin RNA (shRNA; T4 and T5) had strongly reduced proliferation rates compared with control shRNA. Cell cycle analysis demonstrated that TRIP-KD caused growth arrest in the G1/S phase. Keratinocytes with TRIP-KD resembled differentiated cells consistent with the augmented expression of differentiation markers keratin 1 and filaggrin. Luciferase-based reporter assays showed no increase in NF-κB activity in TRIP-KD keratinocytes, indicating that NF-κB activity in keratinocytes is not regulated by TRIP. TRIP expression was increased by ∼2-fold in basal cell carcinomas compared with normal skin. These results underline the important role of TRIP in the regulation of cell cycle progression and the tight linkage of its expression to keratinocyte proliferation.

  7. 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

  8. 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

  9. Modulation of keratinocyte expression of antioxidants by 4-hydroxynonenal, a lipid peroxidation end product.

    PubMed

    Zheng, Ruijin; Heck, Diane E; Mishin, Vladimir; Black, Adrienne T; Shakarjian, Michael P; Kong, Ah-Ng Tony; Laskin, Debra L; Laskin, Jeffrey D

    2014-03-01

    4-Hydroxynonenal (4-HNE) is a lipid peroxidation end product generated in response to oxidative stress in the skin. Keratinocytes contain an array of antioxidant enzymes which protect against oxidative stress. In these studies, we characterized 4-HNE-induced changes in antioxidant expression in mouse keratinocytes. Treatment of primary mouse keratinocytes and PAM 212 keratinocytes with 4-HNE increased mRNA expression for heme oxygenase-1 (HO-1), catalase, NADPH:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) A1-2, GSTA3 and GSTA4. In both cell types, HO-1 was the most sensitive, increasing 86-98 fold within 6h. Further characterization of the effects of 4-HNE on HO-1 demonstrated concentration- and time-dependent increases in mRNA and protein expression which were maximum after 6h with 30 μM. 4-HNE stimulated keratinocyte Erk1/2, JNK and p38 MAP kinases, as well as PI3 kinase. Inhibition of these enzymes suppressed 4-HNE-induced HO-1 mRNA and protein expression. 4-HNE also activated Nrf2 by inducing its translocation to the nucleus. 4-HNE was markedly less effective in inducing HO-1 mRNA and protein in keratinocytes from Nrf2-/- mice, when compared to wild type mice, indicating that Nrf2 also regulates 4-HNE-induced signaling. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that 4-HNE-induced HO-1 is localized in keratinocyte caveolae. Treatment of the cells with methyl-β-cyclodextrin, which disrupts caveolar structure, suppressed 4-HNE-induced HO-1. These findings indicate that 4-HNE modulates expression of antioxidant enzymes in keratinocytes, and that this can occur by different mechanisms. Changes in expression of keratinocyte antioxidants may be important in protecting the skin from oxidative stress. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. T-plastin expression downstream to the calcineurin/NFAT pathway is involved in keratinocyte migration.

    PubMed

    Brun, Cécilia; Demeaux, Agathe; Guaddachi, Frédéric; Jean-Louis, Francette; Oddos, Thierry; Bagot, Martine; Bensussan, Armand; Jauliac, Sébastien; Michel, Laurence

    2014-01-01

    Cutaneous wound healing requires keratinocyte proliferation, migration and differentiation to restore the barrier function of the skin. The calcineurin/nuclear factor of activated-T-cell (NFAT) signaling pathway has been recently shown to be involved in keratinocyte growth, differentiation and migration. It is induced by an increased intracellular calcium rate and its inhibition results in decreased capacities of keratinocytes to migrate. Nevertheless, the link between calcineurin activation and keratinocyte migration remains unknown. Recently, Orai1, a pore subunit of a store-operated calcium channel that favors calcium influx, was shown to play a critical role to control proliferation and migration of basal keratinocytes. Of interest, the actin-bundling T-plastin is crucial in cell motility through cross-linking to actin filament and its synthesis was shown to be induced by calcium influx and regulated by the calcineurin/NFAT pathway in tumor Sezary cells. We investigated herein the role of the calcineurin/NFAT pathway-dependent T-plastin in keratinocyte migration, by quantifying T-plastin expression in keratinocytes and by analyzing their migration under calcineurin inhibition or knockdown of NFAT2 or T-plastin. We did confirm the role of the calcineurin/NFAT pathway in keratinocyte migration as shown by their decreased capacities to migrate after FK506 treatment or siNFAT2 transfection in both scratching and Boyden assays. The expression of NFAT2 and T-plastin in keratinocytes was decreased under FK506 treatment, suggesting that T-plastin plays a role in keratinocyte migration downstream to the calcineurin/NFAT pathway. Accordingly, siRNA knockdown of T-plastin expression also decreased their migration capacities. Actin lamellipodia formation as well as FAK and β6-integrin expression were also significantly decreased after treatment with FK506 or siRNA, reinforcing that NFAT2-dependent T-plastin expression plays a role in keratinocyte migration. These results

  11. 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

  12. 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

  13. Transmembrane Collagen XVII Modulates Integrin Dependent Keratinocyte Migration via PI3K/Rac1 Signaling

    PubMed Central

    Löffek, Stefanie; Sigloch, Florian Christoph; Schilling, Oliver; Tasanen, Kaisa; Bruckner-Tuderman, Leena; Franzke, Claus-Werner

    2014-01-01

    The hemidesmosomal transmembrane component collagen XVII (ColXVII) plays an important role in the anchorage of the epidermis to the underlying basement membrane. However, this adhesion protein seems to be also involved in the regulation of keratinocyte migration, since its expression in these cells is strongly elevated during reepithelialization of acute wounds and in the invasive front of squamous cell carcinoma, while its absence in ColXVII-deficient keratinocytes leads to altered cell motility. Using a genetic model of murine Col17a1−/− keratinocytes we elucidated ColXVII mediated signaling pathways in cell adhesion and migration. Col17a1−/− keratinocytes exhibited increased spreading on laminin 332 and accelerated, but less directed cell motility. These effects were accompanied by increased expression of the integrin subunits β4 and β1. The migratory phenotype, as evidenced by formation of multiple unstable lamellipodia, was associated with enhanced phosphoinositide 3-kinase (PI3K) activity. Dissection of the signaling pathway uncovered enhanced phosphorylation of the β4 integrin subunit and the focal adhesion kinase (FAK) as activators of PI3K. This resulted in elevated Rac1 activity as a downstream consequence. These results provide mechanistic evidence that ColXVII coordinates keratinocyte adhesion and directed motility by interfering integrin dependent PI3K activation and by stabilizing lamellipodia at the leading edge of reepithelializing wounds and in invasive squamous cell carcinoma. PMID:24505282

  14. Modulation of keratinocyte expression of antioxidants by 4-hydroxynonenal, a lipid peroxidation end product

    SciTech Connect

    Zheng, Ruijin; Heck, Diane E.; Mishin, Vladimir; Black, Adrienne T.; Shakarjian, Michael P.; Kong, Ah-Ng Tony; Laskin, Debra L.; Laskin, Jeffrey D.

    2014-03-01

    4-Hydroxynonenal (4-HNE) is a lipid peroxidation end product generated in response to oxidative stress in the skin. Keratinocytes contain an array of antioxidant enzymes which protect against oxidative stress. In these studies, we characterized 4-HNE-induced changes in antioxidant expression in mouse keratinocytes. Treatment of primary mouse keratinocytes and PAM 212 keratinocytes with 4-HNE increased mRNA expression for heme oxygenase-1 (HO-1), catalase, NADPH:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) A1-2, GSTA3 and GSTA4. In both cell types, HO-1 was the most sensitive, increasing 86–98 fold within 6 h. Further characterization of the effects of 4-HNE on HO-1 demonstrated concentration- and time-dependent increases in mRNA and protein expression which were maximum after 6 h with 30 μM. 4-HNE stimulated keratinocyte Erk1/2, JNK and p38 MAP kinases, as well as PI3 kinase. Inhibition of these enzymes suppressed 4-HNE-induced HO-1 mRNA and protein expression. 4-HNE also activated Nrf2 by inducing its translocation to the nucleus. 4-HNE was markedly less effective in inducing HO-1 mRNA and protein in keratinocytes from Nrf2 −/− mice, when compared to wild type mice, indicating that Nrf2 also regulates 4-HNE-induced signaling. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that 4-HNE-induced HO-1 is localized in keratinocyte caveolae. Treatment of the cells with methyl-β-cyclodextrin, which disrupts caveolar structure, suppressed 4-HNE-induced HO-1. These findings indicate that 4-HNE modulates expression of antioxidant enzymes in keratinocytes, and that this can occur by different mechanisms. Changes in expression of keratinocyte antioxidants may be important in protecting the skin from oxidative stress. - Highlights: • Lipid peroxidation generates 4-hydroxynonenal, a reactive aldehyde. • 4-HNE induces antioxidant proteins in mouse keratinocytes. • Induction of

  15. Primary structure of keratinocyte transglutaminase.

    PubMed Central

    Phillips, M A; Stewart, B E; Qin, Q; Chakravarty, R; Floyd, E E; Jetten, A M; Rice, R H

    1990-01-01

    The nucleotide and deduced amino acid sequences of the coding regions of human and rat keratinocyte transglutaminases (protein-glutamine: amine gamma-glutamyltransferase; EC 2.3.2.13) have been determined. These yield proteins of approximately 90 kDa that are 92% identical, indicative of the conservation of important structural features. Alignments of amino acid sequences show substantial similarity among the keratinocyte transglutaminase, human clotting factor XIII catalytic subunit, guinea pig liver tissue transglutaminase, and the human erythrocyte band-4.2 protein. The keratinocyte enzyme is most similar to factor XIII, whereas the band-4.2 protein is most similar to the tissue transglutaminase. A salient feature of the keratinocyte transglutaminase is its 105-residue extension beyond the N terminus of the tissue transglutaminase. This extension and the unrelated activation peptide of factor XIII (a 37-residue extension) appear to be added for specialized functions after divergence of the tissue transglutaminase from their common lineage. Images PMID:1979171

  16. Primary structure of keratinocyte transglutaminase

    SciTech Connect

    Phillips, M.A.; Stewart, B.E.; Qin, Q.; Rice, R.H. ); Chakravarty, R. ); Floyd, E.E.; Jetten, A.M. )

    1990-12-01

    The nucleotide and deduced amino acid sequences of the coding regions of human and rat keratinocyte transglutaminases (protein-glutamine: amine {gamma}-glutamyltransferase; EC 2.3.2.13) have been determined. These yield proteins of {approximately}90 kDa that are 92% identical, indicative of the conservation of important structural features. Alignments of amino acid sequences show substantial similarity among the keratinocyte transglutaminase, human clotting factor XIII catalytic subunit, guinea pig liver tissue transglutaminase, and the human erythrocyte band-4.2 protein. The keratinocyte enzyme is most similar to factor XIII, whereas the band-4.2 protein is most similar to the tissue transglutaminase. A salient feature of the keratinocyte transglutaminase is its 105-residue extension beyond the N terminus of the tissue transglutaminase. This extension and the unreltaed activation peptide of factor XIII (a 37-residue extension) appear to be added for specialized functions after divergence of the tissue transglutaminase from their common lineage.

  17. Valproic Acid Induces Cutaneous Wound Healing In Vivo and Enhances Keratinocyte Motility

    PubMed Central

    Lee, Soung-Hoon; Zahoor, Muhammad; Hwang, Jae-Kwan; Min, Do Sik; Choi, Kang-Yell

    2012-01-01

    Background Cutaneous wound healing is a complex process involving several signaling pathways such as the Wnt and extracellular signal-regulated kinase (ERK) signaling pathways. Valproic acid (VPA) is a commonly used antiepileptic drug that acts on these signaling pathways; however, the effect of VPA on cutaneous wound healing is unknown. Methods and Findings We created full-thickness wounds on the backs of C3H mice and then applied VPA. After 7 d, we observed marked healing and reduced wound size in VPA-treated mice. In the neo-epidermis of the wounds, β-catenin and markers for keratinocyte terminal differentiation were increased after VPA treatment. In addition, α-smooth muscle actin (α-SMA), collagen I and collagen III in the wounds were significantly increased. VPA induced proliferation and suppressed apoptosis of cells in the wounds, as determined by Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining analyses, respectively. In vitro, VPA enhanced the motility of HaCaT keratinocytes by activating Wnt/β-catenin, ERK and phosphatidylinositol 3-kinase (PI3-kinase)/Akt signaling pathways. Conclusions VPA enhances cutaneous wound healing in a murine model and induces migration of HaCaT keratinocytes. PMID:23144972

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. Phosphatidylinositol 3-Kinase and Antiestrogen Resistance in Breast Cancer

    PubMed Central

    Miller, Todd W.; Balko, Justin M.; Arteaga, Carlos L.

    2011-01-01

    Although antiestrogen therapies targeting estrogen receptor (ER) α signaling prevent disease recurrence in the majority of patients with hormone-dependent breast cancer, a significant fraction of patients exhibit de novo or acquired resistance. Currently, the only accepted mechanism linked with endocrine resistance is amplification or overexpression of the ERBB2 (human epidermal growth factor receptor 2 [HER2]) proto-oncogene. Experimental and clinical evidence suggests that hyperactivation of the phosphatidylinositol 3-kinase (PI3K) pathway, the most frequently mutated pathway in breast cancer, promotes antiestrogen resistance. PI3K is a major signaling hub downstream of HER2 and other receptor tyrosine kinases. PI3K activates several molecules involved in cell-cycle progression and survival, and in ER-positive breast cancer cells, it promotes estrogen-dependent and -independent ER transcriptional activity. Preclinical tumor models of antiestrogen-resistant breast cancer often remain sensitive to estrogens and PI3K inhibition, suggesting that simultaneous targeting of the PI3K and ER pathways may be most effective. Herein, we review alterations in the PI3K pathway associated with resistance to endocrine therapy, the state of clinical development of PI3K inhibitors, and strategies for the clinical investigation of such drugs in hormone receptor–positive breast cancer. PMID:22010023

  3. 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.

  4. 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

  5. Phosphoinositide 3-kinase mediated signaling in lobster olfactory receptor neurons

    PubMed Central

    Corey, Elizabeth A.; Bobkov, Yuriy; Pezier, Adeline; Ache, Barry W.

    2010-01-01

    In vertebrates and some invertebrates, odorant molecules bind to G protein-coupled receptors (GPCRs) on olfactory receptor neurons (ORNs) to initiate signal transduction. Phosphoinositide 3-kinase (PI3K) activity has been implicated physiologically in olfactory signal transduction, suggesting a potential role for a GPCR-activated class I PI3K. Using isoform-specific antibodies, we identified a protein in the olfactory signal transduction compartment of lobster ORNs that is antigenically similar to mammalian PI3Kγ and cloned a gene for a PI3K with amino acid homology with PI3Kβ. The lobster olfactory PI3K co-immunoprecipitates with the G protein α and β subunits, and an odorant-evoked increase in phosphatidylinositol (3,4,5)-trisphosphate can be detected in the signal transduction compartment of the ORNs. PI3Kγ and β isoform-specific inhibitors reduce the odorant-evoked output of lobster ORNs in vivo. Collectively, these findings provide evidence that PI3K is indeed activated by odorant receptors in lobster ORNs and further support the potential involvement of G protein activated PI3K signaling in olfactory transduction. PMID:20132480

  6. 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

  7. Role of PI 3-kinase and PIP3 in submandibular gland branching morphogenesis

    PubMed Central

    Larsen, Melinda; Hoffman, Matthew P.; Sakai, Takayoshi; Neibaur, Justin C.; Mitchell, Jonathan M.; Yamada, Kenneth M.

    2007-01-01

    The mouse submandibular gland (SMG) epithelium undergoes extensive morphogenetic branching during embryonic development as the first step in the establishment of its glandular structure. However, the specific signaling pathways required for SMG branching morphogenesis are not well understood. Using E13 mouse SMG organ cultures, we showed that inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase), wortmannin and LY294002, substantially inhibited branching morphogenesis in SMG. Branching morphogenesis of epithelial rudiments denuded of mesenchyme was inhibited similarly, indicating that PI 3-kinase inhibitors act directly on the epithelium. Immunostaining and Western analysis demonstrated that the p85 isoform of PI 3-kinase is expressed in epithelium at levels higher than in the mesenchyme. A target of PI 3-kinase, Akt/protein kinase B (PKB), showed decreased phosphorylation at Ser473 by Western analysis in the presence of PI 3-kinase inhibitors. The major lipid product of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate (PIP3), was added exogenously to SMG via a membrane-transporting carrier in the presence of PI 3-kinase inhibitors and was found to stimulate cleft formation, the first step of branching morphogenesis. Together, these data indicate that PI 3-kinase plays a role in the regulation of epithelial branching morphogenesis in mouse SMG acting through a PIP3 pathway. PMID:12618142

  8. 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

  9. Increased protein glycation in fructosamine 3-kinase-deficient mice

    PubMed Central

    da-Cunha, Maria VEIGA; Jacquemin, Patrick; Delpierre, Ghislain; Godfraind, Catherine; Théate, Ivan; Vertommen, Didier; Clotman, Frédéric; Lemaigre, Frédéric; Devuyst, Olivier; Van Schaftingen, Emile

    2006-01-01

    Amines, including those present on proteins, spontaneously react with glucose to form fructosamines in a reaction known as glycation. In the present paper, we have explored, through a targeted gene inactivation approach, the role of FN3K (fructosamine 3-kinase), an intracellular enzyme that phosphorylates free and protein-bound fructose-ϵ-lysines and which is potentially involved in protein repair. Fn3k−/− mice looked healthy and had normal blood glucose and serum fructosamine levels. However, their level of haemoglobin-bound fructosamines was approx. 2.5-fold higher than that of control (Fn3k+/+) or Fn3k+/− mice. Other intracellular proteins were also significantly more glycated in Fn3k−/− mice in erythrocytes (1.8–2.2-fold) and in brain, kidney, liver and skeletal muscle (1.2–1.8-fold), indicating that FN3K removes fructosamines from intracellular proteins in vivo. The urinary excretion of free fructose-ϵ-lysine was 10–20-fold higher in fed mice compared with mice starved for 36 h, and did not differ between fed Fn3k+/+ and Fn3k−/− mice, indicating that food is the main source of urinary fructose-ϵ-lysine in these mice and that FN3K does not participate in the metabolism of food-derived fructose-ϵ-lysine. However, in starved animals, the urinary excretion of fructose-ϵ-lysine was 2.5-fold higher in Fn3k−/− mice compared with Fn3k+/+ or Fn3k+/− mice. Furthermore, a marked increase (5–13-fold) was observed in the concentration of free fructose-ϵ-lysine in tissues of fed Fn3k−/− mice compared with control mice, indicating that FN3K participates in the metabolism of endogenously produced fructose-ϵ-lysine. Taken together, these data indicate that FN3K serves as a protein repair enzyme and also in the metabolism of endogenously produced free fructose-ϵ-lysine. PMID:16819943

  10. 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.

  11. CCN1, a Pro-Inflammatory Factor, Aggravates Psoriasis Skin Lesions by Promoting Keratinocyte Activation.

    PubMed

    Sun, Yue; Zhang, Jie; Zhou, Zhou; Wu, Pinru; Huo, Rongfen; Wang, Beiqing; Shen, Zhengyu; Li, Huidan; Zhai, Tianhang; Shen, Baihua; Chen, Xiangdong; Li, Ningli

    2015-11-01

    Psoriasis is a common chronic skin disease characterized by epidermal hyperplasia and inflammation. The pathogenesis of psoriasis is multifactorial and is not fully understood. Here we demonstrate that CCN1 (also called Cyr61, which is short for cysteine-rich 61), an extracellular matrix protein that is also considered a pro-inflammatory factor, is highly expressed in the lesional skin of psoriasis patients, as well as in that of imiquimod (IMQ)- and IL-23-treated psoriasis-like mice. Then we show that blocking CCN1 function in vivo attenuates epidermal hyperplasia and inflammation in psoriasis-like mice. Further, in primary cultured normal human keratinocytes and HaCaT (human keratinocyte cell line) cells, CCN1 promotes keratinocyte activation, including the proliferation and expression of immune-related molecules. Finally, we observe that integrin α6β1 is the receptor of CCN1 in keratinocytes, and CCN1 stimulation activates the downstream phosphoinositide-3 kinase/Akt/NF-κB signaling pathway. Taken together, our findings reveal that CCN1 has a critical role in psoriasis pathogenesis. Moreover, as CCN1 is a secreted extracellular matrix (ECM) protein, our study also provides evidence that ECM, which is involved in psoriatic pathogenesis, could be a potent target for psoriasis treatment.

  12. Candida albicans phospholipomannan triggers inflammatory responses of human keratinocytes through Toll-like receptor 2.

    PubMed

    Li, Min; Chen, Qing; Shen, Yongnian; Liu, Weida

    2009-07-01

    The Toll-like receptors (TLRs) play an important role in the recognition of Candida albicans components and activation of innate immunity. Phospholipomannan (PLM), a glycolipid, is expressed at the surface of C. albicans cell wall, which acts as a member of the pathogen-associated molecular patterns family. In this study, we sought to clarify whether C. albicans-native PLM could induce an inflammation response in human keratinocytes and to determine the underlying mechanisms. Exposure of cultured human primary keratinocytes to PLM led to the increased gene expression and secretion of proinflammatory cytokines (IL-6) and chemokines (IL-8). PLM hydrolysed with beta-d-mannoside mannohydrolase failed to induce gene expression and secretion of IL-6 and IL-8. PLM up-regulated the mRNA and protein levels of TLR2, whereas the mRNA level of TLR4 was not altered. Keratinocytes challenged with PLM resulted in the activation of NF-kappaB and mitogen-activated protein kinase (MAPKs) including p38. Anti-TLR2 neutralizing antibody, NFkappaB and p38MAPK inhibitors blocked the PLM-induced secretion of IL-6, IL-8 in keratinocytes, but no such effect was observed in pretreatment with anti-TLR4-neutralizing antibody and lipopolysaccharide inhibitor (polymyxin B). These data suggest C. albicans-native PLM may contribute to the inflammatory responses of cutaneous candidiasis in the TLR2-NF-kappaB and p38MAPK signalling pathway dependent manner.

  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. DNA repair in cultured keratinocytes

    SciTech Connect

    Liu, S.C.; Parsons, S.; Hanawalt, P.C.

    1983-07-01

    Most of our understanding of DNA repair mechanisms in human cells has come from the study of these processes in cultured fibroblasts. The unique properties of keratinocytes and their pattern of terminal differentiation led us to a comparative examination of their DNA repair properties. The relative repair capabilities of the basal cells and the differentiated epidermal keratinocytes as well as possible correlations of DNA repair capacity with respect to age of the donor have been examined. In addition, since portions of human skin are chronically exposed to sunlight, the repair response to ultraviolet (UV) irradiation (254 nm) when the cells are conditioned by chronic low-level UV irradiation has been assessed. The comparative studies of DNA repair in keratinocytes from infant and aged donors have revealed no significant age-related differences for repair of UV-induced damage to DNA. Sublethal UV conditioning of cells from infant skin had no appreciable effect on either the repair or normal replication response to higher, challenge doses of UVL. However, such conditioning resulted in attenuated repair in keratinocytes from adult skin after UV doses above 25 J/m2. In addition, a surprising enhancement in replication was seen in conditioned cells from adult following challenge UV doses.

  15. Kinase Pathway Dependence in Primary Human Leukemias Determined by Rapid Inhibitor Screening

    PubMed Central

    Tyner, Jeffrey W.; Yang, Wayne F.; Bankhead, Armand; Fan, Guang; Fletcher, Luke B.; Bryant, Jade; Glover, Jason M.; Chang, Bill H.; Spurgeon, Stephen E.; Fleming, William H.; Kovacsovics, Tibor; Gotlib, Jason R.; Oh, Stephen T.; Deininger, Michael W.; Zwaan, C. Michel; Den Boer, Monique L.; van den Heuvel-Eibrink, Marry M.; O’Hare, Thomas; Druker, Brian J.; Loriaux, Marc M.

    2012-01-01

    Kinases are dysregulated in most cancer but the frequency of specific kinase mutations is low, indicating a complex etiology in kinase dysregulation. Here we report a strategy to rapidly identify functionally important kinase targets, irrespective of the etiology of kinase pathway dysregulation, ultimately enabling a correlation of patient genetic profiles to clinically effective kinase inhibitors. Our methodology assessed the sensitivity of primary leukemia patient samples to a panel of 66 small-molecule kinase inhibitors over 3 days. Screening of 151 leukemia patient samples revealed a wide diversity of drug sensitivities, with 70% of the clinical specimens exhibiting hypersensitivity to one or more drugs. From this data set, we developed an algorithm to predict kinase pathway dependence based on analysis of inhibitor sensitivity patterns. Applying this algorithm correctly identified pathway dependence in proof-of-principle specimens with known oncogenes, including a rare FLT3 mutation outside regions covered by standard molecular diagnostic tests. Interrogation of all 151 patient specimens with this algorithm identified a diversity of gene targets and signaling pathways that could aid prioritization of deep sequencing data sets, permitting a cumulative analysis to understand kinase pathway dependence within leukemia subsets. In a proof-of-principle case, we showed that in vitro drug sensitivity could predict both a clinical response and the development of drug resistance. Taken together, our results suggested that drug target scores derived from a comprehensive kinase inhibitor panel could predict pathway dependence in cancer cells while simultaneously identifying potential therapeutic options. PMID:23087056

  16. α6 Integrin and CD44 enrich for a primary keratinocyte population that displays resistance to UV-induced apoptosis.

    PubMed

    Wray, Helen; Mackenzie, Ian C; Storey, Alan; Navsaria, Harshad

    2012-01-01

    Epidermal human keratinocytes are exposed to a wide range of environmental genotoxic insults, including the UV component of solar radiation. Epidermal homeostasis in response to cellular or tissue damage is maintained by a population of keratinocyte stem cells (KSC) that reside in the basal layer of the epithelium. Using cell sorting based on cell-surface markers, we have identified a novel α6 integrin(high+)/CD44(+) sub-population of basal keratinocytes. These α6 integrin(high+)/CD44(+) keratinocytes have both high proliferative potential, form colonies in culture that have characteristics of holoclones and have a unique pattern of resistance to apoptosis induced by UVB radiation or by agents that induce single- or double strand DNA breaks. Resistance to UVB induced apoptosis in the α6 integrin(high+)/CD44(+) cells involved increased expression of TAp63 and was overcome by PI-3 kinase inhibition. In marked contrast, the α6 integrin(high+)/CD44(+) cells were sensitive to apoptosis induced by the cross-linking agent cisplatin, and imatinib inhibition of c-Abl blocked the ability of cisplatin to kill α6 integrin(high+)/CD44(+) cells. Our findings reveal a population of basal keratinocytes with long-term proliferative properties that display specific patterns of apoptotic resistance that is dependent upon the genotoxic stimulus, and provide insights into how these cells can be targeted with chemotherapeutic agents.

  17. 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

  18. l-Ascorbic acid 2-phosphate promotes elongation of hair shafts via the secretion of insulin-like growth factor-1 from dermal papilla cells through phosphatidylinositol 3-kinase.

    PubMed

    Kwack, M H; Shin, S H; Kim, S R; Im, S U; Han, I S; Kim, M K; Kim, J C; Sung, Y K

    2009-06-01

    l-Ascorbic acid 2-phosphate (Asc 2-P), a derivative of l-ascorbic acid, promotes elongation of hair shafts in cultured human hair follicles and induces hair growth in mice. To investigate whether the promotion of hair growth by Asc 2-P is mediated by insulin-like growth factor-1 (IGF-1) and, if so, to investigate the mechanism of the Asc 2-P-induced IGF-1 expression. Dermal papilla (DP) cells were cultured and IGF-1 level was measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay after Asc 2-P treatment in the absence or presence of LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. Also, hair shaft elongation in cultured human scalp hair follicles and proliferation of cocultured keratinocytes were examined after Asc 2-P treatment in the absence or presence of neutralizing antibody against IGF-1. In addition, keratinocyte proliferation in cultured hair follicles after Asc 2-P treatment in the absence or presence of LY294002 was examined by Ki-67 immunostaining. IGF-1 mRNA in DP cells was upregulated and IGF-1 protein in the conditioned medium of DP cells was significantly increased after treatment with Asc 2-P. Immunohistochemical staining showed that IGF-1 staining is increased in the DP of cultured human hair follicles by Asc 2-P. The neutralizing antibody against IGF-1 significantly suppressed the Asc 2-P-mediated elongation of hair shafts in hair follicle organ culture and significantly attenuated Asc 2-P-induced growth of cocultured keratinocytes. LY294002 significantly attenuated Asc 2-P-inducible IGF-1 expression and proliferation of follicular keratinocytes in cultured hair follicles. These data show that Asc 2-P-inducible IGF-1 from DP cells promotes proliferation of follicular keratinocytes and stimulates hair follicle growth in vitro via PI3K.

  19. Kindlin-1 Regulates Keratinocyte Electrotaxis.

    PubMed

    Zhang, Gaofeng; Gu, Yu; Begum, Rumena; Chen, Hongduo; Gao, Xinghua; McGrath, John A; Parsons, Maddy; Song, Bing

    2016-11-01

    Kindler syndrome (KS) is an autosomal recessive blistering skin disease resulting from pathogenic mutations in FERMT1. This gene encodes kindlin-1, a focal adhesion protein involved in activation of the integrin family of extracellular matrix receptors. Most cases of KS show a marked reduction or complete absence of the kindlin-1 protein in keratinocytes, resulting in defective cell adhesion and migration. Electric fields also act as intrinsic regulators of adhesion and migration in the skin, but the molecular mechanisms by which this occurs are poorly understood. Here we show that keratinocytes derived from KS patients are unable to undergo electrotaxis, and this defect is restored by overexpression of wild-type kindlin-1 but not a W612A mutation that prevents kindlin-integrin binding. Moreover, deletion of the pleckstrin homology domain of kindlin-1 also failed to rescue electrotaxis in KS cells, indicating that both integrin and lipid binding are required for this function. Kindlin-1 was also required for the maintenance of lamellipodial protrusions during electrotaxis via electric field-activated β1 integrin. Indeed, inhibition of β1 integrins also leads to loss of electrotaxis in keratinocytes. Our data suggest that loss of kindlin-1 function may therefore result in epithelial insensitivity to electric fields and contribute to KS disease pathology. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Aldose reductase in keratinocytes attenuates cellular apoptosis and senescence induced by UV radiation.

    PubMed

    Kang, Eun Sil; Iwata, Kazumi; Ikami, Kanako; Ham, Sun Ah; Kim, Hye Jung; Chang, Ki Churl; Lee, Jae Heun; Kim, Jae-Hwan; Park, Soo-Bong; Kim, Jin-Hoi; Yabe-Nishimura, Chihiro; Seo, Han Geuk

    2011-03-15

    Although aldose reductase (AR) has been implicated in the cellular response to oxidative stress, the role of AR in ultraviolet-B (UVB)-induced cellular injury has not been investigated. Here, we show that an increased expression of AR in human keratinocytes modulates UVB-induced apoptotic cell death and senescence. Overexpression of AR in HaCaT cells significantly attenuated UVB-induced cellular damage and apoptosis, with a decreased generation of reactive oxygen species (ROS) and aldehydes. Ablation of AR with small interfering RNA or inhibition of AR activity abolished these effects. We also show that increased AR activity suppressed UVB-induced activation of the p38 and c-Jun N-terminal kinases, but did not affect the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase pathways. Similarly, UVB-induced translocation of Bax and Bcl-2 to mitochondria and cytosol, respectively, was markedly attenuated in cells overexpressing AR. Knockdown or inhibition of AR activity in primary cultured keratinocytes enhanced UVB-induced cellular senescence and increased the level of a cell-cycle regulatory protein, p53. Finally, cellular apoptosis induced by UVB radiation was significantly reduced in the epidermis of transgenic mice overexpressing human AR. These findings suggest that AR plays an important role in the cellular response to oxidative stress by sequestering ROS and reactive aldehydes generated in keratinocytes. Copyright © 2010 Elsevier Inc. All rights reserved.

  1. Regular exercise enhances insulin activation of IRS-1-associated PI3-kinase in human skeletal muscle.

    PubMed

    Kirwan, J P; del Aguila, L F; Hernandez, J M; Williamson, D L; O'Gorman, D J; Lewis, R; Krishnan, R K

    2000-02-01

    Insulin action in skeletal muscle is enhanced by regular exercise. Whether insulin signaling in human skeletal muscle is affected by habitual exercise is not well understood. Phosphatidylinositol 3-kinase (PI3-kinase) activation is an important step in the insulin-signaling pathway and appears to regulate glucose metabolism via GLUT-4 translocation in skeletal muscle. To examine the effects of regular exercise on PI3-kinase activation, 2-h hyperinsulinemic (40 mU. m(-2). min(-1))-euglycemic (5.0 mM) clamps were performed on eight healthy exercise-trained [24 +/- 1 yr, 71.8 +/- 2.0 kg, maximal O(2) uptake (VO(2 max)) of 56.1 +/- 2.5 ml. kg(-1). min(-1)] and eight healthy sedentary men and women (24 +/- 1 yr, 64.7 +/- 4.4 kg, VO(2 max) of 44.4 +/- 2.7 ml. kg(-1). min(-1)). A [6, 6-(2)H]glucose tracer was used to measure hepatic glucose output. A muscle biopsy was obtained from the vastus lateralis muscle at basal and at 2 h of hyperinsulinemia to measure insulin receptor substrate-1(IRS-1)-associated PI3-kinase activation. Insulin concentrations during hyperinsulinemia were similar for both groups (293 +/- 22 and 311 +/- 22 pM for trained and sedentary, respectively). Insulin-mediated glucose disposal rates (GDR) were greater (P < 0.05) in the exercise-trained compared with the sedentary control group (9.22 +/- 0.95 vs. 6.36 +/- 0.57 mg. kg fat-free mass(-1). min(-1)). Insulin-stimulated PI3-kinase activation was also greater (P < 0.004) in the trained compared with the sedentary group (3.8 +/- 0.5- vs. 1.8 +/- 0.2-fold increase from basal). Endurance capacity (VO(2 max)) was positively correlated with PI3-kinase activation (r = 0.53, P < 0.04). There was no correlation between PI3-kinase and muscle morphology. However, increases in GDR were positively related to PI3-kinase activation (r = 0.60, P < 0.02). We conclude that regular exercise leads to greater insulin-stimulated IRS-1-associated PI3-kinase activation in human skeletal muscle, thus facilitating enhanced

  2. Substrate Stiffness Affects Human Keratinocyte Colony Formation

    PubMed Central

    Zarkoob, Hoda; Bodduluri, Sandeep; Ponnaluri, Sailahari V.; Selby, John C.; Sander, Edward A.

    2015-01-01

    Restoration of epidermal organization and function in response to a variety of pathophysiological insults is critically dependent on coordinated keratinocyte migration, proliferation, and stratification during the process of wound healing. These processes are mediated by the reconfiguration of both cell-cell (desmosomes, adherens junctions) and cell-matrix (focal adhesions, hemidesmosomes) junctions and the cytoskeletal filament networks that they serve to interconnect. In this study, we investigated the role of substrate elasticity (stiffness) on keratinocyte colony formation in vitro during the process of nascent epithelial sheet formation as triggered by the calcium switch model of keratinocyte culture. Keratinocytes cultured on pepsin digested type I collagen coated soft (nominal E = 1.2 kPa) polyacrylamide gels embedded with fluorescent microspheres exhibited (i) smaller spread contact areas, (ii) increased migration velocities, and (iii) increased rates of colony formation with more cells per colony than did keratinocytes cultured on stiff (nominal E = 24 kPa) polyacrylamide gels. As assessed by tracking of embedded microsphere displacements, keratinocytes cultured on soft substrates generated large local substrate deformations that appeared to recruit adjacent keratinocytes into joining an evolving colony. Together with the observed differences in keratinocyte kinematics and substrate deformations, we developed two ad hoc analyses, termed distance rank (DR) and radius of cooperativity (RC), that help to objectively ascribe what we perceive as increasingly cooperative behavior of keratinocytes cultured on soft versus stiff gels during the process of colony formation. We hypothesize that the differences in keratinocyte colony formation observed in our experiments could be due to cell-cell mechanical signaling generated via local substrate deformations that appear to be correlated with the increased expression of β4 integrin within keratinocytes positioned

  3. Mechanisms regulating phosphoinositide 3-kinase signalling in insulin-sensitive tissues.

    PubMed

    Shepherd, P R

    2005-01-01

    A great deal of evidence has accumulated indicating that the activity of PI 3-kinase is necessary, and in some cases sufficient, for a wide range of insulin's actions in the cell. Most biochemical, genetic and pharmacological studies have focused on identifying potential roles for the class-Ia PI 3-kinases which are rapidly activated following insulin stimulation. However, recent evidence indicates the alpha isoform of class-II PI 3-kinase (PI3K-C2alpha) may also play a role as insulin causes a very rapid activation of this as well. The basic mechanisms by which insulin activates the various members of the PI 3-kinase family are increasingly well understood and these studies reveal multiple mechanisms for modulating the activity and functionality of PI 3-kinase and for down regulating the signals they generate. These include inhibitory phosphorylation events, lipid phosphatases such as PTEN and SHIP2 and inhibitor proteins of the suppressors of cytokine signalling (SOCS) family. The current review will focus on these mechanisms and how defects in these might contribute to the development of insulin resistance.

  4. 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

  5. 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.

  6. The Effect of Lipoaspirates on Human Keratinocytes.

    PubMed

    Kim, Bong-Sung; Gaul, Charel; Paul, Nora E; Dewor, Manfred; Stromps, Jan-Philipp; Hwang, Soo Seok; Nourbakhsh, Mahtab; Bernhagen, Jürgen; Rennekampff, Hans-Oliver; Pallua, Norbert

    2016-09-01

    One increasingly important trend in plastic, reconstructive, and aesthetic surgery is the use of fat grafts to improve cutaneous wound healing. In clinical practice, lipoaspirates (adipose tissue harvested by liposuction) are re-injected in a procedure called lipofilling. Previous studies, however, mainly evaluated the regenerative effect of isolated adipocytes, adipose-derived stem cells, and excised en bloc adipose tissue on keratinocytes, whereas no study to date has examined the effect of lipoaspirates. The authors aimed to investigate differences in the regenerative property of en bloc adipose tissue and lipoaspirates on keratinocytes. Human keratinocytes, lipoaspirates, and en bloc adipose tissue from 36 healthy donors were isolated. In vitro proliferation, differentiation, migration, stratification, and wound healing of keratinocyte monolayers were measured. Furthermore, secreted levels of VEGF, bFGF, IGF-1, MMP-9, and MIF were detected by ELISA. Migration, proliferation, and wound healing of keratinocytes were increased by lipoaspirates. Interestingly, the effect of lipoaspirates on keratinocyte proliferation was significantly higher than by en bloc adipose tissue after 5 days. The differentiation of keratinocytes was equally attenuated by lipoaspirates and en bloc adipose tissue. Stratification of keratinocyte layers was enhanced by lipoaspirates and en bloc fat when compared to controls. Lipoaspirates secrete higher levels of bFGF, whereas higher levels of VEGF and IGF-1 are released by en bloc adipose tissue. We show that lipoaspirates and en bloc adipose tissue have a regenerative effect on keratinocytes. One reason for the higher effect of lipoaspirates on keratinocyte proliferation may be the secretion of different cytokines. © 2016 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com.

  7. Bisphosphonates Inhibit Expression of p63 by Oral Keratinocytes

    PubMed Central

    Scheller, E.L.; Baldwin, C.M.; Kuo, S.; D’Silva, N.J.; Feinberg, S.E.; Krebsbach, P.H.; Edwards, P.C.

    2011-01-01

    Osteonecrosis of the jaw (ONJ), a side-effect of bisphosphonate therapy, is characterized by exposed bone that fails to heal within eight weeks. Healing time of oral epithelial wounds is decreased in the presence of amino-bisphosphonates; however, the mechanism remains unknown. We examined human tissue from individuals with ONJ and non-bisphosphonate-treated controlindividuals to identify changes in oral epithelium and connective tissue. Oral and intravenous bisphosphonate-treated ONJ sites had reduced numbers of basal epithelial progenitor cells, as demonstrated by a 13.8 ± 1.1% and 31.9 ± 5.8% reduction of p63 expression, respectively. No significant differences in proliferation rates, vessel density, or macrophage number were noted. In vitro treatment of clonal and primary oral keratinocytes with zoledronic acid (ZA) inhibited p63, and expression was rescued by the addition of mevalonate pathway intermediates. In addition, both ZA treatment and p63 shRNA knock-down impaired formation of 3D Ex Vivo Produced Oral Mucosa Equivalents (EVPOME) and closure of an in vitro scratch assay. Analysis of our data suggests that bisphosphonate treatment may delay oral epithelial healing by interfering with p63-positive progenitor cells in the basal layer of the oral epithelium in a mevalonate-pathway-dependent manner. This delay in healing may increase the likelihood of osteonecrosis developing in already-compromised bone. PMID:21551338

  8. Keratinocyte stem cells: a commentary.

    PubMed

    Potten, Christopher S; Booth, Catherine

    2002-10-01

    For many years it has been widely accepted that stem cells play a crucial role in adult tissue maintenance. The concept that the renewing tissues of the body contain a small subcompartment of self-maintaining stem cells, upon which the entire tissue is dependent, is also now accepted as applicable to all renewing tissues. Gene therapy and tissue engineering are driving considerable interest in the clinical application of such hierarchically organized cellular compartments. Recent initial observations have provided a tantalizing insight into the large pluripotency of these cells. Indeed, scientists are now beginning to talk about the possible totipotency of some adult tissue stem cells. Such work is currently phenomenologic, but analysis of data derived from genomics and proteomics, identifying the crucial control signals involved, will soon provide a further impetus to stem cell biology with far reaching applications. The epidermis with its relatively simple structure, ease of accessibility, and the ability to grow its cells in vitro is one obvious target tissue for testing stem cell manipulation theories. It is crucial, however, that the normal keratinocyte stem cell is thoroughly characterized prior to attempting to manipulate its pluripotency. This commentary assesses the data generated to date and critically discusses the conclusions that have been drawn. Our current level of understanding, or lack of understanding, of the keratinocyte stem cell is reviewed.

  9. TAT-pathway-dependent lipoproteins as a niche-based adaptation in prokaryotes.

    PubMed

    Shruthi, Hamsanathan; Babu, Mohan Madan; Sankaran, Krishnan

    2010-04-01

    Bacterial lipoproteins, characterized by the N-terminal N-acyl S-diacylglyceryl Cysteine, are key membrane proteins in bacterial homeostasis. It is generally thought that during the modification lipoprotein precursors are translocated via the Sec-machinery in an unfolded state. The recent discovery of twin-arginine translocation (TAT) machinery, meant for exporting folded-proteins, and the presence of TAT-type signal sequences in co-factor-containing (hence already folded) lipoproteins, prompted us to investigate its role and significance in lipoprotein biosynthesis. We systematically analyzed 696 prokaryotic genomes using an algorithm based on DOLOP and TatP rules to predict TAT-pathway-dependent lipoprotein substrates. Occurrence of the deduced TAT-pathway-dependent lipoprotein substrates in relation to genome size, presence or absence of TAT machinery, and extent of its usage for lipoprotein export and habitat types revealed that unlike the host-obligates, the free-living prokaryotes in complex hostile environments (e.g., soil) depend more on TAT-exported lipoproteins. Functional classification of the predicted TAT-dependent lipoproteins revealed enrichment in hydrolases and oxido-reductases, which are fast-folding and co-factor-containing proteins. The role of the TAT pathway in the export of folded-lipoproteins and in niche-specific adaptation for survival has important implications not only in lipoprotein biosynthesis, but also for protein and metabolic engineering applications.

  10. Drug-resistant phosphatidylinositol 3-kinase: guidance for the preemptive strike.

    PubMed

    Vogt, Peter K

    2008-08-12

    In this issue of Cancer Cell, Zunder et al. (2008) describe surprising findings from investigating inhibitor-resistant mutations in the affinity pocket of p110 alpha of phosphatidylinositol 3-kinase (PI3K). Information on these critical residues provides a road map for generating novel PI3K inhibitors that can overcome the anticipated resistance mutations.

  11. Interferon regulatory factor 6 regulates keratinocyte migration

    PubMed Central

    Biggs, Leah C.; Naridze, Rachelle L.; DeMali, Kris A.; Lusche, Daniel F.; Kuhl, Spencer; Soll, David R.; Schutte, Brian C.; Dunnwald, Martine

    2014-01-01

    ABSTRACT Interferon regulatory factor 6 (Irf6) regulates keratinocyte proliferation and differentiation. In this study, we tested the hypothesis that Irf6 regulates cellular migration and adhesion. Irf6-deficient embryos at 10.5 days post-conception failed to close their wound compared with wild-type embryos. In vitro, Irf6-deficient murine embryonic keratinocytes were delayed in closing a scratch wound. Live imaging of the scratch showed deficient directional migration and reduced speed in cells lacking Irf6. To understand the underlying molecular mechanisms, cell–cell and cell–matrix adhesions were investigated. We show that wild-type and Irf6-deficient keratinocytes adhere similarly to all matrices after 60 min. However, Irf6-deficient keratinocytes were consistently larger and more spread, a phenotype that persisted during the scratch-healing process. Interestingly, Irf6-deficient keratinocytes exhibited an increased network of stress fibers and active RhoA compared with that observed in wild-type keratinocytes. Blocking ROCK, a downstream effector of RhoA, rescued the delay in closing scratch wounds. The expression of Arhgap29, a Rho GTPase-activating protein, was reduced in Irf6-deficient keratinocytes. Taken together, these data suggest that Irf6 functions through the RhoA pathway to regulate cellular migration. PMID:24777480

  12. 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.

  13. Single cell mechanics of keratinocyte cells.

    PubMed

    Lulevich, Valentin; Yang, Hsin-ya; Isseroff, R Rivkah; Liu, Gang-yu

    2010-11-01

    Keratinocytes represent the major cell type of the uppermost layer of human skin, the epidermis. Using AFM-based single cell compression, the ability of individual keratinocytes to resist external pressure and global rupturing forces is investigated and compared with various cell types. Keratinocytes are found to be 6-70 times stiffer than other cell types, such as white blood, breast epithelial, fibroblast, or neuronal cells, and in contrast to other cell types they retain high mechanic strength even after the cell's death. The absence of membrane rupturing peaks in the force-deformation profiles of keratinocytes and their high stiffness during a second load cycle suggests that their unique mechanical resistance is dictated by the cytoskeleton. A simple analytical model enables the quantification of Young's modulus of keratinocyte cytoskeleton, as high as 120-340 Pa. Selective disruption of the two major cytoskeletal networks, actin filaments and microtubules, does not significantly affect keratinocyte mechanics. F-actin is found to impact cell deformation under pressure. During keratinocyte compression, the plasma membrane stretches to form peripheral blebs. Instead of blebbing, cells with depolymerized F-actin respond to pressure by detaching the plasma membrane from the cytoskeleton underneath. On the other hand, the compression force of keratinocytes expressing a mutated keratin (cell line, KEB-7) is 1.6-2.2 times less than that for the control cell line that has normal keratin networks. Therefore, we infer that the keratin intermediate filament network is responsible for the extremely high keratinocyte stiffness and resilience. This could manifest into the rugged protective nature of the human epidermis.

  14. Exosomes released by keratinocytes modulate melanocyte pigmentation

    PubMed Central

    Cicero, Alessandra Lo; Delevoye, Cédric; Gilles-Marsens, Floriane; Loew, Damarys; Dingli, Florent; Guéré, Christelle; André, Nathalie; Vié, Katell; van Niel, Guillaume; Raposo, Graça

    2015-01-01

    Cells secrete extracellular vesicles (EVs), exosomes and microvesicles, which transfer proteins, lipids and RNAs to regulate recipient cell functions. Skin pigmentation relies on a tight dialogue between keratinocytes and melanocytes in the epidermis. Here we report that exosomes secreted by keratinocytes enhance melanin synthesis by increasing both the expression and activity of melanosomal proteins. Furthermore, we show that the function of keratinocyte-derived exosomes is phototype-dependent and is modulated by ultraviolet B. In sum, this study uncovers an important physiological function for exosomes in human pigmentation and opens new avenues in our understanding of how pigmentation is regulated by intercellular communication in both healthy and diseased states. PMID:26103923

  15. Intrinsic Differences between Oral and Skin Keratinocytes

    PubMed Central

    Turabelidze, Anna; Guo, Shujuan; Chung, Allison Yen; Chen, Lin; Dai, Yang; Marucha, Phillip T.; DiPietro, Luisa A.

    2014-01-01

    Keratinocytes cover both the skin and some oral mucosa, but the morphology of each tissue and the behavior of the keratinocytes from these two sites are different. One significant dissimilarity between the two sites is the response to injury. Oral mucosal wounds heal faster and with less inflammation than equivalent cutaneous wounds. We hypothesized that oral and skin keratinocytes might have intrinsic differences at baseline as well as in the response to injury, and that such differences would be reflected in gene expression profiles. PMID:25198578

  16. Exosomes released by keratinocytes modulate melanocyte pigmentation.

    PubMed

    Lo Cicero, Alessandra; Delevoye, Cédric; Gilles-Marsens, Floriane; Loew, Damarys; Dingli, Florent; Guéré, Christelle; André, Nathalie; Vié, Katell; van Niel, Guillaume; Raposo, Graça

    2015-06-24

    Cells secrete extracellular vesicles (EVs), exosomes and microvesicles, which transfer proteins, lipids and RNAs to regulate recipient cell functions. Skin pigmentation relies on a tight dialogue between keratinocytes and melanocytes in the epidermis. Here we report that exosomes secreted by keratinocytes enhance melanin synthesis by increasing both the expression and activity of melanosomal proteins. Furthermore, we show that the function of keratinocyte-derived exosomes is phototype-dependent and is modulated by ultraviolet B. In sum, this study uncovers an important physiological function for exosomes in human pigmentation and opens new avenues in our understanding of how pigmentation is regulated by intercellular communication in both healthy and diseased states.

  17. Melanosome transfer promoted by keratinocyte growth factor in light and dark skin-derived keratinocytes.

    PubMed

    Cardinali, Giorgia; Bolasco, Giulia; Aspite, Nicaela; Lucania, Giuseppe; Lotti, Lavinia V; Torrisi, Maria R; Picardo, Mauro

    2008-03-01

    The transfer of melanin from melanocytes to keratinocytes is upregulated by UV radiation and modulated by autocrine and paracrine factors. Among them, the keratinocyte growth factor (KGF/FGF7) promotes melanosome transfer acting on the recipient keratinocytes through stimulation of the phagocytic process. To search for possible differences in the melanosome uptake of keratinocytes from different skin color, we analyzed the uptake kinetics and distribution pattern of fluorescent latex beads in primary cultures of light and dark skin-derived keratinocytes stimulated with KGF and we compared the direct effect of KGF on the melanosome transfer in co-cultures of human primary melanocytes with light and dark keratinocytes. KGF-promoted melanosome transfer was more significant in light keratinocytes compared to dark, due to an increased expression of KGF receptor in light skin keratinocytes. Colocalization studies performed by confocal microscopy using FITC-dextran as a phagocytic marker and fluorescent beads as well as inhibition of particle uptake by cytochalasin D, revealed that beads internalization induced by KGF occurs via actin-dependent phagocytosis. 3D image reconstruction by fluorescence microscopy and ultrastructural analysis through transmission electron microscopy showed differences in the distribution pattern of the beads in light and dark keratinocytes, consistent with the different melanosome distribution in human skin.

  18. Role of phosphoinositide 3-kinase in the aggressive tumor growth of HT1080 human fibrosarcoma cells.

    PubMed

    Gupta, S; Stuffrein, S; Plattner, R; Tencati, M; Gray, C; Whang, Y E; Stanbridge, E J

    2001-09-01

    We have developed a model system of human fibrosarcoma cell lines that do or do not possess and express an oncogenic mutant allele of N-ras. HT1080 cells contain an endogenous mutant allele of N-ras, whereas the derivative MCH603 cell line contains only wild-type N-ras. In an earlier study (S. Gupta et al., Mol. Cell. Biol. 20:9294-9306, 2000), we had shown that HT1080 cells produce rapidly growing, aggressive tumors in athymic nude mice, whereas MCH603 cells produced more slowly growing tumors and was termed weakly tumorigenic. An extensive analysis of the Ras signaling pathways (Raf, Rac1, and RhoA) provided evidence for a potential novel pathway that was critical for the aggressive tumorigenic phenotype and could be activated by elevated levels of constitutively active MEK. In this study we examined the role of phosphoinositide 3-kinase (PI 3-kinase) in the regulation of the transformed and aggressive tumorigenic phenotypes expressed in HT1080 cells. Both HT1080 (mutant N-ras) and MCH603 (wild-type N-ras) have similar levels of constitutively active Akt, a downstream target of activated PI 3-kinase. We find that both cell lines constitutively express platelet-derived growth factor (PDGF) and PDGF receptors. Transfection with tumor suppressor PTEN cDNA into HT1080 and constitutively active PI 3-kinase-CAAX cDNA into MCH603 cells, respectively, resulted in several interesting and novel observations. Activation of the PI 3-kinase/Akt pathway, including NF-kappaB, is not required for the aggressive tumorigenic phenotype in HT1080 cells. Activation of NF-kappaB is complex: in MCH603 cells it is mediated by Akt, whereas in HT1080 cells activation also involves other pathway(s) that are activated by mutant Ras. A threshold level of activation of PI 3-kinase is required in MCH603 cells before stimulatory cross talk to the RhoA, Rac1, and Raf pathways occurs, without a corresponding activation of Ras. The increased levels of activation seen were similar to those observed

  19. Conversion from human haematopoietic stem cells to keratinocytes requires keratinocyte secretory factors.

    PubMed

    Fujita, Y; Inokuma, D; Abe, R; Sasaki, M; Nakamura, H; Shimizu, T; Shimizu, H

    2012-08-01

    Recent studies have reported that bone-marrow-derived stem cells (BMSCs), including haematopoietic stem cells (HSCs) and mesenchymal stromal cells, differentiate in order to regenerate various cellular lineages. Based on these findings, it is known that BMSCs can be used clinically to treat various disorders, such as myocardial infarction and neurotraumatic injuries. However, the mechanism of HSC conversion into organ cells is incompletely understood. The mechanism is suspected to involve direct cell-cell interaction between BMSCs, damaged organ cells, and paracrine-regulated soluble factors from the organ, but to date, there have been no investigations into which of these are essential for keratinocyte differentiation from HSCs. To elucidate the mechanism and necessary conditions for HSC differentiation into keratinocytes in vitro. We cultured human (h)HSCs under various conditions to try to elucidate the mechanism and necessary conditions for hHSCs to differentiate into keratinocytes. hHSCs cocultured with mouse keratinocytes induced expression of human keratin 14 and transglutaminase I. Only 0.1% of the differentiated keratinocytes possessed multiple nuclei indicating cell fusion. Coculture of hHSCs with fixed murine keratinocytes (predicted to stabilize cellular components) failed to induce conversion into keratinocytes. Conversely, keratinocyte-conditioned medium from both human and mouse keratinocytes was found to mediate hHSC conversion into keratinocytes. Human HSCs are capable of differentiation into keratinocytes, and cell fusion is extremely rare. This differentiating is mediated by the plasma environment rather than by direct cell-cell interactions. © The Author(s). CED © 2012 British Association of Dermatologists.

  20. Requirement for Interaction of PI3-Kinase p110α with RAS in Lung Tumor Maintenance

    PubMed Central

    Castellano, Esther; Sheridan, Clare; Thin, May Zaw; Nye, Emma; Spencer-Dene, Bradley; Diefenbacher, Markus E.; Moore, Christopher; Kumar, Madhu S.; Murillo, Miguel M.; Grönroos, Eva; Lassailly, Francois; Stamp, Gordon; Downward, Julian

    2013-01-01

    Summary RAS proteins directly activate PI3-kinases. Mice bearing a germline mutation in the RAS binding domain of the p110α subunit of PI3-kinse are resistant to the development of RAS-driven tumors. However, it is unknown whether interaction of RAS with PI3-kinase is required in established tumors. The need for RAS interaction with p110α in the maintenance of mutant Kras-driven lung tumors was explored using an inducible mouse model. In established tumors, removal of the ability of p110α to interact with RAS causes long-term tumor stasis and partial regression. This is a tumor cell-autonomous effect, which is improved significantly by combination with MEK inhibition. Total removal of p110α expression or activity has comparable effects, albeit with greater toxicities. PMID:24229709

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

    SciTech Connect

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

    2007-04-27

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

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. Phosphoinositide 3-kinase controls early and late events in mammalian cell division.

    PubMed

    García, Zaira; Kumar, Amit; Marqués, Miriam; Cortés, Isabel; Carrera, Ana C

    2006-02-22

    Phosphoinositide 3-kinase (PI3K) plays a crucial role in triggering cell division. To initiate this process, PI3K induces two distinct routes, of which one promotes cell growth and the other regulates cyclin-dependent kinases. Fine-tuned PI3K regulation is also required for later cell cycle phases. Here, we review the multiple points at which PI3K controls cell division and discuss its impact on human cancer.

  7. PI-3 kinase-PTEN signaling node: an intercept point for the control of angiogenesis.

    PubMed

    Castellino, R C; Muh, C R; Durden, D L

    2009-01-01

    Angiogenesis is tightly regulated by opposing mechanisms in mammalian cells and is controlled by the angiogenic switch. Other review articles have described a central role for the PTEN/PI-3 kinase/AKT signaling node in the coordinate control of cell division, tumor growth, apoptosis, invasion and cellular metabolism [1, 2]. In this review, we focus on literature that supports the PTEN/PI-3 kinase/AKT signaling node as a major control point for the angiogenic switch in both the on and off positions. We also discuss the rationale for designing small molecule drugs that target the PTEN/PI-3 kinase/AKT signaling node for therapeutic intervention. Our hypothesis is that, instead of inhibiting one cell surface receptor, such as VEGFR2 with bevacizumab (Avastin), thereby leaving a significant number of receptors free to pulse angiogenic signals, a more effective strategy may be to regulate signaling through an intercept node where redundant cell surface receptor signals converge to transmit important signaling events within the cell. This therapeutic configuration brings coordinate control over multiple cell surface receptors in concert with a physiologic response which may combine arrest of cell cycle progression with growth inhibition and the induction of genes involved in specialized functions such as movement, which are all required for the complex process of angiogenesis to occur in a temporal-spatial paradigm.

  8. 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.

  9. 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.

  10. Myricetin inhibits UVB-induced angiogenesis by regulating PI-3 kinase in vivo

    PubMed Central

    Jung, Sung Keun; Lee, Ki Won; Byun, Sanguine; Lee, Eun Jung; Kim, Jong-Eun; Bode, Ann M.; Dong, Zigang

    2010-01-01

    Myricetin is one of the principal phytochemicals in onions, berries and red wine. Previous studies showed that myricetin exhibits potent anticancer and chemopreventive effects. The present study examined the effect of myricetin on ultraviolet (UV) B-induced angiogenesis in an SKH-1 hairless mouse skin tumorigenesis model. Topical treatment with myricetin inhibited repetitive UVB-induced neovascularization in SKH-1 hairless mouse skin. The induction of vascular endothelial growth factor, matrix metalloproteinase (MMP)-9 and MMP-13 expression by chronic UVB irradiation was significantly suppressed by myricetin treatment. Immunohistochemical and western blot analyses revealed that myricetin inhibited UVB-induced hypoxia inducible factor-1α expression in mouse skin. Western blot analysis and kinase assay data revealed that myricetin suppressed UVB-induced phosphatidylinositol-3 (PI-3) kinase activity and subsequently attenuated the UVB-induced phosphorylation of Akt/p70S6K in mouse skin lysates. A pull-down assay revealed the direct binding of PI-3 kinase and myricetin in mouse skin lysates. Our results indicate that myricetin suppresses UVB-induced angiogenesis by regulating PI-3 kinase activity in vivo in mouse skin. PMID:20008033

  11. Investigating the role of class-IA PI 3-kinase isoforms in adipocyte differentiation

    SciTech Connect

    Kim, Ji Eun; Shepherd, Peter R. Chaussade, Claire

    2009-02-20

    PI 3-kinases, in particular class-IA, are key signalling molecules controlling many cellular processes including growth, proliferation, migration and differentiation. In this study, we have used a collection of isoform selective PI 3-kinase inhibitors to determine whether attenuation of signalling through class-IA PI 3-kinase isoforms will impact adipocyte differentiation. First, we analysed the expression profiles and found that fibroblastic pre-adipocytes express detectable levels of p110{alpha} and p110{delta} and that after differentiation, p110{delta} levels fall while p110{alpha} levels rise, together with C/EBP{alpha} and PPAR{gamma}. When using specific inhibitors during the differentiation process, we observed that neither p110{beta} nor p110{delta} inhibition, had any significant effect. In contrast PIK-75, a selective p110{alpha} inhibitor completely abolished adipocyte differentiation as assessed by morphology, transcript and protein levels of adipocyte markers. These results indicate that long term treatment with p110{alpha} inhibitors could potentially have a severe impact on fat cell numbers in vivo.

  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. 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

  14. Role of Phosphoinositide 3-Kinase in the Aggressive Tumor Growth of HT1080 Human Fibrosarcoma Cells

    PubMed Central

    Gupta, Swati; Stuffrein, Selma; Plattner, Rina; Tencati, Michael; Gray, Christa; Whang, Young E.; Stanbridge, Eric J.

    2001-01-01

    We have developed a model system of human fibrosarcoma cell lines that do or do not possess and express an oncogenic mutant allele of N-ras. HT1080 cells contain an endogenous mutant allele of N-ras, whereas the derivative MCH603 cell line contains only wild-type N-ras. In an earlier study (S. Gupta et al., Mol. Cell. Biol. 20:9294–9306, 2000), we had shown that HT1080 cells produce rapidly growing, aggressive tumors in athymic nude mice, whereas MCH603 cells produced more slowly growing tumors and was termed weakly tumorigenic. An extensive analysis of the Ras signaling pathways (Raf, Rac1, and RhoA) provided evidence for a potential novel pathway that was critical for the aggressive tumorigenic phenotype and could be activated by elevated levels of constitutively active MEK. In this study we examined the role of phosphoinositide 3-kinase (PI 3-kinase) in the regulation of the transformed and aggressive tumorigenic phenotypes expressed in HT1080 cells. Both HT1080 (mutant N-ras) and MCH603 (wild-type N-ras) have similar levels of constitutively active Akt, a downstream target of activated PI 3-kinase. We find that both cell lines constitutively express platelet-derived growth factor (PDGF) and PDGF receptors. Transfection with tumor suppressor PTEN cDNA into HT1080 and constitutively active PI 3-kinase–CAAX cDNA into MCH603 cells, respectively, resulted in several interesting and novel observations. Activation of the PI 3-kinase/Akt pathway, including NF-κB, is not required for the aggressive tumorigenic phenotype in HT1080 cells. Activation of NF-κB is complex: in MCH603 cells it is mediated by Akt, whereas in HT1080 cells activation also involves other pathway(s) that are activated by mutant Ras. A threshold level of activation of PI 3-kinase is required in MCH603 cells before stimulatory cross talk to the RhoA, Rac1, and Raf pathways occurs, without a corresponding activation of Ras. The increased levels of activation seen were similar to those observed

  15. Analyses of the secondary particle radiation and the DNA damage it causes to human keratinocytes

    SciTech Connect

    Lebel E.; Rusek A.; Sivertz, M.; Yip, K.; Thompson, K.; Tafrov, S.

    2011-11-22

    High-energy protons, and high mass and energy ions, along with the secondary particles they produce, are the main contributors to the radiation hazard during space explorations. Skin, particularly the epidermis, consisting mainly of keratinocytes with potential for proliferation and malignant transformation, absorbs the majority of the radiation dose. Therefore, we used normal human keratinocytes to investigate and quantify the DNA damage caused by secondary radiation. Its manifestation depends on the presence of retinol in the serum-free media, and is regulated by phosphatidylinositol 3-kinases. We simulated the generation of secondary radiation after the impact of protons and iron ions on an aluminum shield. We also measured the intensity and the type of the resulting secondary particles at two sample locations; our findings agreed well with our predictions. We showed that secondary particles inflict DNA damage to different extents, depending on the type of primary radiation. Low-energy protons produce fewer secondary particles and cause less DNA damage than do high-energy protons. However, both generate fewer secondary particles and inflict less DNA damage than do high mass and energy ions. The majority of cells repaired the initial damage, as denoted by the presence of 53BPI foci, within the first 24 hours after exposure, but some cells maintained the 53BP1 foci longer.

  16. Analyses of the Secondary Particle Radiation and the DNA Damage it Causes to Human Keratinocytes

    SciTech Connect

    Lebel E. A.; Tafrov S.; Rusek, A.; Sivertz, M. B.; Yip, K.; Thompson, K. H.

    2011-11-01

    High-energy protons, and high mass and energy ions, along with the secondary particles they produce, are the main contributors to the radiation hazard during space explorations. Skin, particularly the epidermis, consisting mainly of keratinocytes with potential for proliferation and malignant transformation, absorbs the majority of the radiation dose. Therefore, we used normal human keratinocytes to investigate and quantify the DNA damage caused by secondary radiation. Its manifestation depends on the presence of retinol in the serum-free media, and is regulated by phosphatidylinositol 3-kinases. We simulated the generation of secondary radiation after the impact of protons and iron ions on an aluminum shield. We also measured the intensity and the type of the resulting secondary particles at two sample locations; our findings agreed well with our predictions. We showed that secondary particles inflict DNA damage to different extents, depending on the type of primary radiation. Low-energy protons produce fewer secondary particles and cause less DNA damage than do high-energy protons. However, both generate fewer secondary particles and inflict less DNA damage than do high mass and energy ions. The majority of cells repaired the initial damage, as denoted by the presence of 53BPI foci, within the first 24 hours after exposure, but some cells maintained the 53BP1 foci longer.

  17. 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

  18. Comparative metabolomics and structural characterizations illuminate colibactin pathway-dependent small molecules.

    PubMed

    Vizcaino, Maria I; Engel, Philipp; Trautman, Eric; Crawford, Jason M

    2014-07-02

    The gene cluster responsible for synthesis of the unknown molecule "colibactin" has been identified in mutualistic and pathogenic Escherichia coli. The pathway endows its producer with a long-term persistence phenotype in the human bowel, a probiotic activity used in the treatment of ulcerative colitis, and a carcinogenic activity under host inflammatory conditions. To date, functional small molecules from this pathway have not been reported. Here we implemented a comparative metabolomics and targeted structural network analyses approach to identify a catalog of small molecules dependent on the colibactin pathway from the meningitis isolate E. coli IHE3034 and the probiotic E. coli Nissle 1917. The structures of 10 pathway-dependent small molecules are proposed based on structural characterizations and network relationships. The network will provide a roadmap for the structural and functional elucidation of a variety of other small molecules encoded by the pathway. From the characterized small molecule set, in vitro bacterial growth inhibitory and mammalian CNS receptor antagonist activities are presented.

  19. Dopamine Depletion Impairs Bilateral Sensory Processing in the Striatum in a Pathway-Dependent Manner.

    PubMed

    Ketzef, Maya; Spigolon, Giada; Johansson, Yvonne; Bonito-Oliva, Alessandra; Fisone, Gilberto; Silberberg, Gilad

    2017-05-17

    Parkinson's disease (PD) is a movement disorder caused by the loss of dopaminergic innervation, particularly to the striatum. PD patients often exhibit sensory impairments, yet the underlying network mechanisms are unknown. Here we examined how dopamine (DA) depletion affects sensory processing in the mouse striatum. We used the optopatcher for online identification of direct and indirect pathway projection neurons (MSNs) during in vivo whole-cell recordings. In control mice, MSNs encoded the laterality of sensory inputs with larger and earlier responses to contralateral than ipsilateral whisker deflection. This laterality coding was lost in DA-depleted mice due to adaptive changes in the intrinsic and synaptic properties, mainly, of direct pathway MSNs. L-DOPA treatment restored laterality coding by increasing the separation between ipsilateral and contralateral responses. Our results show that DA depletion impairs bilateral tactile acuity in a pathway-dependent manner, thus providing unexpected insights into the network mechanisms underlying sensory deficits in PD. VIDEO ABSTRACT. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Mesoscale Phenomena Associated with Mineral Surfaces and Pathway-Dependent Chemical Processes

    NASA Astrophysics Data System (ADS)

    Brown, G. E.; Johnson, N. C.; Garcia Del Real, P.; Maher, K.; Bird, D. K.; Rosenbauer, R. J.; Thomas, B.; Levard, C.

    2012-12-01

    Multiphase physicochemical transport and interfacial processes in natural and synthetic permeable media are pervasive in energy and Earth systems, where interfacial chemical reactions play an enormous role. These coupled reactions control the composition of our environment, including the atmosphere, oceans, and groundwaters, and the soils derived from interactions of atmospheric gases and natural waters with solid phases. Aqueous fluids, liquid hydrocarbons, and gases flow through permeable geological media along pathways that can be exceedingly complex at the nano- to microscales. Adding to this complexity are the chemical reactions occurring along these pathways that can irreversibly alter permeability and porosity as well as the compositions of fluid, gas, and solid phases, depending on physicochemical conditions. This talk will discuss the role of chemical reactions on mineral surfaces in several areas, including the structure of the electrical double layer at mineral/water interfaces and how it changes as a function of solution conditions, sequestration and transformation of environmental contaminants on mineral surfaces, mineral carbonation reactions and CO2 sequestration, and nanoparticle stability and transformations in natural systems. It will also include examples of pathway-dependent mesoscale chemical processes in the synthetic world involving energy materials. Examples in this area will include a synchrotron-based high-resolution 3D tomography study of Li-NiO battery electrodes under in operando conditions and metal-organic framework structures that can be used for hydrogen storage, separation, catalysis, and sequestration.

  1. Modulation of calprotectin in human keratinocytes by keratinocyte growth factor and interleukin-1alpha.

    PubMed

    Bando, Mika; Hiroshima, Yuka; Kataoka, Masatoshi; Herzberg, Mark C; Ross, Karen F; Shinohara, Yasuo; Yamamoto, Takenori; Nagata, Toshihiko; Kido, Jun-ichi

    2010-01-01

    Calprotectin is an antimicrobial complex composed of the S100A8 and S100A9 protein family subunits. Contributing to innate immunity, calprotectin expression is increased by interleukin-1alpha (IL-1alpha), which modulates keratinocyte differentiation. Keratinocyte growth factor (KGF) is produced by mesenchymal cells and has a mitogenic activity for epithelial cells. In this study, we investigated the effect of KGF on calprotectin expression in keratinocytes and modulation by IL-1alpha. Human keratinocytes were cultured with KGF in the presence or absence of a KGF receptor (KGFR) inhibitor or mitogen-activated protein kinase (MAPK) inhibitors. Calprotectin (S100A8/S100A9) expression was determined by northern blotting and enzyme-linked immunosorbent assay, respectively, whereas MAPK phosphorylation was analyzed by western blot analysis. KGF significantly decreased the expression of S100A8/S100A9-specific mRNAs and calprotectin protein. In the presence of KGF, KGFR inhibitor or extracellular-regulated kinase inhibitor restored KGF-downregulated expression of S100A8/S100A9. KGF increased IL-1alpha expression in keratinocytes, whereas IL-1alpha increased KGF expression in fibroblasts. Cocultured fibroblast and keratinocytes showed lower S100A8/S100A9 mRNA expression than keratinocytes alone in the presence or absence of IL-1alpha or KGF. These results suggest that fibroblast-derived KGF reduces or restricts calprotectin expression in keratinocytes, which supports our hypothesis that calprotectin expression in keratinocytes is modulated by factors associated with epithelial-mesenchymal interactions.

  2. Enhancement of Morphological Plasticity in Hippocampal Neurons by a Physically Modified Saline via Phosphatidylinositol-3 Kinase

    PubMed Central

    Roy, Avik; Modi, Khushbu K.; Khasnavis, Saurabh; Ghosh, Supurna; Watson, Richard; Pahan, Kalipada

    2014-01-01

    Increase of the density of dendritic spines and enhancement of synaptic transmission through ionotropic glutamate receptors are important events, leading to synaptic plasticity and eventually hippocampus-dependent spatial learning and memory formation. Here we have undertaken an innovative approach to upregulate hippocampal plasticity. RNS60 is a 0.9% saline solution containing charge-stabilized nanobubbles that are generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not NS (normal saline), PNS60 (saline containing a comparable level of oxygen without the TCP modification), or RNS10.3 (TCP-modified normal saline without excess oxygen), stimulated morphological plasticity and synaptic transmission via NMDA- and AMPA-sensitive calcium influx in cultured mouse hippocampal neurons. Using mRNA-based targeted gene array, real-time PCR, immunoblot, and immunofluorescence analyses, we further demonstrate that RNS60 stimulated the expression of many plasticity-associated genes in cultured hippocampal neurons. Activation of type IA, but not type IB, phosphatidylinositol-3 (PI-3) kinase by RNS60 together with abrogation of RNS60-mediated upregulation of plasticity-related proteins (NR2A and GluR1) and increase in spine density, neuronal size, and calcium influx by LY294002, a specific inhibitor of PI-3 kinase, suggest that RNS60 upregulates hippocampal plasticity via activation of PI-3 kinase. Finally, in the 5XFAD transgenic model of Alzheimer’s disease (AD), RNS60 treatment upregulated expression of plasticity-related proteins PSD95 and NR2A and increased AMPA- and NMDA-dependent hippocampal calcium influx. These results describe a novel property of RNS60 in stimulating hippocampal plasticity, which may help AD and other dementias. PMID:25007337

  3. Fructosamine 3-kinase is involved in an intracellular deglycation pathway in human erythrocytes.

    PubMed Central

    Delpierre, Ghislain; Collard, François; Fortpied, Juliette; Van Schaftingen, Emile

    2002-01-01

    Fructosamine 3-kinase, which phosphorylates low-molecular-mass and protein-bound fructosamines on the third carbon of their deoxyfructose moiety, is quite active in erythrocytes, and was proposed to initiate a process removing fructosamine residues from proteins. In the present study, we show that incubation of human erythrocytes with 200 mM glucose not only caused the progressive formation of glycated haemoglobin, but also increased the level of an anionic form of haemoglobin containing alkali-labile phosphate, to approx. 5% of total haemoglobin. 1-Deoxy-1-morpholinofructose (DMF), a substrate and competitive inhibitor of fructosamine 3-kinase, doubled the rate of accumulation of glycated haemoglobin, but markedly decreased the amount of haemoglobin containing alkali-labile phosphate. The latter corresponds therefore to haemoglobin bound to a fructosamine 3-phosphate group (FN3P-Hb). Returning erythrocytes incubated with 200 mM glucose and DMF to a low-glucose medium devoid of DMF caused a decrease in the amount of glycated haemoglobin, a transient increase in FN3P-Hb and a net decrease in the sum (glycated haemoglobin+FN3P-Hb). These effects were prevented by DMF, indicating that fructosamine 3-kinase is involved in the removal of fructosamine residues. The second step of this 'deglycation' process is most likely a spontaneous decomposition of the fructosamine 3-phosphate residues to a free amine, 3-deoxyglucosone and P(i). This is consistent with the findings that 2-oxo-3-deoxygluconate, the product of 3-deoxyglucosone oxidation, is formed in erythrocytes incubated for 2 days with 200 mM glucose in a sufficient amount to account for the removal of fructosamine residues from proteins, and that DMF appears to inhibit the formation of 2-oxo-3-deoxygluconate from elevated glucose concentrations. PMID:11975663

  4. 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.

  5. 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.

  6. Modulation of phosphatidylinositol 3-kinase signaling reduces intimal hyperplasia in aortocoronary saphenous vein grafts.

    PubMed

    Hata, Jonathan A; Petrofski, Jason A; Schroder, Jacob N; Williams, Matthew L; Timberlake, Sarah H; Pippen, Anne; Corwin, Michael T; Solan, Amy K; Jakoi, Andre; Gehrig, Thomas R; Kontos, Christopher D; Milano, Carmelo A

    2005-06-01

    Fifty percent of human aortocoronary saphenous vein grafts are occluded after 10 years. Intimal hyperplasia is an initial step in graft occlusion and consists of vascular smooth muscle cell proliferation. Phosphatidylinositol 3-kinase and its downstream regulator, the inositol 3-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10), are important regulators of vascular smooth muscle cell proliferation, migration, and cell death. This study tests whether overexpression of PTEN in aortocoronary saphenous vein grafts can reduce intimal hyperplasia. Adult dogs underwent aortocoronary bypass grafting to the left anterior descending artery by using the autologous saphenous vein. Saphenous vein grafts were treated with phosphate-buffered saline (n = 9), empty adenovirus (n = 8), or adenovirus encoding for PTEN (n = 8). Arteriography at 30 and 90 days assessed saphenous vein graft patency. A subset received saphenous vein grafts treated with a marker transgene (beta-galactosidase, n = 3), empty adenovirus (n = 4), or adenovirus encoding for PTEN (n = 4) and were killed on postoperative day 3 to confirm expression. Vascular smooth muscle cells were isolated from canine saphenous vein infected with adenovirus encoding for PTEN, and immunoblotting and proliferation assays were performed. Saphenous vein graft transgene expression was confirmed by means of immunohistochemistry, immunoblotting, and polymerase chain reaction. Arteriograms revealed all saphenous vein grafts to be patent. Saphenous vein grafts treated with adenovirus encoding for PTEN demonstrated reduced intimal area compared with those treated with empty adenovirus and phosphate-buffered saline (1.39 +/- 0.11 vs 2.35 +/- 0.3 and 2.57 +/- 0.4 mm 2 , P < .05), and the intima/media ratio was lower in saphenous vein grafts treated with adenovirus encoding for PTEN (0.50 +/- 0.05 vs 1.43 +/- 0.18 and 1.11 +/- 0.14, P < .005). PTEN overexpression in vascular smooth muscle cells inhibited platelet

  7. 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

  8. PI3-kinase and mTOR inhibitors differently modulate the function of the ABCG2 multidrug transporter.

    PubMed

    Hegedüs, Csilla; Truta-Feles, Krisztina; Antalffy, Géza; Brózik, Anna; Kasza, Ildikó; Német, Katalin; Orbán, Tamás I; Özvegy-Laczka, Csilla; Váradi, András; Sarkadi, Balázs

    2012-04-20

    The ATP-binding cassette (ABC) transporter ABCG2 plays an important role in tissue detoxification and confers multidrug resistance to cancer cells. Identification of expressional and functional cellular regulators of this multidrug transporter is therefore intensively pursued. The PI3-kinase/Akt signaling axis has been implicated as a key element in regulating various cellular functions, including the expression and plasma membrane localization of ABCG2. Here we demonstrate that besides inhibiting their respective target kinases, the pharmacological PI3-kinase inhibitor LY294002 and the downstream mTOR kinase inhibitor rapamycin also directly inhibit ABCG2 function. In contrast, wortmannin, another commonly used pharmacological inhibitor of PI3-kinase does not interact with the transporter. We suggest that direct functional modulation of ABCG2 should be taken into consideration when pharmacological agents are applied to dissect the specific role of PI3-kinase/Akt/mTOR signaling in cellular functions.

  9. 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

  10. Identification and Targeting of Upstream Tyrosine Kinases Mediating PI3 Kinase Activation in PTEN-Deficient Prostate Cancer

    DTIC Science & Technology

    2009-06-01

    by Ras, and is amplified by PTEN loss in the majority of advanced prostate cancers (PCa). We found that RTK inhibitors lapatinib and sorafenib could...PI3 kinase activity, but combined treatment with lapatinib and sorafenib (a multi-kinase inhibitor) was as effective as a direct PI3 kinase... sorafenib , contributed to PI3K pathway activation in PTEN deficient LNCaP cells. Moreover, this inhibition correlated with loss of tyrosine

  11. 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.

  12. Small molecule inhibitors of phosphoinositide 3-kinase (PI3K) delta and gamma.

    PubMed

    Ameriks, Michael K; Venable, Jennifer D

    2009-01-01

    In recent years, pharmaceutical companies have increasingly focused on phosphoinositide 3-kinases delta (PI3Kdelta) and gamma (PI3Kgamma) as therapeutic targets for the treatment of inflammatory and autoimmune diseases. All class 1 PI3-kinases (alpha/beta/gamma/delta) generate phospholipid second messengers that help govern cellular processes such as migration, proliferation, and apoptosis. PI3K delta/ gamma lipid kinases are mainly restricted to the hematopoetic system whereas PI3K alpha/beta are ubiquitously expressed, thus raising potential toxicity concerns for chronic indications such as asthma and rheumatoid arthritis. Therefore, the challenge in developing a small molecule inhibitor of PI3K is to define and attain the appropriate isoform selectivity profile. Significant advances in the design of such compounds have been achieved by utilizing x-ray crystal structures of various inhibitors bound to PI3Kgamma in conjunction with pharmacophore modeling and high-throughput screening. Herein, we review the history and challenges involved with the discovery of small molecule isoform-specific PI3K inhibitors. Recent progress in the design of selective PI3Kdelta, PI3Kgamma, and PI3Kdelta/gamma dual inhibitors will be presented.

  13. Anaplasma phagocytophilum infection modulates expression of megakaryocyte cell cycle genes through phosphatidylinositol-3-kinase signaling.

    PubMed

    Khanal, Supreet; Sultana, Hameeda; Catravas, John D; Carlyon, Jason A; Neelakanta, Girish

    2017-01-01

    Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis infects neutrophils and other cells from hematopoietic origin. Using human megakaryocytic cell line, MEG-01, we show that expression of cell cycle genes in these cells are altered upon A. phagocytophilum infection. Expression of several cell cycle genes in MEG-01 cells was significantly up regulated at early and then down regulated at later stages of A. phagocytophilum infection. Lactate dehydrogenase (LDH) assays revealed reduced cellular cytotoxicity in MEG-01 cells upon A. phagocytophilum infection. The levels of both PI3KCA (p110 alpha, catalytic subunit) and PI3KR1 (p85, regulatory subunit) of Class I PI3 kinases and phosphorylated protein kinase B (Akt/PKB) and inhibitory kappa B (IκB) were elevated at both early and late stages of A. phagocytophilum infection. Inhibition of PI3 kinases with LY294002 treatment resulted in significant reduction in the expression of tested cell cycle genes, A. phagocytophilum burden and phosphorylated Akt levels in these MEG-01 cells. Collectively, these results suggest a role for PI3K-Akt-NF-κB signaling pathway in the modulation of megakaryocyte cell cycle genes upon A. phagocytophilum infection.

  14. 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.

  15. Advances in the researches on the biological activities and inhibitors of phosphatidylinositol 3-kinase.

    PubMed

    Tang, Jian-Feng; Wen, Qing; Sun, Jian; Zhang, Wei-Ming; Zhu, Hai-Liang

    2014-06-01

    The PI3K/AKT/mTOR pathway is an intracellular signaling pathway, being important in apoptosis hence cancer such as breast cancer and non-small-cell lung cancer. It signaling axis controls cell proliferation and survival and has achieved major importance as a target for cancer therapy. The serine/threonine kinase Akt (also known as protein kinase B or PKB), since its initial discovery as a protooncogene, has become a major focus of attention because of its critical regulatory role in diverse cellular processes, including cancer progression and insulin metabolism. The Akt cascade is activated by receptor tyrosine kinases, integrins, B and T cell receptors, cytokine receptors, G protein coupled receptors and other stimuli that induce the production of phosphatidylinositol 3,4,5 triphosphates (PtdIns(3,4,5)P3) by phosphoinositide 3-kinase (PI3K). Therefore, PI3K plays an important role in in numerous cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking. In this review, we introduced the structure of the PI3K, and then focused on its biological activities. In addition, we reviewed the advances in the researches of PI3K as well as related inhibitors over the last couple of decades. Finally, we also discussed the prospect and developmental trend of phosphatidylinositol 3-kinase as antitumor agents.

  16. 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.

  17. Identification of Small Molecule Inhibitors of Phosphatidylinositol 3-Kinase and Autophagy*

    PubMed Central

    Farkas, Thomas; Daugaard, Mads; Jäättelä, Marja

    2011-01-01

    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. PMID:21930714

  18. 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

  19. 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.

  20. 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.

  1. Phosphatidylinositol 3-kinase and dynamics of insulin resistance in denervated slow and fast muscles in vivo.

    PubMed

    Elmendorf, J S; Damrau-Abney, A; Smith, T R; David, T S; Turinsky, J

    1997-04-01

    Regulation of glucose uptake by 1- and 3-day denervated soleus (slow-twitch) and plantaris (fast-twitch) muscles in vivo was investigated. One day after denervation, soleus and plantaris muscles exhibited 62 and 65% decreases in insulin-stimulated 2-deoxyglucose uptake, respectively, compared with corresponding control muscles. At this interval, denervated muscles showed no alterations in insulin receptor binding and activity, amount and activity of phosphatidylinositol 3-kinase, and amounts of GLUT-1 and GLUT-4. Three days after denervation, there was no increase in 2-deoxyglucose uptake in response to insulin in soleus muscle, whereas plantaris muscle exhibited a 158% increase in basal and an almost normal absolute increment in insulin-stimulated uptake. Despite these differences, denervated soleus and plantaris muscles exhibited comparable decreases in insulin-stimulated activities of the insulin receptor (approximately 40%) and phosphatidylinositol 3-kinase (approximately 50%) and a pronounced decrease in GLUT-4. An increase in GLUT-1 in plantaris, but not soleus, muscle 3 days after denervation is consistent with augmented basal 2-deoxyglucose uptake in plantaris muscle at this interval. These results demonstrate that, in denervated muscles, there is a clear dissociation between insulin-stimulated 2-deoxyglucose uptake and upstream events involved in insulin-stimulated glucose uptake.

  2. Omega-3 polyunsaturated fatty acids selectively inhibit growth in neoplastic oral keratinocytes by differentially activating ERK1/2

    PubMed Central

    Parkinson, Eric Kenneth

    2013-01-01

    The long-chain omega-3 polyunsaturated fatty acids (n-3 PUFAs)—eicosapentaenoic acid (EPA) and its metabolite docosahexaenoic acid (DHA)—inhibit cancer formation in vivo, but their mechanism of action is unclear. Extracellular signal-regulated kinase 1/2 (ERK1/2) activation and inhibition have both been associated with the induction of tumour cell apoptosis by n-3 PUFAs. We show here that low doses of EPA, in particular, inhibited the growth of premalignant and malignant keratinocytes more than the growth of normal counterparts by a combination of cell cycle arrest and apoptosis. The growth inhibition of the oral squamous cell carcinoma (SCC) lines, but not normal keratinocytes, by both n-3 PUFAs was associated with epidermal growth factor receptor (EGFR) autophosphorylation, a sustained phosphorylation of ERK1/2 and its downstream target p90RSK but not with phosphorylation of the PI3 kinase target Akt. Inhibition of EGFR with either the EGFR kinase inhibitor AG1478 or an EGFR-blocking antibody inhibited ERK1/2 phosphorylation, and the blocking antibody partially antagonized growth inhibition by EPA but not by DHA. DHA generated more reactive oxygen species and activated more c-jun N-terminal kinase than EPA, potentially explaining its increased toxicity to normal keratinocytes. Our results show that, in part, EPA specifically inhibits SCC growth and development by creating a sustained signalling imbalance to amplify the EGFR/ERK/p90RSK pathway in neoplastic keratinocytes to a supraoptimal level, supporting the chemopreventive potential of EPA, whose toxicity to normal cells might be reduced further by blocking its metabolism to DHA. Furthermore, ERK1/2 phosphorylation may have potential as a biomarker of n-3 PUFA function in vivo. PMID:23892603

  3. Intracellular Localization of Phosphatidylinositide 3-kinase and Insulin Receptor Substrate-1 in Adipocytes: Potential Involvement of a Membrane Skeleton

    PubMed Central

    Clark, Sharon F.; Martin, Sally; Carozzi, Amanda J.; Hill, Michelle M.; James, David E.

    1998-01-01

    Phosphatidylinositide (PI) 3-kinase binds to tyrosyl-phosphorylated insulin receptor substrate-1 (IRS-1) in insulin-treated adipocytes, and this step plays a central role in the regulated movement of the glucose transporter, GLUT4, from intracellular vesicles to the cell surface. PDGF, which also activates PI 3-kinase in adipocytes, has no significant effect on GLUT4 trafficking in these cells. We propose that this specificity may be mediated by differential localization of PI 3-kinase in response to insulin versus PDGF activation. Using subcellular fractionation in 3T3-L1 adipocytes, we show that insulin- and PDGF-stimulated PI 3-kinase activities are located in an intracellular high speed pellet (HSP) and in the plasma membrane (PM), respectively. The HSP is also enriched in IRS-1, insulin-stimulated tyrosyl-phosphorylated IRS-1 and intracellular GLUT4-containing vesicles. Using sucrose density gradient sedimentation, we have been able to segregate the HSP into two separate subfractions: one enriched in IRS-1, tyrosyl-phosphorylated IRS-1, PI 3-kinase as well as cytoskeletal elements, and another enriched in membranes, including intracellular GLUT4 vesicles. Treatment of the HSP with nonionic detergent, liberates all membrane constituents, whereas IRS-1 and PI 3-kinase remain insoluble. Conversely, at high ionic strength, membranes remain intact, whereas IRS-1 and PI 3-kinase become freely soluble. We further show that this IRS-1–PI 3-kinase complex exists in CHO cells overexpressing IRS-1 and, in these cells, the cytosolic pool of IRS-1 and PI 3-kinase is released subsequent to permeabilization with Streptolysin-O, whereas the particulate fraction of these proteins is retained. These data suggest that IRS-1, PI 3-kinase, as well as other signaling intermediates, may form preassembled complexes that may be associated with the actin cytoskeleton. This complex must be in close apposition to the cell surface, enabling access to the insulin receptor and presumably

  4. Methicillin-resistant Staphylococcus aureus adaptation to human keratinocytes.

    PubMed

    Soong, Grace; Paulino, Franklin; Wachtel, Sarah; Parker, Dane; Wickersham, Matthew; Zhang, Dongni; Brown, Armand; Lauren, Christine; Dowd, Margaret; West, Emily; Horst, Basil; Planet, Paul; Prince, Alice

    2015-04-21

    Skin is the most common site of Staphylococcus aureus infection. While most of these infections are self-limited, recurrent infections are common. Keratinocytes and recruited immune cells participate in skin defense against infection. We postulated that S. aureus is able to adapt to the milieu within human keratinocytes to avoid keratinocyte-mediated clearance. From a collection of S. aureus isolated from chronically infected patients with atopic dermatitis, we noted 22% had an agr mutant-like phenotype. Using several models of human skin infection, we demonstrate that toxin-deficient, agr mutants of methicillin-resistant S. aureus (MRSA) USA300 are able to persist within keratinocytes by stimulating autophagy and evading caspase-1 and inflammasome activation. MRSA infection induced keratinocyte autophagy, as evidenced by galectin-8 and LC3 accumulation. Autophagy promoted the degradation of inflammasome components and facilitated staphylococcal survival. The recovery of more than 58% agr or RNAIII mutants (P < 0.0001) of an inoculum of wild-type (WT) MRSA from within wortmannin-treated keratinocytes compared to control keratinocytes reflected the survival advantage for mutants no longer expressing agr-dependent toxins. Our results illustrate the dynamic interplay between S. aureus and keratinocytes that can result in the selection of mutants that have adapted specifically to evade keratinocyte-mediated clearance mechanisms. Human skin is a major site of staphylococcal infection, and keratinocytes actively participate in eradication of these pathogens. We demonstrate that methicillin-resistant Staphylococcus aureus (MRSA) is ingested by keratinocytes and activates caspase-1-mediated clearance through pyroptosis. Toxin-deficient MRSA mutants are selected within keratinocytes that fail to induce caspase-1 activity and keratinocyte-mediated clearance. These intracellular staphylococci induce autophagy that enhances their intracellular survival by diminishing

  5. RAS/MAPK Activation Drives Resistance to Smo Inhibition, Metastasis, and Tumor Evolution in Shh Pathway-Dependent Tumors.

    PubMed

    Zhao, Xuesong; Ponomaryov, Tatyana; Ornell, Kimberly J; Zhou, Pengcheng; Dabral, Sukriti K; Pak, Ekaterina; Li, Wei; Atwood, Scott X; Whitson, Ramon J; Chang, Anne Lynn S; Li, Jiang; Oro, Anthony E; Chan, Jennifer A; Kelleher, Joseph F; Segal, Rosalind A

    2015-09-01

    Aberrant Shh signaling promotes tumor growth in diverse cancers. The importance of Shh signaling is particularly evident in medulloblastoma and basal cell carcinoma (BCC), where inhibitors targeting the Shh pathway component Smoothened (Smo) show great therapeutic promise. However, the emergence of drug resistance limits long-term efficacy, and the mechanisms of resistance remain poorly understood. Using new medulloblastoma models, we identify two distinct paradigms of resistance to Smo inhibition. Sufu mutations lead to maintenance of the Shh pathway in the presence of Smo inhibitors. Alternatively activation of the RAS-MAPK pathway circumvents Shh pathway dependency, drives tumor growth, and enhances metastatic behavior. Strikingly, in BCC patients treated with Smo inhibitor, squamous cell cancers with RAS/MAPK activation emerged from the antecedent BCC tumors. Together, these findings reveal a critical role of the RAS-MAPK pathway in drug resistance and tumor evolution of Shh pathway-dependent tumors.

  6. RAS/MAPK activation drives resistance to Smo inhibition, metastasis and tumor evolution in Shh pathway-dependent tumors

    PubMed Central

    Zhao, Xuesong; Ponomaryov, Tatyana; Ornell, Kimberly J.; Zhou, Pengcheng; Dabral, Sukriti K.; Pak, Ekaterina; Li, Wei; Atwood, Scott X.; Whitson, Ramon J.; Chang, Anne Lynn S.; Li, Jiang; Oro, Anthony E.; Chan, Jennifer A.; Kelleher, Joseph F.; Segal, Rosalind A.

    2015-01-01

    Aberrant Shh signaling promotes tumor growth in diverse cancers. The importance of Shh signaling is particularly evident in medulloblastoma and basal cell carcinoma (BCC), where inhibitors targeting the Shh pathway component Smoothened (Smo) show great therapeutic promise. However, the emergence of drug resistance limits long-term efficacy and the mechanisms of resistance remain poorly understood. Using new medulloblastoma models, we identify two distinct paradigms of resistance to Smo inhibition. Sufu mutations lead to maintenance of the Shh pathway in the presence of Smo inhibitors. Alternatively activation of the RAS/MAPK pathway circumvents Shh pathway-dependency, drives tumor growth and enhances metastatic behavior. Strikingly, in BCC patients treated with Smo inhibitor, squamous cell cancers with RAS/MAPK activation emerged from the antecedent BCC tumors. Together these findings reveal a critical role of RAS/MAPK pathway in drug resistance and tumor evolution of Shh pathway-dependent tumors. PMID:26130651

  7. 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

  8. 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.

  9. The p110α isoform of phosphoinositide 3-kinase is essential for cone photoreceptor survival.

    PubMed

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

    2015-05-01

    Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that phosphorylates the 3'OH of the inositol ring of phosphoinositides (PIs). They are responsible for coordinating a diverse range of cellular functions. Class IA PI3K is a heterodimeric protein composed of a regulatory p85 and a catalytic p110 subunit. In this study, we conditionally deleted the p110α-subunit of PI3K in cone photoreceptor cells using the Cre-loxP system. Cone photoreceptors allow for color vision in bright light (daylight vision). Cone-specific deletion of p110α resulted in cone degeneration. Our studies suggest that PI3K signaling is essential for cone photoreceptor functions. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  10. 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.

  11. 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.

  12. Keratinocyte dysfunction in vitiligo epidermis: cytokine microenvironment and correlation to keratinocyte apoptosis.

    PubMed

    Moretti, Silvia; Fabbri, Paolo; Baroni, Gianna; Berti, Samantha; Bani, Daniele; Berti, Emilio; Nassini, Romina; Lotti, Torello; Massi, Daniela

    2009-07-01

    Vitiligo is a skin disorder characterized by loss of functional melanocytes. Keratinocytes contribute to melanocyte homeostasis, and keratinocyte alteration may play a role in melanocyte dysfunction in vitiligo. In particular, the release of melanogenic mediators and the level of functioning keratinocytes may affect melanocyte dysfunction in vitiligo epidermis. Keratinocyte-derived mediators involved in pigmentation, analysed by in situ hybridization, and epidermal apoptosis, detected by TUNEL assay and electron microscopy, were evaluated in lesional and perilesional skin biopsies from 15 patients with active vitiligo and in 5 control subjects. Among the melanogenic mediators, stem cell factor (SCF) and endothelin-1 (ET-1) mRNA were significantly reduced in lesional as compared to perilesional epidermis, whereas no difference was observed in mRNA of basic fibroblastic growth factor (bFGF) and granulocyte-monocyte colony stimulating factor (GM-CSF). The expression of mRNA for tumor necrosis factor (TNF)-alpha and interleukin-6 (IL-6), two pro-inflammatory cytokines with an inhibitory effect on pigmentation, was increased in the epidermis from vitiligo biopsies, whereas their expression was practically undetectable in the skin of control subjects. Apoptotic keratinocytes were more abundant in lesional vs. perilesional skin of vitiligo patients and were absent in the epidermis of control subjects. Changes in expression of keratinocyte-derived mediators observed in the present study are consistent with their differential functions in melanocyte regulation. In particular, increased TNF-alpha could contribute to keratinocyte apoptosis, which results in reduced release of melanogenic cytokines and ultimately in melanocyte disappearance.

  13. Enhancement of UVB-induced apoptosis by apigenin in human keratinocytes and organotypic keratinocyte cultures.

    PubMed

    Abu-Yousif, Adnan O; Smith, Kimberly A; Getsios, Spiro; Green, Kathleen J; Van Dross, Rukiyah T; Pelling, Jill C

    2008-04-15

    Topical application of the bioflavonoid 4',5,7-trihydroxyflavone (apigenin) to mouse skin effectively reduces the incidence and size of skin tumors caused by UVB exposure. The ability to act as a chemopreventive compound indicates that apigenin treatment alters the molecular events initiated by UVB exposure; however, the effects of apigenin treatment on UVB-irradiated keratinocytes are not fully understood. In the present study, we have used three models of human keratinocytes to study the effect of apigenin treatment on UVB-induced apoptosis: HaCaT human keratinocyte cells, primary keratinocyte cultures isolated from human neonatal foreskin, and human organotypic keratinocyte cultures. Each keratinocyte model was exposed to a moderate dose of UVB (300-1,000 J/m(2)), then treated with apigenin (0-50 micromol/L), and harvested to assess apoptosis by Western blot analysis for poly(ADP)ribose polymerase cleavage, annexin-V staining by flow cytometry, and/or the presence of sunburn cells. Apigenin treatment enhanced UVB-induced apoptosis >2-fold in each of the models tested. When keratinocytes were exposed to UVB, apigenin treatment stimulated changes in Bax localization and increased the release of cytochrome c from the mitochondria compared with UVB exposure alone. Overexpression of the antiapoptotic protein Bcl-2 and expression of a dominant-negative form of Fas-associated death domain led to a reduction in the ability of apigenin to enhance UVB-induced apoptosis. These results suggest that enhancement of UVB-induced apoptosis by apigenin treatment involves both the intrinsic and extrinsic apoptotic pathways. The ability of apigenin to enhance UVB-induced apoptosis may explain, in part, the photochemopreventive effects of apigenin.

  14. 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.

  15. Targeting Glutamatergic Signaling and the PI3 Kinase Pathway to Halt Melanoma Progression.

    PubMed

    Rosenberg, Stephen A; Niglio, Scot A; Salehomoum, Negar; Chan, Joseph L-K; Jeong, Byeong-Seon; Wen, Yu; Li, Jiadong; Fukui, Jami; Chen, Suzie; Shin, Seung-Shick; Goydos, James S

    2015-02-01

    Our group has previously reported that the majority of human melanomas (>60%) express the metabotropic glutamate receptor 1 (GRM1) and that the glutamate release inhibitor riluzole, a drug currently used to treat amyotrophic lateral sclerosis, can induce apoptosis in GRM1-expressing melanoma cells. Our group previously reported that in vitro riluzole treatment reduces cell growth in three-dimensional (3D) soft agar colony assays by 80% in cells with wildtype phosphoinositide 3-kinase (PI3K) pathway activation. However, melanoma cell lines harboring constitutive activating mutations of the PI3K pathway (PTEN and NRAS mutations) showed only a 35% to 40% decrease in colony formation in soft agar in the presence of riluzole. In this study, we have continued our preclinical studies of riluzole and its effect on melanoma cells alone and in combination with inhibitors of the PI3 kinase pathway: the AKT inhibitor, API-2, and the mammalian target of rapamycin (mTOR) inhibitor, rapamycin. We modeled these combinatorial therapies on various melanoma cell lines in 3D and 2D systems and in vivo. Riluzole combined with mTOR inhibition is more effective at halting melanoma anchorage-independent growth and xenograft tumor progression than either agent alone. PI3K signaling changes associated with this combinatorial treatment shows that 3D (nanoculture) modeling of cell signaling more closely resembles in vivo signaling than monolayer models. Riluzole combined with mTOR inhibition is effective at halting tumor cell progression independent of BRAF mutational status. This makes this combinatorial therapy a potentially viable alternative for metastatic melanoma patients who are BRAF WT and are therefore ineligible for vemurafenib therapy. Copyright © 2014. Published by Elsevier Inc.

  16. Targeting Glutamatergic Signaling and the PI3 Kinase Pathway to Halt Melanoma Progression12

    PubMed Central

    Rosenberg, Stephen A.; Niglio, Scot A.; Salehomoum, Negar; Chan, Joseph L.-K.; Jeong, Byeong-Seon; Wen, Yu; Li, Jiadong; Fukui, Jami; Chen, Suzie; Shin, Seung-Shick; Goydos, James S.

    2015-01-01

    Our group has previously reported that the majority of human melanomas (> 60%) express the metabotropic glutamate receptor 1 (GRM1) and that the glutamate release inhibitor riluzole, a drug currently used to treat amyotrophic lateral sclerosis, can induce apoptosis in GRM1-expressing melanoma cells. Our group previously reported that in vitro riluzole treatment reduces cell growth in three-dimensional (3D) soft agar colony assays by 80% in cells with wildtype phosphoinositide 3-kinase (PI3K) pathway activation. However, melanoma cell lines harboring constitutive activating mutations of the PI3K pathway (PTEN and NRAS mutations) showed only a 35% to 40% decrease in colony formation in soft agar in the presence of riluzole. In this study, we have continued our preclinical studies of riluzole and its effect on melanoma cells alone and in combination with inhibitors of the PI3 kinase pathway: the AKT inhibitor, API-2, and the mammalian target of rapamycin (mTOR) inhibitor, rapamycin. We modeled these combinatorial therapies on various melanoma cell lines in 3D and 2D systems and in vivo. Riluzole combined with mTOR inhibition is more effective at halting melanoma anchorage-independent growth and xenograft tumor progression than either agent alone. PI3K signaling changes associated with this combinatorial treatment shows that 3D (nanoculture) modeling of cell signaling more closely resembles in vivo signaling than monolayer models. Riluzole combined with mTOR inhibition is effective at halting tumor cell progression independent of BRAF mutational status. This makes this combinatorial therapy a potentially viable alternative for metastatic melanoma patients who are BRAF WT and are therefore ineligible for vemurafenib therapy. PMID:25749171

  17. 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

  18. Rotation is the primary motion of paired human epidermal keratinocytes.

    PubMed

    Tate, Sota; Imai, Matome; Matsushita, Natsuki; Nishimura, Emi K; Higashiyama, Shigeki; Nanba, Daisuke

    2015-09-01

    Collective motion of keratinocytes is involved in morphogenesis, homeostasis, and wound healing of the epidermis. Yet how the collective motion of keratinocytes emerges from the behavior of individual cells is still largely unknown. The aim of this study was to find the cellular behavior that links single and collective motion of keratinocytes. We investigated the behavior of two-cell colonies of HaCaT keratinocytes by a combination of time-lapse imaging and image processing. The two-cell colonies of HaCaT cells were formed as a contacted pair of keratinocyte clones. Image analysis and cell culture experiments revealed that the rotational speed of two-cell colonies was positively associated with their proliferative capacity. α6 integrin was required for the rotational motion of two-cell keratinocyte colonies. We also confirmed that two-cell colonies of keratinocytes predominantly exhibited the rotational, but not translational, motion, two modes of motion in a contact pair of rotating objects. The rotational motion is the primary motion of two-cell keratinocyte colonies and its speed is positively associated with their proliferative capacity. This study suggests that the assembly of rotating keratinocytes generates the collective motion of proliferative keratinocytes during morphogenesis and wound healing of the epidermis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  19. Keratinocyte growth factor protected cultured human keratinocytes exposed to oxidative stress.

    PubMed

    Gragnani, Alfredo; Rossi, Marina Bertelli; Albuquerque, Janne Cely Medeiros; Brito, Gabriela Soares Silva; Aloise, Antonio Carlos; Ferreira, Lydia Masako

    2010-02-01

    To evaluate effects of oxidative stress and supplementation of keratinocyte growth factor (KGF) on cultivated human keratinocytes. Oxidative stress was produced through addition of hydrogen peroxide (H(2)O(2)) to the culture medium. Cultivated human keratinocytes were divided in 4 groups: Group control (G C), Group KGF (G KGF), Group H(2)O(2) (G H(2)O(2)), Group H(2)O(2) and KGF (G H(2)O(2)-KGF). Each experiment was accomplished with the same lineage cultivated keratinocytes, in triplicate. Cell viability was evaluated by trypan blue exclusion assay. The results showed that the culture medium supplemented with KGF presented a small rate of cell viability when compared to cells only in culture medium (p<0,001). It demonstrated that only the growth factor does not have protector effects for cells in vitro. However, in front of the oxidative stress produced by addition of hydrogen peroxide to the medium, KGF showed a beneficial effect, protecting cells when compared to the group that suffered hydrogen peroxide action but had not been exposed to KGF (p<0,001). KGF determined protection to the primary human keratinocytes exposed to oxidative stress.

  20. Death penalty for keratinocytes: apoptosis versus cornification.

    PubMed

    Lippens, S; Denecker, G; Ovaere, P; Vandenabeele, P; Declercq, W

    2005-11-01

    Homeostasis implies a balance between cell growth and cell death. This balance is essential for the development and maintenance of multicellular organisms. Homeostasis is controlled by several mechanisms including apoptosis, a process by which cells condemned to death are completely eliminated. However, in some cases, total destruction and removal of dead cells is not desirable, as when they fulfil a specific function such as formation of the skin barrier provided by corneocytes, also known as terminally differentiated keratinocytes. In this case, programmed cell death results in accumulation of functional cell corpses. Previously, this process has been associated with apoptotic cell death. In this overview, we discuss differences and similarities in the molecular regulation of epidermal programmed cell death and apoptosis. We conclude that despite earlier confusion, apoptosis and cornification occur through distinct molecular pathways, and that possibly antiapoptotic mechanisms are implicated in the terminal differentiation of keratinocytes.

  1. Automated identification of epidermal keratinocytes in reflectance confocal microscopy

    NASA Astrophysics Data System (ADS)

    Gareau, Dan

    2011-03-01

    Keratinocytes in skin epidermis, which have bright cytoplasmic contrast and dark nuclear contrast in reflectance confocal microscopy (RCM), were modeled with a simple error function reflectance profile: erf( ). Forty-two example keratinocytes were identified as a training set which characterized the nuclear size a = 8.6+/-2.8 μm and reflectance gradient b = 3.6+/-2.1 μm at the nuclear/cytoplasmic boundary. These mean a and b parameters were used to create a rotationally symmetric erf( ) mask that approximated the mean keratinocyte image. A computer vision algorithm used an erf( ) mask to scan RCM images, identifying the coordinates of keratinocytes. Applying the mask to the confocal data identified the positions of keratinocytes in the epidermis. This simple model may be used to noninvasively evaluate keratinocyte populations as a quantitative morphometric diagnostic in skin cancer detection and evaluation of dermatological cosmetics.

  2. Differentiation of cultured keratinocytes promotes the adherence of Streptococcus pyogenes.

    PubMed Central

    Darmstadt, G L; Fleckman, P; Jonas, M; Chi, E; Rubens, C E

    1998-01-01

    Based on a consideration of the histopathology of nonbullous impetigo that shows localization of Streptococcus pyogenes to highly differentiated, subcorneal keratinocytes, we hypothesized that adherence of an impetigo strain of S. pyogenes would be promoted by terminal differentiation of keratinocytes. An assay was developed in which S. pyogenes adhered via pilus-like projections from the cell wall to the surface of cultured human keratinocytes in a time- and inoculum-dependent manner suggestive of a receptor-mediated process. Terminal differentiation of keratinocytes was induced by increasing the calcium concentration in the growth medium, and was confirmed by morphologic analysis using electron microscopy. Adherence of S. pyogenes was three and fourfold greater to keratinocytes differentiated in 1.0 and 1.5 mM calcium, respectively, compared with undifferentiated keratinocytes in 0.15 mM calcium. The presence of calcium during the adherence assay further enhanced adherence nearly twofold. Adherence occurred preferentially to sites of contact between adjacent keratinocytes, suggesting that the keratinocyte receptor may be a molecule involved in cell-to-cell adhesion. In contrast, nonpathogenic Streptococcus gordonii adhered poorly to keratinocytes regardless of their state of terminal differentiation, and adherence of a pharyngeal strain of S. pyogenes was twofold greater to undifferentiated than differentiated keratinocytes. This is the first report of in vitro adherence of S. pyogenes to keratinocytes in a manner that emulates human impetigo. Adherence of only the impetigo strain, and not the pharyngeal strain of S. pyogenes or the nonpathogenic S. gorgonii isolate, was promoted by keratinocyte differentiation. This result provides a model system for investigating the molecular pathogenesis of streptococcal skin infections. PMID:9421474

  3. Roles of transient receptor potential proteins (TRPs) in epidermal keratinocytes.

    PubMed

    Denda, Mitsuhiro; Tsutsumi, Moe

    2011-01-01

    Epidermal keratinocytes are the epithelial cells of mammalian skin. At the basal layer of the epidermis, these cells proliferate strongly, and as they move towards the skin surface, differentiation proceeds. At the uppermost layer of the epidermis, keratinocytes undergo apoptosis and die, forming a thin, water-impermeable layer called the stratum corneum. Peripheral blood vessels do not reach the epidermis, but peripheral nerve fibers do penetrate into it. Until recently, it was considered that the main role of epidermal keratinocytes was to construct and maintain the water-impermeable barrier function. However, since the functional existence of TRPV1, which is activated by heat and low pH, in epidermal keratinocytes was identified, our understanding of the role of keratinocytes has changed enormously. It has been found that many TRP channels are expressed in epidermal keratinocytes, and play important roles in differentiation, proliferation and barrier homeostasis. Moreover, because TRP channels expressed in keratinocytes have the ability to sense a variety of environmental factors, such as temperature, mechanical stress, osmotic stress and chemical stimuli, epidermal keratinocytes might form a key part of the sensory system of the skin. The present review deals with the potential roles of TRP channels expressed in epidermal keratinocytes and focuses on the concept of the epidermis as an active interface between the body and the environment.

  4. Methicillin-Resistant Staphylococcus aureus Adaptation to Human Keratinocytes

    PubMed Central

    Soong, Grace; Paulino, Franklin; Wachtel, Sarah; Parker, Dane; Wickersham, Matthew; Zhang, Dongni; Brown, Armand; Lauren, Christine; Dowd, Margaret; West, Emily; Horst, Basil; Planet, Paul

    2015-01-01

    ABSTRACT Skin is the most common site of Staphylococcus aureus infection. While most of these infections are self-limited, recurrent infections are common. Keratinocytes and recruited immune cells participate in skin defense against infection. We postulated that S. aureus is able to adapt to the milieu within human keratinocytes to avoid keratinocyte-mediated clearance. From a collection of S. aureus isolated from chronically infected patients with atopic dermatitis, we noted 22% had an agr mutant-like phenotype. Using several models of human skin infection, we demonstrate that toxin-deficient, agr mutants of methicillin-resistant S. aureus (MRSA) USA300 are able to persist within keratinocytes by stimulating autophagy and evading caspase-1 and inflammasome activation. MRSA infection induced keratinocyte autophagy, as evidenced by galectin-8 and LC3 accumulation. Autophagy promoted the degradation of inflammasome components and facilitated staphylococcal survival. The recovery of more than 58% agr or RNAIII mutants (P < 0.0001) of an inoculum of wild-type (WT) MRSA from within wortmannin-treated keratinocytes compared to control keratinocytes reflected the survival advantage for mutants no longer expressing agr-dependent toxins. Our results illustrate the dynamic interplay between S. aureus and keratinocytes that can result in the selection of mutants that have adapted specifically to evade keratinocyte-mediated clearance mechanisms. PMID:25900653

  5. Enhancement of Keratinocyte Differentiation by Rose Absolute Oil

    PubMed Central

    Kim, Jin-Hwa; Choi, Dae-Kyoung; Lee, Sang-Sin; Choi, Sun Ja; Kim, Chang Deok; Yoon, Tae-Jin

    2010-01-01

    Background Through differentiation processes, keratinocytes provide a physical barrier to our bodies and control skin features such as moisturization, wrinkles and pigmentation. Keratinocyte differentiation is disturbed in several skin diseases such as psoriasis and atopic dermatitis. Objective The aim of this study is to evaluate the keratinocyte differentiation-enhancing effect of rose absolute oil (RAO). Methods Primary cultured human normal keratinocytes were treated with RAO, and differentiation then checked by the expression of marker genes. Results RAO did not induce cytotoxicity on cultured keratinocytes at a dose of 10µM. The level of involucrin, an early marker for keratinocyte differentiation, was significantly increased by RAO. Concomitantly, RAO increased involucrin promoter activity, indicating that RAO increased involucrin gene expression at the mRNA level. Furthermore, RAO increased the level of filaggrin in cultured keratinocytes, and in the granular layer of mouse skin. In line with these results, RAO decreased the proliferation of keratinocytes cultured in vitro. When RAO was applied topically on the tape-stripped mouse skins, it accelerated the recovery of disturbed barrier function. Conclusion These results suggest that RAO may be applicable for the control of skin texture and keratinocyte differentiation-related skin diseases. PMID:20711260

  6. Keratinocyte growth factor and the expression of wound-healing-related genes in primary human keratinocytes from burn patients.

    PubMed

    Chomiski, Verônica; Gragnani, Alfredo; Bonucci, Jéssica; Correa, Silvana Aparecida Alves; Noronha, Samuel Marcos Ribeiro de; Ferreira, Lydia Masako

    2016-08-01

    To evaluate the effect of keratinocyte growth factor (KGF) treatment on the expression of wound-healing-related genes in cultured keratinocytes from burn patients. Keratinocytes were cultured and divided into 4 groups (n=4 in each group): TKB (KGF-treated keratinocytes from burn patients), UKB (untreated keratinocytes from burn patients), TKC (KGF-treated keratinocytes from controls), and UKC (untreated keratinocytes from controls). Gene expression analysis using quantitative polymerase chain reaction (qPCR) array was performed to compare (1) TKC versus UKC, (2) UKB versus UKC, (3) TKB versus UKC, (4) TKB versus UKB, (5) TKB versus TKC, and (6) UKB versus TKC. Comparison 1 showed one down-regulated and one up-regulated gene; comparisons 2 and 3 resulted in the same five down-regulated genes; comparison 4 had no significant difference in relative gene expression; comparison 5 showed 26 down-regulated and 7 up-regulated genes; and comparison 6 showed 25 down-regulated and 11 up-regulated genes. There was no differential expression of wound-healing-related genes in cultured primary keratinocytes from burn patients treated with keratinocyte growth factor.

  7. Hyaluronic acid inhibits the adherence and growth of monolayer keratinocytes but does not affect the growth of keratinocyte epithelium.

    PubMed

    Harvima, Ilkka T; Heikura, Hanna; Hyttinen, Mika; Naukkarinen, Anita

    2006-10-01

    Hyaluronic acid (HA) is involved in epidermal biology but evidence for its functional significance is sparse. In this study, low-calcium monolayer and high-calcium epithelium cultures of human keratinocytes were used to study the effect of up to four different HA preparations on keratinocyte growth and on the adherence of proliferating keratinocytes onto the plastic surface coated with different matrix proteins. In suboptimally growing monolayer culture, up to 1,000 microg/ml rooster comb HA and streptococcus equi HA inhibited keratinocyte growth. Instead, all HA preparations tested did not affect the growth and migration of keratinocyte epithelium using optimal or suboptimal growth conditions. In the cell adherence assays, up to 1,000 microg/ml rooster comb HA and streptococcus equi HA inhibited the keratinocyte adherence onto the fibronectin- and collagen-coated substratum. In contrast to other HA preparations, HA from human umbilical cord did not affect the growth of monolayer keratinocytes and it increased markedly the cell adherence onto the collagen-coated substratum. This increase, however, can be attributed to chonroitin sulphate proteoglycan contaminant present in this HA preparation. In conclusion, HA can inhibit the growth and adherence of proliferating monolayer keratinocytes, but it has no apparent effect on the growth and migration of keratinocyte epithelium.

  8. 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.

  9. 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.

  10. 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.

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

    PubMed

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

    2010-01-01

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

  12. RAS and RHO Families of GTPases Directly Regulate Distinct Phosphoinositide 3-Kinase Isoforms

    PubMed Central

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

    2013-01-01

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

  13. The role of phosphoinositide 3-kinase in adhesion of oral epithelial cells to titanium.

    PubMed

    Atsuta, Ikiru; Ayukawa, Yasunori; Yamaza, Takayoshi; Furuhashi, Akihiro; Koyano, Kiyoshi

    2013-11-01

    Oral epithelial cells (OECs) adhesion to titanium may improve the success rate of implant restoration. We investigated the mechanism by which OECs adhere to titanium dental implants. (1) After culturing rat OECs on titanium plates (Ti) or culture dishes in the presence or absence of a phosphoinositide 3-kinase (PI3K) activator or inhibitors and/or growth factors, and OEC morphology under these conditions were analyzed. (2) Right maxillary first molars were extracted and replaced with experimental implants. The rats were treated with or without growth factors. (1) Cell adherence was lower of OECs on Ti than in those on culture dishes, as were the levels of integrin β4 and the continuity of F-actin structures. After PI3K inhibition, markedly reducing adherence to both substrates. In contrast, PI3K activation with activator or insulin-like growth factor restored the OEC adherence and the expression of adhesion molecules on Ti to the levels seen in OECs cultured on dishes. Cell migration was inhibited by PI3K activation. (2) High expression of integrin β4 was observed in the peri-implant epithelia of PI3K-activated rats. These findings suggest that PI3K plays an important role in the adhesion of OECs to Ti. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Phosphatidylinositol 3-Kinase γ Is Required for the Development of Experimental Cerebral Malaria

    PubMed Central

    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. PMID:25775137

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

    SciTech Connect

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

    2009-09-18

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

  16. 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

  17. 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.

  18. Sulforaphane prevents human platelet aggregation through inhibiting the phosphatidylinositol 3-kinase/Akt pathway.

    PubMed

    Chuang, Wen-Ying; Kung, Po-Hsiung; Kuo, Chih-Yun; Wu, Chin-Chung

    2013-06-01

    Sulforaphane, a dietary isothiocyanate found in cruciferous vegetables, has been shown to exert beneficial effects in animal models of cardiovascular diseases. However, its effect on platelet aggregation, which is a critical factor in arterial thrombosis, is still unclear. In the present study, we show that sulforaphane inhibited human platelet aggregation caused by different receptor agonists, including collagen, U46619 (a thromboxane A2 mimic), protease-activated receptor 1 agonist peptide (PAR1-AP), and an ADP P2Y12 receptor agonist. Moreover, sulforaphane significantly reduced thrombus formation on a collagen-coated surface under whole blood flow conditions. In exploring the underlying mechanism, we found that sulforaphane specifically prevented phosphatidylinositol 3-kinase (PI3K)/Akt signalling, without markedly affecting other signlaling pathways involved in platelet aggregation, such as protein kinase C activation, calcium mobilisation, and protein tyrosine phosphorylation. Although sulforaphane did not directly inhibit the catalytic activity of PI3K, it caused ubiquitination of the regulatory p85 subunit of PI3K, and prevented PI3K translocation to membranes. In addition, sulforaphane caused ubiquitination and degradation of phosphoinositide-dependent kinase 1 (PDK1), which is required for Akt activation. Therefore, sulforaphane is able to inhibit the PI3K/Akt pathway at two distinct sites. In conclusion, we have demonstrated that sulforaphane prevented platelet aggregation and reduced thrombus formation in flow conditions; our data also support that the inhibition of the PI3K/Akt pathway by sulforaphane contributes it antiplatelet effects.

  19. 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.

  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. 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.

  2. Par3 integrates Tiam1 and phosphatidylinositol 3-kinase signaling to change apical membrane identity.

    PubMed

    Ruch, Travis R; Bryant, David M; Mostov, Keith E; Engel, Joanne N

    2017-01-15

    Pathogens can alter epithelial polarity by recruiting polarity proteins to the apical membrane, but how a change in protein localization is linked to polarity disruption is not clear. In this study, we used chemically induced dimerization to rapidly relocalize proteins from the cytosol to the apical surface. We demonstrate that forced apical localization of Par3, which is normally restricted to tight junctions, is sufficient to alter apical membrane identity through its interactions with phosphatidylinositol 3-kinase (PI3K) and the Rac1 guanine nucleotide exchange factor Tiam1. We further show that PI3K activity is required upstream of Rac1, and that simultaneously targeting PI3K and Tiam1 to the apical membrane has a synergistic effect on membrane remodeling. Thus, Par3 coordinates the action of PI3K and Tiam1 to define membrane identity, revealing a signaling mechanism that can be exploited by human mucosal pathogens. © 2017 Ruch et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  3. 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.

  4. Ablation of Phosphoinositide 3-Kinase-γ Reduces the Severity of Acute Pancreatitis

    PubMed Central

    Lupia, Enrico; Goffi, Alberto; De Giuli, Paolo; Azzolino, Ornella; Bosco, Ornella; Patrucco, Enrico; Vivaldo, Maria Cristina; Ricca, Marco; Wymann, Matthias P.; Hirsch, Emilio; Montrucchio, Giuseppe; Emanuelli, Giorgio

    2004-01-01

    In pancreatic acini, the G-protein-activated phosphoinositide 3-kinase-γ (PI3Kγ) regulates several key pathological responses to cholecystokinin hyperstimulation in vitro. Thus, using mice lacking PI3Kγ, we studied the function of this enzyme in vivo in two different models of acute pancreatitis. The disease was induced by supramaximal concentrations of cerulein and by feeding mice a choline-deficient/ethionine-supplemented diet. Although the secretive function of isolated pancreatic acini was identical in mutant and control samples, in both models, genetic ablation of PI3Kγ significantly reduced the extent of acinar cell injury/necrosis. In agreement with a protective role of apoptosis in pancreatitis, PI3Kγ-deficient pancreata showed an increased number of apoptotic acinar cells, as determined by terminal dUTP nick-end labeling and caspase-3 activity. In addition, neutrophil infiltration within the pancreatic tissue was also reduced, suggesting a dual action of PI3Kγ, both in the triggering events within acinar cells and in the subsequent neutrophil recruitment and activation. Finally, the lethality of the choline-deficient/ethionine-supplemented diet-induced pancreatitis was significantly reduced in mice lacking PI3Kγ. Our results thus suggest that inhibition of PI3Kγ may be of therapeutic value in acute pancreatitis. PMID:15579443

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

    PubMed Central

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

    2011-01-01

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

  6. High fat diet induced obesity alters ovarian phosphatidylinositol-3 kinase signaling gene expression.

    PubMed

    Nteeba, J; Ross, J W; Perfield, J W; Keating, A F

    2013-12-01

    Insulin regulates ovarian phosphatidylinositol-3-kinase (PI3 K) signaling, important for primordial follicle viability and growth activation. This study investigated diet-induced obesity impacts on: (1) insulin receptor (Insr) and insulin receptor substrate 1 (Irs1); (2) PI3K components (Kit ligand (Kitlg), kit (c-Kit), protein kinase B alpha (Akt1) and forkhead transcription factor subfamily 3 (Foxo3a)); (3) xenobiotic biotransformation (microsomal epoxide hydrolase (Ephx1), Cytochrome P450 isoform 2E1 (Cyp2e1), Glutathione S-transferase (Gst) isoforms mu (Gstm) and pi (Gstp)) and (4) microRNA's 184, 205, 103 and 21 gene expression. INSR, GSTM and GSTP protein levels were also measured. Obese mouse ovaries had decreased Irs1, Foxo3a, Cyp2e1, MiR-103, and MiR-21 but increased Kitlg, Akt1, and miR-184 levels relative to lean littermates. These results support that diet-induced obesity potentially impairs ovarian function through aberrant gene expression. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. High fat diet induced obesity alters ovarian phosphatidylinositol-3 kinase signaling gene expression

    PubMed Central

    Nteeba, J.; Ross, J.W.; Perfield, J.W.; Keating, A.F.

    2013-01-01

    Insulin regulates ovarian phosphatidylinositol-3-kinase (PI3K) signaling, important for primordial follicle viability and growth activation. This study investigated diet-induced obesity impacts on: 1) insulin receptor (Insr) and insulin receptor substrate 1 (Irs1); 2) PI3K components (Kit ligand (Kitlg), kit (c-Kit), protein kinase B alpha (Akt1) and forkhead transcription factor subfamily 3 (Foxo3a)); 3) xenobiotic biotransformation (microsomal epoxide hydrolase (Ephx1), Cytochrome P450 isoform 2E1 (Cyp2e1), Glutathione S-transferase (Gst) isoforms mu (Gstm) and pi (Gstp)) and 4) microRNA’s 184, 205, 103 and 21 gene expression. INSR, GSTM and GSTP protein levels were also measured. Obese mouse ovaries had decreased Irs1, Foxo3a, Cyp2e1, MiR-103, and MiR-21 but increased Kitlg, Akt1, and miR-184 levels relative to lean littermates. These results support that diet-induced obesity potentially impairs ovarian function through aberrant gene expression. PMID:23954404

  8. 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

  9. 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

  10. 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.

  11. Eupafolin suppresses prostate cancer by targeting phosphatidylinositol 3-kinase-mediated Akt signaling.

    PubMed

    Liu, Kangdong; Park, Chanmi; Chen, Hanyong; Hwang, Joonsung; Thimmegowda, N R; Bae, Eun Young; Lee, Ki Won; Kim, Hong-Gyum; Liu, Haidan; Soung, Nak Kyun; Peng, Cong; Jang, Jae Hyuk; Kim, Kyoon Eon; Ahn, Jong Seog; Bode, Ann M; Dong, Ziming; Kim, Bo Yeon; Dong, Zigang

    2015-09-01

    Phosphatase and tensin homolog (PTEN) loss or mutation consistently activates the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling pathway, which contributes to the progression and invasiveness of prostate cancer. Furthermore, the PTEN/PI3-K/Akt and Ras/MAPK pathways cooperate to promote the epithelial-mesenchymal transition (EMT) and metastasis initiated from prostate stem/progenitor cells. For these reasons, the PTEN/PI3-K/Akt pathway is considered as an attractive target for both chemoprevention and chemotherapy. Herein we report that eupafolin, a natural compound found in common sage, inhibited proliferation of prostate cancer cells. Protein content analysis indicated that phosphorylation of Akt and its downstream kinases was inhibited by eupafolin treatment. Pull-down assay and in vitro kinase assay results indicated that eupafolin could bind with PI3-K and attenuate its kinase activity. Eupafolin also exhibited tumor suppressive effects in vivo in an athymic nude mouse model. Overall, these results suggested that eupafolin exerts antitumor effects by targeting PI3-K. © 2014 Wiley Periodicals, Inc.

  12. Eupafolin suppresses prostate cancer by targeting phosphatidylinositol 3-kinase-mediated Akt signaling

    PubMed Central

    Liu, Kangdong; Park, Chanmi; Chen, Hanyong; Hwang, Joonsung; Thimmegowda, N.R.; Bae, Eun Young; Lee, Ki Won; Kim, Hong-Gyum; Liu, Haidan; Soung, Nak Kyun; Peng, Cong; Jang, Jae Hyuk; Kim, Kyoon Eon; Ahn, Jong Seog; Bode, Ann M.; Dong, Ziming; Kim, Bo Yeon; Dong, Zigang

    2014-01-01

    Phosphatase and tensin homolog (PTEN) loss or mutation consistently activates the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling pathway, which contributes to the progression and invasiveness of prostate cancer. Furthermore, the PTEN/PI3-K/Akt and Ras/MAPK pathways cooperate to promote the epithelial-mesenchymal transition (EMT) and metastasis initiated from prostate stem/progenitor cells. For these reasons, the PTEN/PI3-K/Akt pathway is considered as an attractive target for both chemoprevention and chemotherapy. Herein we report that eupafolin, a natural compound found in common sage, inhibited proliferation of prostate cancer cells. Protein content analysis indicated that phosphorylation of Akt and its downstream kinases was inhibited by eupafolin treatment. Pull-down assay and in vitro kinase assay results indicated that eupafolin could bind with PI3-K and attenuate its kinase activity. Eupafolin also exhibited tumor suppressive effects in vivo in an athymic nude mouse model. Overall, these results suggested that eupafolin exerts antitumor effects by targeting PI3-K. PMID:24700667

  13. 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

  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. Class I and class III phosphoinositide 3-kinases are required for actin polymerization that propels phagosomes

    PubMed Central

    Bohdanowicz, Michal; Cosío, Gabriela; Backer, Jonathan M.

    2010-01-01

    Actin polymerization drives the extension of pseudopods that trap and engulf phagocytic targets. The polymerized actin subsequently dissociates as the phagocytic vacuole seals and detaches from the plasma membrane. We found that phagosomes formed by engagement of integrins that serve as complement receptors (CR3) undergo secondary waves of actin polymerization, leading to the formation of “comet tails” that propel the vacuoles inside the cells. Actin tail formation was accompanied by and required de novo formation of PI(3,4)P2 and PI(3,4,5)P3 on the phagosomal membrane by class I phosphoinositide 3-kinases (PI3Ks). Although the phosphatidylinositide phosphatase Inpp5B was recruited to nascent phagosomes, it rapidly detached from the membrane after phagosomes sealed. Detachment of Inpp5B required the formation of PI(3)P. Thus, class III PI3K activity was also required for the accumulation of PI(4,5)P2 and PI(3,4,5)P3 and for actin tail formation. These experiments reveal a new PI(3)P-sensitive pathway leading to PI(3,4)P2 and PI(3,4,5)P3 formation and signaling in endomembranes. PMID:21115805

  16. 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.

  17. 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

  18. A chemical proteomics approach to phosphatidylinositol 3-kinase signaling in macrophages.

    PubMed

    Pasquali, Christian; Bertschy-Meier, Dominique; Chabert, Christian; Curchod, Marie-Laure; Arod, Christian; Booth, Randy; Mechtler, Karl; Vilbois, Francis; Xenarios, Ioannis; Ferguson, Colin G; Prestwich, Glenn D; Camps, Montserrat; Rommel, Christian

    2007-11-01

    Prior work using lipid-based affinity matrices has been done to investigate distinct sets of lipid-binding proteins, and one series of experiments has proven successful in mammalian cells for the proteome-wide identification of lipid-binding proteins. However, most lipid-based proteomics screens require scaled up sample preparation, are often composed of multiple cell types, and are not adapted for simultaneous signal transduction studies. Herein we provide a chemical proteomics strategy that uses cleavable lipid "baits" with broad applicability to diverse biological samples. The novel baits were designed to avoid preparative steps to allow functional proteomics studies when the biological source is a limiting factor. Validation of the chemical baits was first confirmed by the selective isolation of several known endogenous phosphatidylinositol 3-kinase signaling proteins using primary bone marrow-derived macrophages. The use of this technique for cellular proteomics and MS/MS analysis was then demonstrated by the identification of known and potential novel lipid-binding proteins that was confirmed in vitro for several proteins by direct lipid-protein interactions. Further to the identification, the method is also compatible with subsequent signal transduction studies, notably for protein kinase profiling of the isolated lipid-bound protein complexes. Taken together, this integration of minimal scale proteomics, lipid chemistry, and activity-based readouts provides a significant advancement in the ability to identify and study the lipid proteome of single, relevant cell types.

  19. 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

  20. Design and purification of active truncated phosphoinositide 3-kinase gamma protein constructs for structural studies.

    PubMed

    Vujičić Žagar, A; Scapozza, L; Vadas, O

    2017-07-01

    Phosphoinositide 3-kinase gamma (PI3Kγ) is a lipid kinase that plays a crucial role in cell migration, chemotaxis, oxidative burst and myocardial contractility. It is activated downstream of G protein-coupled receptors (GPCRs) and small GTPases of Ras superfamily. PI3Kγ is a heterodimer composed of a catalytic and a regulatory subunit that is expressed mostly in hematopoietic cells and in the heart. Although it has attracted a lot of attention because of its link with tumor inflammation and heart diseases, its regulation is still not fully understood. This can be attributed to the absence of high-resolution structural details of the PI3Kγ heterodimer. Here we describe the design and purification of PI3Kγ constructs where flexible loops in the regulatory subunit have been removed based on structural information obtained by hydrogen/deuterium exchange - mass spectrometry (HDX-MS). The soluble constructs retain both basal activity and sensitivity to GPCR stimulation, and are thus an optimal tool to further explore their regulation using a structure-based approach. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. 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

  2. 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

  3. 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

  4. 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

  5. AMPK signaling in neuronal polarization: Putting the brakes on axonal traffic of PI3-Kinase.

    PubMed

    Amato, Stephen; Man, Heng-Ye

    2012-03-01

    Neuronal polarization, the process by which neurons form multiple dendrites and an axon from the soma, is the first critical step in the formation and function of neural networks. Polarization begins with the rapid extension of a single neurite to produce an axon of impressive size and complex geometry, while the remaining sister neurites differentiate into dendrites. The extensive biosynthesis required to produce an axon therefore necessitates coordination with cellular energy status to ensure an ample energy supply. Our recent work shows that activity of the AMP-activated protein kinase (AMPK), the bio-energy sensor responsible for maintaining cellular energy homeostasis in all eukaryotic cells, plays an important role in the initiation of axonal growth. AMPK phosphorylates the cargo-binding light chain of the Kif5 motor protein, leading to dissociation of the phosphatidylinositol 3-Kinase (PI3K) from the motor complex. The mislocation of PI3K, which is normally enriched at the axonal tip for extension and differentiation, results in a lack of neurite specification and neuron polarization. These findings reveal a link between cellular bioenergy homeostasis and neuron morphogenesis, and suggest a novel cellular mechanism underlying the long-term neurological abnormalities as a consequence of bioenergy deficiency during early brain development.

  6. 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.

  7. 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.

  8. Inhibition of Human Class I vs Class III Phosphatidylinositol 3'-Kinases.

    PubMed

    Hassett, Matthew R; Sternberg, Anna R; Roepe, Paul D

    2017-08-22

    Most investigations of phosphatidylinositol 3'-kinase (PI3K) drug inhibition have been via assays based on ADP appearance or ATP consumption (e.g., Liu, Q., et al. ( 2011 ) J. Med. Chem. 54 , 1473 - 1480 ). However, at least some PI3K isoforms show basal ATPase activity in the absence of PI lipid substrate(s), which may complicate quantification of drug potency, isoform specificity of some drugs, and synergy for drug combinations. In this study, we probe the class I vs class III isoform specificity of a selected set of PI3K inhibitors using a simple, inexpensive, semi high-throughput assay that quantifies production of phosphatidylinositol 3'-phosphate (PI3P) from phosphatidylinositol. Results are compared to previous data largely generated using ATPase activity assays. Good agreement between EC50 values computed via ATPase assays vs the reported PI3P formation assay is found for most drugs, but with a few exceptions. Furthermore, for the first time, drug inhibition of class I vs class III enzymes is compared side-by-side with the same assay for the important class I-specific inhibitors GSK2126458 ("Omipalisib") and NVP-BGT226 ("BGT226") currently in clinical development for advanced solid tumors.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. Intracellular Movement of Green Fluorescent Protein–Tagged Phosphatidylinositol 3-Kinase in Response to Growth Factor Receptor Signaling

    PubMed Central

    Gillham, Helen; Golding, Matthew C.H.M.; Pepperkok, Rainer; Gullick, William J.

    1999-01-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase which has been implicated in mitogenesis, protein trafficking, inhibition of apoptosis, and integrin and actin functions. Here we show using a green fluorescent protein–tagged p85 subunit that phosphatidylinositol 3-kinase is distributed throughout the cytoplasm and is localized to focal adhesion complexes in resting NIH-3T3, A431, and MCF-7 cells. Ligand stimulation of an epidermal growth factor receptor/c-erbB-3 chimera expressed in these cells results in a redistribution of p85 to the cell membrane which is independent of the catalytic activity of the enzyme and the integrity of the actin cytoskeleton. The movement is, however, dependent on the phosphorylation status of the erbB-3 chimera. Using rhodamine-labeled epidermal growth factor we show that the phosphatidylinositol 3-kinase and the receptors colocalize in discrete patches on the cell surface. Low concentrations of ligand cause patching only at the periphery of the cells, whereas at high concentrations patches were seen over the whole cell surface. Using green fluorescent protein–tagged fragments of p85 we show that binding to the receptor requires the NH2-terminal part of the protein as well as its SH2 domains. PMID:10459020

  17. SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

    PubMed Central

    McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A

    1992-01-01

    The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. Hyaluronan-Phosphatidylethanolamine Polymers Form Pericellular Coats on Keratinocytes and Promote Basal Keratinocyte Proliferation

    PubMed Central

    Symonette, Caitlin J.; Tan, Xiao Cherie; Tolg, Cornelia; Ma, Jenny; Perera, Francisco; Turley, Eva A.

    2014-01-01

    Aged keratinocytes have diminished proliferative capacity and hyaluronan (HA) cell coats, which are losses that contribute to atrophic skin characterized by reduced barrier and repair functions. We formulated HA-phospholipid (phosphatidylethanolamine, HA-PE) polymers that form pericellular coats around cultured dermal fibroblasts independently of CD44 or RHAMM display. We investigated the ability of these HA-PE polymers to penetrate into aged mouse skin and restore epidermal function in vivo. Topically applied Alexa647-HA-PE penetrated into the epidermis and dermis, where it associated with both keratinocytes and fibroblasts. In contrast, Alexa647-HA was largely retained in the outer cornified layer of the epidermis and quantification of fluorescence confirmed that significantly more Alexa647-HA-PE penetrated into and was retained within the epidermis than Alexa647-HA. Multiple topical applications of HA-PE to shaved mouse skin significantly stimulated basal keratinocyte proliferation and epidermal thickness compared to HA or vehicle cream alone. HA-PE had no detectable effect on keratinocyte differentiation and did not promote local or systemic inflammation. These effects of HA-PE polymers are similar to those reported for endogenous epidermal HA in youthful skin and show that topical application of HA-PE polymers can restore some of the impaired functions of aged epidermis. PMID:25276814

  3. Hyaluronan-phosphatidylethanolamine polymers form pericellular coats on keratinocytes and promote basal keratinocyte proliferation.

    PubMed

    Symonette, Caitlin J; Kaur Mann, Aman; Tan, Xiao Cherie; Tolg, Cornelia; Ma, Jenny; Perera, Francisco; Yazdani, Arjang; Turley, Eva A

    2014-01-01

    Aged keratinocytes have diminished proliferative capacity and hyaluronan (HA) cell coats, which are losses that contribute to atrophic skin characterized by reduced barrier and repair functions. We formulated HA-phospholipid (phosphatidylethanolamine, HA-PE) polymers that form pericellular coats around cultured dermal fibroblasts independently of CD44 or RHAMM display. We investigated the ability of these HA-PE polymers to penetrate into aged mouse skin and restore epidermal function in vivo. Topically applied Alexa(647)-HA-PE penetrated into the epidermis and dermis, where it associated with both keratinocytes and fibroblasts. In contrast, Alexa(647)-HA was largely retained in the outer cornified layer of the epidermis and quantification of fluorescence confirmed that significantly more Alexa(647)-HA-PE penetrated into and was retained within the epidermis than Alexa(647)-HA. Multiple topical applications of HA-PE to shaved mouse skin significantly stimulated basal keratinocyte proliferation and epidermal thickness compared to HA or vehicle cream alone. HA-PE had no detectable effect on keratinocyte differentiation and did not promote local or systemic inflammation. These effects of HA-PE polymers are similar to those reported for endogenous epidermal HA in youthful skin and show that topical application of HA-PE polymers can restore some of the impaired functions of aged epidermis.

  4. 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.

  5. 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.

  6. UVB light upregulates prostaglandin synthases and prostaglandin receptors in mouse keratinocytes

    SciTech Connect

    Black, Adrienne T.; Gray, Joshua P.; Shakarjian, Michael P.; Mishin, Vladimir; Laskin, Debra L.; Heck, Diane E.; Laskin, Jeffrey D.

    2008-10-01

    Prostaglandins belong to a class of cyclic lipid-derived mediators synthesized from arachidonic acid via COX-1, COX-2 and various prostaglandin synthases. Members of this family include prostaglandins such as PGE{sub 2}, PGF{sub 2{alpha}}, PGD{sub 2} and PGI{sub 2} (prostacyclin) as well as thromboxane. In the present studies we analyzed the effects of UVB on prostaglandin production and prostaglandin synthase expression in primary cultures of undifferentiated and calcium-differentiated mouse keratinocytes. Both cell types were found to constitutively synthesize PGE{sub 2}, PGD{sub 2} and the PGD{sub 2} metabolite PGJ{sub 2}. Twenty-four hours after treatment with UVB (25 mJ/cm{sup 2}), production of PGE{sub 2} and PGJ{sub 2} increased, while PGD{sub 2} production decreased. This was associated with increased expression of COX-2 mRNA and protein. UVB (2.5-25 mJ/cm{sup 2}) also caused marked increases in mRNA expression for the prostanoid synthases PGDS, mPGES-1, mPGES-2, PGFS and PGIS, as well as expression of receptors for PGE{sub 2} (EP1 and EP2), PGD{sub 2} (DP and CRTH2) and prostacyclin (IP). UVB was more effective in inducing COX-2 and DP in differentiated cells and EP1 and IP in undifferentiated cells. UVB readily activated keratinocyte PI-3-kinase (PI3K)/Akt, JNK and p38 MAP signaling pathways which are known to regulate COX-2 expression. While inhibition of PI3K suppressed UVB-induced mPGES-1 and CRTH2 expression, JNK inhibition suppressed mPGES-1, PGIS, EP2 and CRTH2, and p38 kinase inhibition only suppressed EP1 and EP2. These data indicate that UVB modulates expression of prostaglandin synthases and receptors by distinct mechanisms. Moreover, both the capacity of keratinocytes to generate prostaglandins and their ability to respond to these lipid mediators are stimulated by exposure to UVB.

  7. A comparison of pathway-independent and pathway-dependent methods in the calculation of conformational free enthalpy differences.

    PubMed

    Lin, Zhixiong; van Gunsteren, Wilfred F

    2016-01-01

    The multistep umbrella sampling method, which belongs to pathway-dependent methods to calculate conformational free enthalpy differences, is used to calculate the free enthalpy difference between a right-handed 2.710/12 -helix and a left-handed 314 -helix of a hexa-β-peptide in methanol solution. The same conformational free enthalpy difference was previously investigated using pathway-independent methods such as direct counting and enveloping distribution sampling. Our results show that the pathway-dependent simulations are sensitive to the choice of the pathway and its parameter values. A pathway based on restraining distances of hydrogen-bonding atom pairs shows poor sampling for two different values of the restraining force constant. Another pathway based on restraining backbone dihedral angles did smoothly sample the transition between the two helical conformations, but only with a proper choice of the restraining force constant. The results illustrate that if, and only if, a proper pathway and proper parameters are chosen, the multistep umbrella sampling can be almost 50 times more efficient than the pathway-independent methods in this case. The analysis illustrates the advantages and pitfalls of the much used multistep umbrella sampling methodology. © 2015 The Protein Society.

  8. 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

  9. 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

  10. 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.

  11. Decorin gene expression and its regulation in human keratinocytes

    SciTech Connect

    Velez-DelValle, Cristina; Marsch-Moreno, Meytha; Castro-Munozledo, Federico; Kuri-Harcuch, Walid

    2011-07-22

    Highlights: {yields} We showed that cultured human diploid epidermal keratinocytes express and synthesize decorin. {yields} Decorin is found intracytoplasmic in suprabasal cells of cultures and in human epidermis. {yields} Decorin mRNA expression in cHEK is regulated by pro-inflammatory and proliferative cytokines. {yields} Decorin immunostaining of psoriatic lesions showed a lower intensity and altered intracytoplasmic arrangements. -- Abstract: In various cell types, including cancer cells, decorin is involved in regulation of cell attachment, migration and proliferation. In skin, decorin is seen in dermis, but not in keratinocytes. We show that decorin gene (DCN) is expressed in the cultured keratinocytes, and the protein is found in the cytoplasm of differentiating keratinocytes and in suprabasal layers of human epidermis. RT-PCR experiments showed that DCN expression is regulated by pro-inflammatory and proliferative cytokines. Our data suggest that decorin should play a significant role in keratinocyte terminal differentiation, cutaneous homeostasis and dermatological diseases.

  12. 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.

  13. 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.

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

    PubMed Central

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

    2014-01-01

    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. PMID:24280724

  15. 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.

  16. 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

  17. 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

  18. Phosphatidylinositol 3-kinase inhibition potentiates glucocorticoid response in B-cell acute lymphoblastic leukemia.

    PubMed

    Evangelisti, Cecilia; Cappellini, Alessandra; Oliveira, Mariana; Fragoso, Rita; Barata, João T; Bertaina, Alice; Locatelli, Franco; Simioni, Carolina; Neri, Luca M; Chiarini, Francesca; Lonetti, Annalisa; Buontempo, Francesca; Orsini, Ester; Pession, Andrea; Manzoli, Lucia; Martelli, Alberto Maria; Evangelisti, Camilla

    2017-08-04

    Despite remarkable progress in polychemotherapy protocols, pediatric B-cell acute lymphoblastic leukemia (B-ALL) remains fatal in around 20% of cases. Hence, novel targeted therapies are needed for patients with poor prognosis. Glucocorticoids (GCs) are drugs commonly administrated for B-ALL treatment. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin signaling pathway is frequently observed in B-ALL and contributes to GC-resistance. Here, we analyzed for the first time to our knowledge, the therapeutic potential of pan and isoform-selective PI3K p110 inhibitors, alone or combined with dexamethasone (DEX), in B-ALL leukemia cell lines and patient samples. We found that a pan PI3K p110 inhibitor displayed the most powerful cytotoxic effects in B-ALL cells, by inducing cell cycle arrest and apoptosis. Both a pan PI3K p110 inhibitor and a dual γ/δ PI3K p110 inhibitor sensitized B-ALL cells to DEX by restoring nuclear translocation of the GC receptor and counteracted stroma-induced DEX-resistance. Finally, gene expression analysis documented that, on one hand the combination consisting of a pan PI3K p110 inhibitor and DEX strengthened the DEX-induced up- or down-regulation of several genes involved in apoptosis, while on the other, it rescued the effects of genes that might be involved in GC-resistance. Overall, our findings strongly suggest that PI3K p110 inhibition could be a promising strategy for treating B-ALL patients by improving GC therapeutic effects and/or overcoming GC-resistance. © 2017 Wiley Periodicals, Inc.

  19. Phosphatidylinositol 3-kinase inhibition broadly sensitizes glioblastoma cells to death receptor- and drug-induced apoptosis.

    PubMed

    Opel, Daniela; Westhoff, Mike-Andrew; Bender, Ariane; Braun, Veit; Debatin, Klaus-Michael; Fulda, Simone

    2008-08-01

    The aberrant activity of the phosphatidylinositol 3-kinase (PI3K) pathway has been reported to correlate with adverse clinical outcome in human glioblastoma in vivo. However, the question of how this survival network can be successfully targeted to restore the sensitivity of glioblastoma to apoptosis induction has not yet been answered. Here, we report that inhibition of PI3K by LY294002 broadly sensitizes wild-type and mutant PTEN glioblastoma cells to both death receptor- and chemotherapy-induced apoptosis, whereas mammalian target of rapamycin (mTOR) inhibition is not sufficient to restore apoptosis sensitivity. LY294002 significantly enhances apoptosis triggered by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), agonistic anti-CD95 antibodies, or several anticancer drugs (i.e., doxorubicin, etoposide, and vincristine) in a highly synergistic manner. In addition, LY294002 cooperates with TRAIL or doxorubicin to suppress colony formation, thus also showing a strong effect on long-term survival. Similarly, genetic knockdown of PI3K subunits p110alpha and/or p110beta by RNA interference (RNAi) primes glioblastoma cells for TRAIL- or doxorubicin-mediated apoptosis. In contrast to PI3K inhibition, pharmacologic or genetic blockade of mTOR by RAD001 (everolimus), rapamycin, or RNAi fails to enhance TRAIL- or doxorubicin-induced apoptosis. Analysis of apoptosis pathways reveals that PI3K inhibition acts in concert with TRAIL or doxorubicin to trigger mitochondrial membrane permeabilization, caspase activation, and caspase-dependent apoptosis, which are abolished by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Most importantly, PI3K inhibition by LY294002 sensitizes primary cultured glioblastoma cells obtained from surgical specimens to TRAIL- or chemotherapy-induced cell death. By showing that PI3K inhibition broadly primes glioblastoma cells for apoptosis, our findings provide the rationale for using PI3K inhibitors in

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. Protein Kinase Activity of Phosphoinositide 3-Kinase Regulates Cytokine-Dependent Cell Survival

    PubMed Central

    Green, Benjamin D.; Barry, Emma F.; Ma, Yuefang; Woodcock, Joanna; Fitter, Stephen; Zannettino, Andrew C. W.; Pitson, Stuart M.; Hughes, Timothy P.; Lopez, Angel F.; Shepherd, Peter R.; Wei, Andrew H.; Ekert, Paul G.; Guthridge, Mark A.

    2013-01-01

    The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in

  6. 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.

  7. Phosphoinositide 3-kinase isoforms selectively couple receptors to vascular L-type Ca(2+) channels.

    PubMed

    Macrez, N; Mironneau, C; Carricaburu, V; Quignard, J F; Babich, A; Czupalla, C; Nürnberg, B; Mironneau, J

    2001-10-12

    Heterodimeric class I phosphoinositide 3-kinase (PI3K) has been shown to be involved in the stimulation of voltage-gated Ca(2+) channels by various mediators. In this study, we bring evidences that vascular L-type Ca(2+) channels can be modulated by both tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks. Purified recombinant PI3Ks increased the peak Ca(2+) channel current density when applied intracellularly. Furthermore, PI3Kalpha-, beta-, and delta-mediated stimulations of Ca(2+) channel currents were increased by preactivation by a phosphotyrosyl peptide, whereas PI3Kgamma- and beta-mediated effects were increased by Gbetagamma. In freshly isolated and cultured vascular myocytes, angiotensin II and Gbetagamma stimulated L-type Ca(2+) channel current. In contrast, platelet-derived growth factor (PDGF)-BB and the phosphotyrosyl peptide did not stimulate Ca(2+) channel current in freshly isolated cells despite the presence of endogenous PDGF receptors and PI3Kalpha and PI3Kgamma. Interestingly, when endogenous PI3Kbeta expression arose in cultured myocytes, both PDGF and phosphotyrosyl peptide stimulated Ca(2+) channels through PI3Kbeta, as revealed by the inhibitory effect of an anti-PI3Kbeta antibody. These results suggest that endogenous PI3Kbeta but not PI3Kalpha is specifically involved in PDGF receptor-induced stimulation of Ca(2+) channels and that different isoforms of PI3K regulate physiological increases of Ca(2+) influx in vascular myocytes stimulated by vasoconstrictor or growth factor.

  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. 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

  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. 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.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. Inhibitory effects of nicotine derived from cigarette smoke on thymic stromal lymphopoietin production in epidermal keratinocytes.

    PubMed

    Dong, Jiangxu; Segawa, Ryosuke; Mizuno, Natsumi; Hiratsuka, Masahiro; Hirasawa, Noriyasu

    2016-04-01

    Thymic stromal lymphopoietin (TSLP) is regarded as the main factor responsible for the pathogenesis of atopic dermatitis (AD). Cigarette smoke is an aggravating factor for allergies, but has been reported to decrease the risk of AD. In the present study, we evaluated the role of nicotine, the main constituent in cigarette smoke extract, and its underlying mechanism of action in the regulation of TSLP expression. We found that nicotine significantly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced TSLP expression in BALB/c mice and the mouse keratinocyte cell line PAM212. Nicotine inhibition of TSLP production was abolished by pretreatments with α7 nicotinic acetylcholine receptor (α7 nAChR) antagonists, AMP-activated protein kinase (AMPK) inhibitor, and phosphoinositide 3-kinase (PI3K) inhibitors. The same inhibitors abolished inhibition of nuclear factor-κB (NF-κB) activation by nicotine. These results suggest that nicotine inhibits the expression of TSLP by suppressing the activation of NF-κB through the α7 nAChR-PI3K-AMPK signaling pathway.

  19. A new strategy of using keratinocytes for skin graft: animal experimental study of keratinocyte suspension combined with the dermal substitutes.

    PubMed

    Xiao, S C; Zhu, S H; Li, H Y; Yang, J; Xia, Z F

    2009-01-01

    To provide a new and simple strategy for keratinocyte transplantation. Keratinocyte suspension and dermal substitutes including acellular dermal matrix (ADM) and collagen membrane were prepared. Full-thickness skin defect wounds of Balb/c-nu mice were created and the mice were assigned into 5 groups: the control group, where the wound was grafted with keratinocyte suspension only; the one-step ADM and collagen membrane groups, where the wound was grafted with ADM or collagen membrane plus dripping of keratinocyte suspension; the two-step ADM and collagen membrane groups, where keratinocyte suspension was dripped 2 weeks after ADM or collagen membrane grafting. The percentage of epithelium was observed. Two weeks after grafting, no evidence of re-epithelialization was seen in the one-step ADM group, and the re-epithelialization rate in the one-step collagen membrane group, two-step ADM group and two-step collagen membrane group was 54.1, 41.8 and 76.8%, respectively, indicating that the re-epithelialization rate in the two-step collagen membrane group was higher than that in the other two groups (p < 0.01). Keratinocyte suspension combined with dermal substitutes grafting offers a more flexible way of making use of keratinocytes. Compared with ADM, microporous collagen membrane is a more preferable option for combined use with keratinocyte suspension for skin grafting. Copyright (c) 2009 S. Karger AG, Basel.

  20. Keratinocyte cytogenetics in 10 patients with pigmentary mosaicism: identification of one case of trisomy 20 mosaicism confined to keratinocytes.

    PubMed

    Taibjee, S M; Hall, D; Balderson, D; Larkins, S; Stubbs, T; Moss, C

    2009-10-01

    Hypomelanosis of Ito and linear and whorled hypermelanosis are pigmentary disorders that follow Blaschko's lines and are associated with cytogenetic mosaicism. However, mosaicism cannot always be shown using conventional karyotyping of blood lymphocytes or skin fibroblasts. This may be because these cell lines originate from mesoderm, whereas Blaschko's lines are an ectodermal phenomenon. To investigate the diagnostic value of keratinocyte cytogenetics in patients with pigmentary mosaicism (PM). We undertook a prospective study of 10 patients with clinically suspected PM. Previous karyotyping of blood, and in some cases skin fibroblasts, was normal in all cases. Keratinocytes and fibroblasts were cultured from skin biopsies taken from light and dark skin, and examined for cytogenetic abnormalities. In 9 of 10 cases both keratinocyte and fibroblast cytogenetic analyses were normal. The remaining patient showed trisomy 20 mosaicism confined to keratinocytes from hypopigmented skin. Fluorescent in situ hybridization using a probe for 20q confirmed trisomy 20 mosaicism in keratinocytes but not fibroblasts, with higher signal expression in hypopigmented compared with normal skin. In patients with clinically suspected PM but normal blood cytogenetics, keratinocytes may be more sensitive than skin fibroblasts in identifying cytogenetic mosaicism in selected patients. However, the additional diagnostic yield appears to be insufficient to justify routine keratinocyte cytogenetic investigation. Our findings indirectly support the hypothesis that Blaschko's lines delineate the embryonal migration paths taken by ectodermal cells including keratinocytes and melanocytes.

  1. Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation.

    PubMed

    Nakamura, Takashi; Yoshitomi, Yasuo; Sakai, Kiyoshi; Patel, Vyomesh; Fukumoto, Satoshi; Yamada, Yoshihiko

    2014-12-15

    The basal layer of the epidermis contains stem cells and transit amplifying cells that rapidly proliferate and differentiate further into the upper layers of the epidermis. A number of molecules have been identified as regulators of this process, including p63 (also known as tumor protein 63) and Notch1. However, little is known about the mechanisms that regulate the transitions from stem cell to proliferating or differentiating transit amplifying cell. Here, we demonstrate that epiprofin (Epfn, also known as Sp6) plays crucial distinct roles in these transition stages as a cell cycle regulator and a transcription factor. Epfn knockout mice have a thickened epidermis, in which p63-expressing basal cells form multiple layers owing to the accumulation of premature transit amplifying cells with reduced proliferation and a reduction in the number of differentiating keratinocytes expressing Notch1. We found that low levels of Epfn expression increased the proliferation of human immortalized keratinocyte (HaCaT) cells by increasing EGF responsiveness and superphosphorylation of Rb. By contrast, high levels of Epfn expression promoted cell cycle exit and differentiation, by reducing E2F transactivation and inducing Notch1 expression. Our findings identify multiple novel functions of Epfn in epidermal development.

  2. Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation

    PubMed Central

    Nakamura, Takashi; Yoshitomi, Yasuo; Sakai, Kiyoshi; Patel, Vyomesh; Fukumoto, Satoshi; Yamada, Yoshihiko

    2014-01-01

    ABSTRACT The basal layer of the epidermis contains stem cells and transit amplifying cells that rapidly proliferate and differentiate further into the upper layers of the epidermis. A number of molecules have been identified as regulators of this process, including p63 (also known as tumor protein 63) and Notch1. However, little is known about the mechanisms that regulate the transitions from stem cell to proliferating or differentiating transit amplifying cell. Here, we demonstrate that epiprofin (Epfn, also known as Sp6) plays crucial distinct roles in these transition stages as a cell cycle regulator and a transcription factor. Epfn knockout mice have a thickened epidermis, in which p63-expressing basal cells form multiple layers owing to the accumulation of premature transit amplifying cells with reduced proliferation and a reduction in the number of differentiating keratinocytes expressing Notch1. We found that low levels of Epfn expression increased the proliferation of human immortalized keratinocyte (HaCaT) cells by increasing EGF responsiveness and superphosphorylation of Rb. By contrast, high levels of Epfn expression promoted cell cycle exit and differentiation, by reducing E2F transactivation and inducing Notch1 expression. Our findings identify multiple novel functions of Epfn in epidermal development. PMID:25344255

  3. 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.

  4. [The role of phosphoinositide-3-kinase in controlling the shape and directional movement in Physarum polycephalum plasmodium].

    PubMed

    Matveeva, N B; Beĭlina, S I; Teplov, V A

    2008-01-01

    The influence of wortmannin and LY294002, specific inhibitors of phosphoinosite-3-kinase, on the shape, motile behavior, and chemotaxis toward glucose has been investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the autooscillatory mode of motion. Both inhibitors were shown to cause a reduction of the plasmodium frontal edge and a decrease in the efficiency of mass transfer during migration. They also suppress chemotaxis toward glucose and eliminate characteristic changes in autooscillatory behavior normally observed in response to the treatment of the whole plasmodium with glucose. The manifestation of these effects depends on the inhibitor concentration, the duration of treatment, and the size of plasmodium. The involvement of phosphoinosite-3-kinase in creating the frontal edge and in controlling the chemotaxis of Physarum plasmodium suggests that the interrelation of polar shape and directional movement of amoeboid cells with the distribution of phosphoinositides in the plasma membrane has the universal nature.

  5. Insulin-stimulated phosphatidylinositol 3-kinase activity and 2-deoxy-D-glucose uptake in rat skeletal muscles.

    PubMed

    Elmendorf, J S; Damrau-Abney, A; Smith, T R; David, T S; Turinsky, J

    1995-03-28

    To date there is suggestive evidence that phosphatidylinositol 3-kinase participates in insulin-stimulated glucose transport. However, its involvement in skeletal muscle, a major site of insulin-stimulated glucose disposal, has not been addressed. Therefore, we tested the effects of wortmannin, a known inhibitor of phosphatidylinositol 3-kinase, on insulin-stimulated 2-deoxyglucose uptake by rat soleus muscle in vitro: Wortmannin (1 microM) reversibly inhibited insulin-induced 2-deoxyglucose uptake in soleus muscle by 44%. Inclusion of 5 microM wortmannin in the incubation medium completely abolished the insulin-induced increment in 2-deoxyglucose uptake. In conclusion, the insulin-signaling cascade linking insulin-receptor tyrosine kinase activation to glucose uptake in skeletal muscle.

  6. [Significance of phosphoinositide 3 kinase/AKT pathway alterations in endometrial carcinoma].

    PubMed

    Yang, Xi; Dong, Ying; Zhang, Xiao-ming; Liang, Ying; Zhang, Ying; Meng, Yi-ting; Wang, Ying; Wang, Wei; Nong, Lin; Li, Ting; Liao, Qin-Ping

    2011-12-01

    To investigate the clinicopathologic and prognostic implications of phosphoinositide 3 kinase (PI3K)/AKT pathway alterations in endometrial cancers of Chinese women. The expression of PTEN, p-AKT, and ER/PR was assessed in 71 cases of endometrial carcinoma by immunohistochemistry (EnVision method). The PIK3CA mutation at exon 9 and exon 20 was analyzed by PCR and direct sequencing in 34 tumors. (1) Of the 71 cases of endometrial carcinoma, 65 cases were endometrioid adenocarcinoma (EEC) and 6 cases were nonendometrioid adenocarcinoma (NEEC). PTEN loss of expression was found in 63.4% (45/71) of tumors, and more commonly occurred in EEC (66.2%, 43/65) than that in NEEC (2/6, P = 0.18). Patients with PTEN loss in their tumors (45 cases) had a better survival than those without (26 cases, P = 0.07). In ER negative subgroup, the patients with PTEN loss of expression (12 cases) had longer survival than those with normal PTEN expression (7 cases; P = 0.04). (2) The frequency of PIK3CA mutation was 41.2% (14/34) with a hot mutation spot at T544 in exon 9. PIK3CA mutations more commonly occurred in EEC (44.8%, 13/29) than in NEEC (1/5, P > 0.05). The mutations at exon 9 more commonly occurred in EEC, well- and moderately-differentiated EEC, and tumors at early stage (P > 0.05). On the contrary, in tumors at early stages, the frequency of mutations in exon 20 (14.3%, 4/28) was significantly lower than that at late stages (4/6, P = 0.01). (3) p-AKT was positive in 59.2% (42/71) of tumors that were more frequently found in EEC (60.0%, 39/65) than that in NEEC (3/6, P = 0.68). However, the significant difference of p-AKT expression was found between well- and moderately-differentiated EEC (75.0%, 21/28; 53.6%, 15/28) and poorly-differentiated EEC (3/9, P = 0.02). Moreover, p-AKT expression was significantly correlated with positive ER (r = 0.339, P = 0.00). Endometrial carcinoma patients with loss of PTEN and p-AKT positivity have a favorable prognosis. PIK3CA mutations at

  7. 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

  8. 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

  9. 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

  10. 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

  11. Genetic alterations in the phosphatidylinositol-3 kinase/Akt pathway in thyroid cancer.

    PubMed

    Xing, Mingzhao

    2010-07-01

    Aberrant activation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway plays a fundamental role in thyroid tumorigenesis, particularly in follicular thyroid cancer (FTC) and aggressive thyroid cancer, such as anaplastic thyroid cancer (ATC). As the drivers of this process, many genetic alterations activating the PI3K/Akt pathway have been identified in thyroid cancer in recent years. This review summarizes the current knowledge on major genetic alterations in the PI3K/Akt pathway. These include PIK3CA mutations and genomic amplification/copy gain, Ras mutations, PTEN mutations, RET/PTC and PPARgamma/Pax8 rearrangements, as well as amplification/copy gain of PIK3CB, PDK1, Akt, and various receptor tyrosine kinase genes. Most of these genetic alterations are particularly common in FTC and many of them are even more common in ATC; they are generally less common in papillary thyroid cancer (PTC), in which the MAP kinase (MAPK) pathway activated by the BRAF mutation instead plays a major role. Methylation and, thus, epigenetic silencing of PTEN, a major negative regulator of the PI3K/Akt pathway, occurs in close association with activating genetic alterations of the PI3K/Akt pathway, constituting a unique self-enhancement mechanism for this pathway. Many of these genetic alterations are mutually exclusive in differentiated thyroid tumors, but with increasing concurrence from benign tumors to FTC to ATC. RET/PTC, Ras, and receptor tyrosine kinase could dually activate the PI3K/Akt and MAPK pathways. Most cases of ATC harbor genetic alterations in these genes or other genetic combinations that can activate both pathways. It is proposed that genetic alterations in the PI3K/Akt pathway promote thyroid cell transformation to FTC and that genetic alterations in the MAPK pathway promote cell transformation to PTC; accumulation of multiple genetic alterations that can activate both pathways promotes thyroid cancer aggressiveness and progression to ATC. Genetic alterations

  12. Anti-inflammation effects of naloxone involve phosphoinositide 3-kinase delta and gamma.

    PubMed

    Wang, Tao-Yeuan; Su, Nuan-Yen; Shih, Ping-Cheng; Tsai, Pei-Shan; Huang, Chun-Jen

    2014-12-01

    Phosphoinositide 3-kinase (PI3K) delta and gamma (the p110δ and p110γ isoforms of PI3K) actively participate in the process of inflammation. We sought to elucidate the possible roles of PI3Kδ and PI3Kγ in mediating the anti-inflammation effects of naloxone. Murine macrophages were treated with endotoxin, endotoxin plus naloxone, or endotoxin plus naloxone plus the PI3K inhibitors (the PI3Kδ inhibitor IC87114, the PI3Kγ inhibitor AS252424, or IC87114 plus AS252424) and denoted as the LPS, LPS + N, LPS + N + IC, LPS + N + AS, and LPS + N + IC + AS group, respectively. Differences in inflammatory molecules and levels of nuclear factor-κB (NF-κB) activation and Akt activation (indicator of PI3K activity) among these groups were compared. The concentrations of inflammatory molecules (macrophage inflammatory protein 2, tumor necrosis factor-α, interleukin-1β, and cyclooxygenase-2/prostaglandin E2) and the levels of NF-κB activation (p-NF-κB p65 and p-inhibitor-κB concentrations and NF-κB-DNA binding activity) of the LPS + N group were significantly lower than those of the LPS group (all P < 0.001). These data confirmed the anti-inflammation effects of naloxone. Moreover, the anti-inflammation effects of naloxone could be counteracted by the inhibitors of PI3Kδ and PI3Kγ, as the concentrations of inflammatory molecules and the levels of NF-κB activation of the LPS + N group were significantly lower than those of the LPS + N + IC, LPS + N + AS, and LPS + N + IC + AS groups (all P < 0.05). In contrast, the concentration of phosphorylated Akt of the LPS + N group was significantly higher than those of the LPS, LPS + N + IC, LPS + N + AS, and LPS + N + IC + AS groups (all P < 0.05). PI3Kδ and PI3Kγ play crucial roles in mediating the anti-inflammation effects of naloxone. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Evolutionary history of phosphatidylinositol- 3-kinases: ancestral origin in eukaryotes and complex duplication patterns.

    PubMed

    Philippon, Héloïse; Brochier-Armanet, Céline; Perrière, Guy

    2015-10-19

    Phosphatidylinositol-3-kinases (PI3Ks) are a family of eukaryotic enzymes modifying phosphoinositides in phosphatidylinositols-3-phosphate. Located upstream of the AKT/mTOR signalling pathway, PI3Ks activate secondary messengers of extracellular signals. They are involved in many critical cellular processes such as cell survival, angiogenesis and autophagy. PI3K family is divided into three classes, including 14 human homologs. While class II enzymes are composed of a single catalytic subunit, class I and III also contain regulatory subunits. Here we present an in-depth phylogenetic analysis of all PI3K proteins. We confirmed that PI3K catalytic subunits form a monophyletic group, whereas regulatory subunits form three distinct groups. The phylogeny of the catalytic subunits indicates that they underwent two major duplications during their evolutionary history: the most ancient arose in the Last Eukaryotic Common Ancestor (LECA) and led to the emergence of class III and class I/II, while the second - that led to the separation between class I and II - occurred later, in the ancestor of Unikonta (i.e., the clade grouping Amoebozoa, Fungi, and Metazoa). These two major events were followed by many lineage specific duplications in particular in vertebrates, but also in various protist lineages. Major loss events were also detected in Vidiriplantae and Fungi. For the regulatory subunits, we identified homologs of class III in all eukaryotic groups indicating that, for this class, both the catalytic and the regulatory subunits were presents in LECA. In contrast, homologs of the regulatory class I have a more recent origin. The phylogenetic analysis of the PI3K shed a new light on the evolutionary history of these enzymes. We found that LECA already contained a PI3K class III composed of a catalytic and a regulatory subunit. Absence of class II regulatory subunits and the recent origin of class I regulatory subunits is puzzling given that the class I/II catalytic subunit

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

    PubMed

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

    2013-12-01

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

  15. Targeting Phosphoinositide-3-Kinase-δ with Theophylline Reverses Corticosteroid Insensitivity in Chronic Obstructive Pulmonary Disease

    PubMed Central

    To, Yasuo; Ito, Kazuhiro; Kizawa, Yasuo; Failla, Marco; Ito, Misako; Kusama, Tadashi; Elliott, W. Mark; Hogg, James C.; Adcock, Ian M.; Barnes, Peter J.

    2010-01-01

    Rationale: Patients with chronic obstructive pulmonary disease (COPD) show a poor response to corticosteroids. This has been linked to a reduction of histone deacetylase-2 as a result of oxidative stress and is reversed by theophylline. Objectives: To determine the role of phosphoinositide-3-kinase-delta (PI3K-δ) on the development of corticosteroid insensitivity in COPD and under oxidative stress, and as a target for theophylline. Methods: Corticosteroid sensitivity was determined as the 50% inhibitory concentration of dexamethasone on tumor necrosis factor-α–induced interleukin-8 release in peripheral blood mononuclear cells from patients with COPD (n = 17) and compared with that of nonsmoking (n = 8) and smoking (n = 7) control subjects. The effect of theophylline and a selective PI3K-δ inhibitor (IC87114) on restoration of corticosteroid sensitivity was confirmed in cigarette smoke–exposed mice. Measurements and Main Results: Peripheral blood mononuclear cells of COPD (50% inhibitory concentration of dexamethasone: 156.8 ± 32.6 nM) were less corticosteroid sensitive than those of nonsmoking (41.2 ± 10.5 nM; P = 0.018) and smoking control subjects (47.5 ± 19.6 nM; P = 0.031). Corticosteroid insensitivity and reduced histone deacetylase-2 activity after oxidative stress were reversed by a non-selective PI3K inhibitor (LY294002) and low concentrations of theophylline. Theophylline was a potent selective inhibitor of oxidant-activated PI3K-δ, which was up-regulated in peripheral lung tissue of patients with COPD. Furthermore, cells with knock-down of PI3K-δ failed to develop corticosteroid insensitivity with oxidative stress. Both theophylline and IC87114, combined with dexamethasone, inhibited corticosteroid-insensitive lung inflammation in cigarette–smoke-exposed mice in vivo. Conclusions: Inhibition of oxidative stress dependent PI3K-δ activation by a selective inhibitor or theophylline provides a novel approach to reversing corticosteroid

  16. Genetic Alterations in the Phosphatidylinositol-3 Kinase/Akt Pathway in Thyroid Cancer

    PubMed Central

    2010-01-01

    Background Aberrant activation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway plays a fundamental role in thyroid tumorigenesis, particularly in follicular thyroid cancer (FTC) and aggressive thyroid cancer, such as anaplastic thyroid cancer (ATC). As the drivers of this process, many genetic alterations activating the PI3K/Akt pathway have been identified in thyroid cancer in recent years. Summary This review summarizes the current knowledge on major genetic alterations in the PI3K/Akt pathway. These include PIK3CA mutations and genomic amplification/copy gain, Ras mutations, PTEN mutations, RET/PTC and PPARγ/Pax8 rearrangements, as well as amplification/copy gain of PIK3CB, PDK1, Akt, and various receptor tyrosine kinase genes. Most of these genetic alterations are particularly common in FTC and many of them are even more common in ATC; they are generally less common in papillary thyroid cancer (PTC), in which the MAP kinase (MAPK) pathway activated by the BRAF mutation instead plays a major role. Methylation and, thus, epigenetic silencing of PTEN, a major negative regulator of the PI3K/Akt pathway, occurs in close association with activating genetic alterations of the PI3K/Akt pathway, constituting a unique self-enhancement mechanism for this pathway. Many of these genetic alterations are mutually exclusive in differentiated thyroid tumors, but with increasing concurrence from benign tumors to FTC to ATC. RET/PTC, Ras, and receptor tyrosine kinase could dually activate the PI3K/Akt and MAPK pathways. Most cases of ATC harbor genetic alterations in these genes or other genetic combinations that can activate both pathways. It is proposed that genetic alterations in the PI3K/Akt pathway promote thyroid cell transformation to FTC and that genetic alterations in the MAPK pathway promote cell transformation to PTC; accumulation of multiple genetic alterations that can activate both pathways promotes thyroid cancer aggressiveness and progression to ATC

  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. Molecular explorations of substituted 2-(4-phenylquinolin-2-yl) phenols as phosphoinositide 3-kinase inhibitors and anticancer agents.

    PubMed

    Alagumuthu, Manikandan; Arumugam, Sivakumar

    2017-02-01

    Substituted 2-(4-phenylquinolin-2-yl) phenols (PQPDs) emerged as the inhibitors of phosphoinositide 3-kinase (PI3K) and anticancer agents. PI3K inhibition was assessed by competitive ELISA. Anticancer activity was evaluated against breast cancer (MCF-7), skin cancer (G-361), and colon cancer (HCT 116) cell lines. In PI3 Kinase assay, PQPDs 4c, 4d, and 4k were inactive with IC50 >5 µM. IC50 for 4a, 4b, 4f-h, and 4j was ≥0.05 µM. Rest PQPDs IC50 was <1.0 µM. Anticancer activity found selective toward breast cancer (MCF-7); 4a, 4b, and 4j were showed excellent inhibitory (73.95, 68.36, and 70.06%) and IC50 1.16 µM (4a), 2.07 µM (4b), 1.021 µM (4f) and 1.981 µM (4j) while the standard (Doxorubicin) found with IC50 1.812 µM (72% inhibition). PQPDs were docked into the active site of PI3 Kinase p110α (PDB ID: 2RD0). Docking results suggested the hydrophobic interactions in PI3K binding pocket conquered affinity of the most favorable binding ligands [4a, 4b: inhibitory constant (ki) = 53.33, 41.23 pM]. PI3K assay and cancer cell line experimental results ensured that the inhibitory and anticancer activity potentials of PQPDs are more selective toward breast cancer treatments. PQPDs 4a, 4b, 4f, 4g, and 4j were displayed potent PI3 Kinase and anticancer activities. SAR studies demonstrated PQPDs as the PI3K precise inhibitors with the impending to treat various cancers.

  19. Stathmin regulates keratinocyte proliferation and migration during cutaneous regeneration.

    PubMed

    Schmitt, Sabrina; Safferling, Kai; Westphal, Kathi; Hrabowski, Manuel; Müller, Ute; Angel, Peter; Wiechert, Lars; Ehemann, Volker; Müller, Benedikt; Holland-Cunz, Stefan; Stichel, Damian; Harder, Nathalie; Rohr, Karl; Germann, Günter; Matthäus, Franziska; Schirmacher, Peter; Grabe, Niels; Breuhahn, Kai

    2013-01-01

    Cutaneous regeneration utilizes paracrine feedback mechanisms to fine-tune the regulation of epidermal keratinocyte proliferation and migration. However, it is unknown how fibroblast-derived hepatocyte growth factor (HGF) affects these mutually exclusive processes in distinct cell populations. We here show that HGF stimulates the expression and phosphorylation of the microtubule-destabilizing factor stathmin in primary human keratinocytes. Quantitative single cell- and cell population-based analyses revealed that basal stathmin levels are important for the migratory ability of keratinocytes in vitro; however, its expression is moderately induced in the migration tongue of mouse skin or organotypic multi-layered keratinocyte 3D cultures after full-thickness wounding. In contrast, clearly elevated stathmin expression is detectable in hyperproliferative epidermal areas. In vitro, stathmin silencing significantly reduced keratinocyte proliferation. Automated quantitative and time-resolved analyses in organotypic cocultures demonstrated a high correlation between Stathmin/phospho-Stathmin and Ki67 positivity in epidermal regions with proliferative activity. Thus, activation of stathmin may stimulate keratinocyte proliferation, while basal stathmin levels are sufficient for keratinocyte migration during cutaneous regeneration.

  20. Acute and chronic wound fluids influence keratinocyte function differently.

    PubMed

    Thamm, Oliver C; Koenen, Paola; Bader, Nicola; Schneider, Alina; Wutzler, Sebastian; Neugebauer, Edmund A M; Spanholtz, Timo A

    2015-04-01

    Wound healing requires a proper functioning of keratinocytes that migrate, proliferate and lead to a competent wound closure. Impaired wound healing might be due to a disturbed keratinocyte function caused by the wound environment. Basically, chronic wound fluid (CWF) differs from acute wound fluid (AWF). The aim of this study was to analyse the effects of AWF and CWF on keratinocyte function. We therefore investigated keratinocyte migration and proliferation under the influence of AWF and CWF using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test and scratch assay. We further measured the gene expression by qRT-PCR regarding growth factors and matrixmetalloproteinases (MMPs) involved in regeneration processes. AWF had a positive impact on keratinocyte proliferation over time, whereas CWF had an anti-proliferative effect. Keratinocyte migration was significantly impaired by CWF in contrast to an undisturbed wound closure under the influence of AWF. MMP-9 expression was strongly upregulated by CWF compared with AWF. Keratinocyte function was significantly impaired by CWF. An excessive induction of MMP-9 by CWF might lead to a permanent degradation of extracellular matrix and thereby prevent wounds from healing. © 2013 The Authors. International Wound Journal © 2013 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

  1. Lipin-1 expression is critical for keratinocyte differentiation.

    PubMed

    Chae, Minjung; Jung, Ji-Yong; Bae, Il-Hong; Kim, Hyoung-June; Lee, Tae Ryong; Shin, Dong Wook

    2016-04-01

    Lipin-1 is an Mg(2+)-dependent phosphatidate phosphatase that facilitates the dephosphorylation of phosphatidic acid to generate diacylglycerol. Little is known about the expression and function of lipin-1 in normal human epidermal keratinocytes (NHEKs). Here, we demonstrate that lipin-1 is present in basal and spinous layers of the normal human epidermis, and lipin-1 expression is gradually downregulated during NHEK differentiation. Interestingly, lipin-1 knockdown (KD) inhibited keratinocyte differentiation and caused G1 arrest by upregulating p21 expression. Cell cycle arrest by p21 is required for commitment of keratinocytes to differentiation, but must be downregulated for the progress of keratinocyte differentiation. Therefore, reduced keratinocyte differentiation results from sustained upregulation of p21 by lipin-1 KD. Lipin-1 KD also decreased the phosphorylation/activation of protein kinase C (PKC)α, whereas lipin-1 overexpression increased PKCα phosphorylation. Treatment with PKCα inhibitors, like lipin-1 KD, stimulated p21 expression, while lipin-1 overexpression reduced p21 expression, implicating PKCα in lipin-1-induced regulation of p21 expression. Taken together, these results suggest that lipin-1-mediated downregulation of p21 is critical for the progress of keratinocyte differentiation after the initial commitment of keratinocytes to differentiation induced by p21, and that PKCα is involved in p21 expression regulation by lipin-1. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

  2. Stathmin Regulates Keratinocyte Proliferation and Migration during Cutaneous Regeneration

    PubMed Central

    Schmitt, Sabrina; Safferling, Kai; Westphal, Kathi; Hrabowski, Manuel; Müller, Ute; Angel, Peter; Wiechert, Lars; Ehemann, Volker; Müller, Benedikt; Holland-Cunz, Stefan; Stichel, Damian; Harder, Nathalie; Rohr, Karl; Germann, Günter; Matthäus, Franziska; Schirmacher, Peter; Grabe, Niels; Breuhahn, Kai

    2013-01-01

    Cutaneous regeneration utilizes paracrine feedback mechanisms to fine-tune the regulation of epidermal keratinocyte proliferation and migration. However, it is unknown how fibroblast-derived hepatocyte growth factor (HGF) affects these mutually exclusive processes in distinct cell populations. We here show that HGF stimulates the expression and phosphorylation of the microtubule-destabilizing factor stathmin in primary human keratinocytes. Quantitative single cell- and cell population-based analyses revealed that basal stathmin levels are important for the migratory ability of keratinocytes in vitro; however, its expression is moderately induced in the migration tongue of mouse skin or organotypic multi-layered keratinocyte 3D cultures after full-thickness wounding. In contrast, clearly elevated stathmin expression is detectable in hyperproliferative epidermal areas. In vitro, stathmin silencing significantly reduced keratinocyte proliferation. Automated quantitative and time-resolved analyses in organotypic cocultures demonstrated a high correlation between Stathmin/phospho-Stathmin and Ki67 positivity in epidermal regions with proliferative activity. Thus, activation of stathmin may stimulate keratinocyte proliferation, while basal stathmin levels are sufficient for keratinocyte migration during cutaneous regeneration. PMID:24066165

  3. 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.

  4. 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.

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

    PubMed Central

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

    2009-01-01

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

  6. Induction of senescence pathways in Kindler syndrome primary keratinocytes.

    PubMed

    Piccinni, E; Di Zenzo, G; Maurelli, R; Dellambra, E; Teson, M; Has, C; Zambruno, G; Castiglia, D

    2013-05-01

    Individuals with Kindler syndrome (KS) have loss-of-function mutations in the FERMT1 gene that encodes the focal adhesion component kindlin-1. The major clinical manifestation of KS is epidermal atrophy (premature skin ageing). This phenotypic feature is thought to be related to the decreased proliferation rate of KS keratinocytes; nevertheless, molecular mediators of such abnormal behaviour have not been fully elucidated. To investigate how kindlin-1 deficiency affects the proliferative potential of primary human keratinocytes. We serially cultivated nine primary KS keratinocyte strains until senescence and determined their lifespan and colony-forming efficiency (CFE) at each serial passage. The expression of molecular markers of stemness and cellular senescence were investigated by immunoblotting using cell extracts of primary keratinocyte cultures from patients with KS and healthy donors. In another set of experiments, kindlin-1 downregulation in normal keratinocytes was obtained by small interfering RNA (siRNA) technology. We found that KS keratinocytes exhibited a precocious senescence and strongly reduced clonogenic potential. Moreover, KS cultures showed a strikingly increased percentage of aborted colonies (paraclones) already at early passages indicating an early depletion of stem cells. Immunoblotting analysis of KS keratinocyte extracts showed reduced levels of the stemness markers p63 and Bmi-1, upregulation of p16 and scant amounts of hypophosphorylated Rb protein, which indicated cell cycle-arrested status. Treatment of normal human primary keratinocytes with siRNA targeting kindlin-1 proved that its deficiency was directly responsible for p63, Bmi-1 and pRb downregulation and p16 induction. Our data directly implicate kindlin-1 in preventing premature senescence of keratinocytes. © 2013 The Authors. BJD © 2013 British Association of Dermatologists.

  7. Stereotyped distribution of proliferating keratinocytes in disorders affecting the epidermis

    SciTech Connect

    Pierard-Franchimont, C.; Pierard, G.E.

    1989-06-01

    We used the technique of autoradiography after incorporation of tritiated thymidine (/sup 3/H-TdR) to evaluate keratinocyte proliferation in basal, epibasal, and other epidermal layers in 30 diseases affecting the epidermis. The number and proportion of /sup 3/H-TdR-labeled keratinocytes were counted in the different layers of the epidermis. Significant correlations were found between the proliferative indices of the different epidermal layers. Such links indicate that the epidermis responds in a rather stereotyped way to various pathological conditions. There exists some regulation in the distribution, number, and proportion of /sup 3/H-TdR-labeled keratinocytes in the various layers of the epidermis.

  8. Gene network dynamics controlling keratinocyte migration

    PubMed Central

    Busch, Hauke; Camacho-Trullio, David; Rogon, Zbigniew; Breuhahn, Kai; Angel, Peter; Eils, Roland; Szabowski, Axel

    2008-01-01

    Translation of large-scale data into a coherent model that allows one to simulate, predict and control cellular behavior is far from being resolved. Assuming that long-term cellular behavior is reflected in the gene expression kinetics, we infer a dynamic gene regulatory network from time-series measurements of DNA microarray data of hepatocyte growth factor-induced migration of primary human keratinocytes. Transferring the obtained interactions to the level of signaling pathways, we predict in silico and verify in vitro the necessary and sufficient time-ordered events that control migration. We show that pulse-like activation of the proto-oncogene receptor Met triggers a responsive state, whereas time sequential activation of EGF-R is required to initiate and maintain migration. Context information for enhancing, delaying or stopping migration is provided by the activity of the protein kinase A signaling pathway. Our study reveals the complex orchestration of multiple pathways controlling cell migration. PMID:18594517

  9. Scanning Ion Conductance Microscopy of Live Keratinocytes

    NASA Astrophysics Data System (ADS)

    Hegde, V.; Mason, A.; Saliev, T.; Smith, F. J. D.; McLean, W. H. I.; Campbell, P. A.

    2012-07-01

    Scanning ion conductance microscopy (SICM) is perhaps the least well known technique from the scanning probe microscopy (SPM) family of instruments. As with its more familiar counterpart, atomic force microscopy (AFM), the technique provides high-resolution topographic imaging, with the caveat that target structures must be immersed in a conducting solution so that a controllable ion current may be utilised as the basis for feedback. In operation, this non-contact characteristic of SICM makes it ideal for the study of delicate structures, such as live cells. Moreover, the intrinsic architecture of the instrument, incorporating as it does, a scanned micropipette, lends itself to combination approaches with complementary techniques such as patch-clamp electrophysiology: SICM therefore boasts the capability for both structural and functional imaging. For the present observations, an ICnano S system (Ionscope Ltd., Melbourn, UK) operating in 'hopping mode' was used, with the objective of assessing the instrument's utility for imaging live keratinocytes under physiological buffers. In scans employing cultured HaCaT cells (spontaneously immortalised, human keratinocytes), we compared the qualitative differences of live cells imaged with SICM and AFM, and also with their respective counterparts after chemical fixation in 4% paraformaldehyde. Characteristic surface microvilli were particularly prominent in live cell imaging by SICM. Moreover, time lapse SICM imaging on live cells revealed that changes in the pattern of microvilli could be tracked over time. By comparison, AFM imaging on live cells, even at very low contact forces (

  10. Sphingolipid metabolism in organotypic mouse keratinocyte cultures

    SciTech Connect

    Madison, K.C.; Swartzendruber, D.C.; Wertz, P.W.; Downing, D.T. )

    1990-12-01

    Ceramides are the dominant component of the stratum corneum intercellular lipid lamellae, which constitute the epidermal permeability barrier. Only pig and human epidermal ceramides have been extensively characterized and the structures of the ceramides of cultured keratinocytes have not been previously investigated. In the present studies, we have characterized the ceramides synthesized by organotypic lifted mouse keratinocyte cultures for the first time and compared them to the ceramides of intact mouse epidermis. Both mouse epidermis and cultures contained five ceramides, ceramide 1 being the least polar and ceramide 5 the most polar. Ceramide 1 was a group of acylceramides, i.e., very-long-chain omega-hydroxyceramides with an ester-linked nonhydroxy fatty acid. Ceramide 2 contained medium-length saturated nonhydroxy fatty acids. (In culture, the ceramide 2 band was split into two parts with the slightly more polar ceramide 2' containing short-chain saturated nonhydroxy fatty acids.) Ceramide 5 contained short-chain alpha-hydroxy fatty acids. The structures of ceramides 1, 2, and 5 were analagous to those of pig and human epidermis. Mouse epidermal ceramide 3 was quite unusual, containing beta-hydroxy fatty acids, a structure not previously identified among mammalian ceramides. In contrast, culture ceramide 3 was composed of omega-hydroxy fatty acids with a chain-length distribution similar to that of ceramide 1. Mouse ceramide 4 was composed of fatty acids with chromatographic mobility similar to hydroxy fatty acids but with different chemical reactivity; it remains only partially characterized. Culture ceramide 4 was present in quantities too small for analysis. All ceramides in mouse epidermis and cultures contained only sphingosine bases, whereas pig and human ceramides also contain phytosphingosine.

  11. 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

  12. 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

  13. 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.

  14. Advances in keratinocyte delivery in burn wound care.

    PubMed

    Ter Horst, Britt; Chouhan, Gurpreet; Moiemen, Naiem S; Grover, Liam M

    2017-06-28

    This review gives an updated overview on keratinocyte transplantation in burn wounds concentrating on application methods and future therapeutic cell delivery options with a special interest in hydrogels and spray devices for cell delivery. To achieve faster re-epithelialisation of burn wounds, the original autologous keratinocyte culture and transplantation technique was introduced over 3 decades ago. Application types of keratinocytes transplantation have improved from cell sheets to single-cell solutions delivered with a spray system. However, further enhancement of cell culture, cell viability and function in vivo, cell carrier and cell delivery systems remain themes of interest. Hydrogels such as chitosan, alginate, fibrin and collagen are frequently used in burn wound care and have advantageous characteristics as cell carriers. Future approaches of keratinocyte transplantation involve spray devices, but optimisation of application technique and carrier type is necessary. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  15. Cyclic AMP mediates keratinocyte directional migration in an electric field.

    PubMed

    Pullar, Christine E; Isseroff, R Rivkah

    2005-05-01

    Re-epithelialization of wounded skin is necessary for wound closure and restoration of barrier function and requires directional keratinocyte migration towards the center of the wound. The electric field (EF) generated immediately upon wounding could be the earliest signal keratinocytes receive to initiate directional migration and healing. Keratinocytes express many beta2-adrenergic receptors (beta2-ARs), but their role in the epidermis is unknown. We have previously shown that beta-AR agonists decrease keratinocyte migration in a cyclic AMP (cAMP) independent mechanism involving the activation of protein phosphatase 2A (PP2A). Here, we ask whether beta2-ARs play a role in keratinocyte galvanotaxis. We report a bimodal response. When keratinocytes were exposed to higher concentrations of beta-AR agonist (0.1 microM), their tracked migratory speed was inhibited, in both the presence (directional migration) and the absence (random migration) of a 100 mV mm(-1) EF, as expected. At lower agonist concentrations (0.1 pM to 0.1 nM), there was no effect on migratory speed; however, all directionality was lost - essentially, cells were 'blinded' to the directional cue. Preincubating the cells with beta-antagonist restored directional migration, demonstrating that the 'blindness' was beta2-AR mediated. Incubation of keratinocytes with agents known to increase intracellular cAMP levels, such as sp-cAMP, pertussis toxin and forskolin, resulted in similar 'blinding' to the EF, whereas random migration was unaffected. The inactive cAMP analog rp-cAMP had no effect on keratinocyte migration, whether directional or random. However, rp-cAMP pretreatment before beta-agonist addition fully restored galvanotaxis, demonstrating the complete cAMP dependence of the attenuation of keratinocyte directional migration. This is the first report that cAMP is capable of mediating keratinocyte galvanotaxis. beta-AR agonists and antagonists could be valuable tools for modulating re

  16. Cellular and molecular facets of keratinocyte reepithelization during wound healing

    SciTech Connect

    Santoro, Massimo M. . E-mail: msantoro@unipmn.it; Gaudino, Giovanni

    2005-03-10

    Cutaneous wound healing is a highly coordinated physiological process that rapidly and efficiently restores skin integrity. Reepithelization is a crucial step during wound healing, which involves migration and proliferation of keratinocytes to cover the denuded dermal surface. Recent advances in wound biology clarified the molecular pathways governing keratinocyte reepithelization at wound sites. These new findings point towards novel therapeutic targets and provide suitable methods to promote faster tissue regeneration in vivo.

  17. Knockdown of PKD1 in normal human epidermal keratinocytes increases mRNA expression of keratin 10 and involucrin: early markers of keratinocyte differentiation.

    PubMed

    Ivanova, Petya; Atanasova, Ganka; Poumay, Yves; Mitev, Vanyo

    2008-03-01

    Subconfluent normal human keratinocytes exhibit autonomous (autocrine growth factor driven) proliferation and express the specific markers for keratinocyte proliferation K5 (keratin 5) and K14 (keratin 14). Utilizing this model the effects of PKD1 (Protein kinase D1) knockdown on activation of differentiation was studied. siRNA approach was applied to achieve specific knockdown of PKD1 and the mRNA levels of different keratinocyte markers -- K14 and PCNA (markers of basal proliferating keratinocytes), involucrin and K10 (early differentiation markers) were analyzed. Treatment of cultured keratinocytes with siRNA for PKD1 resulted in reduction of mRNA levels of PKD1, altered cell phenotype and promotion of keratinocyte differentiation, demonstrated by increased expression of involucrin and K10 mRNAs. No significant changes in K14 mRNA expression levels were detected, but the expression of PCNA mRNA was markedly diminished. This study was the first to show that mRNA expression of PKD1 in subconfluent normal human keratinocytes is very low, the PKD1 mRNA levels were more than 8-fold lower than the same ones in hTert keratinocytes. These findings suggest antidifferentiative role of PKD1 in normal human keratinocytes, contrary to the prodiferentiative role of PKD1 in human hTert keratinocytes. We came to the conclusion that there are differences between transduction pathways involving PKD1 in primary human keratinocyte cultures and these in immortalized hTert keratinocytes.

  18. Distinctive molecular responses to ultraviolet radiation between keratinocytes and melanocytes

    PubMed Central

    Sun, Xiaoyun; Kim, Arianna; Nakatani, Masashi; Shen, Yao; Liu, Liang

    2017-01-01

    Solar ultraviolet radiation (UVR) is the major risk factor for skin carcinogenesis. To gain new insights into the molecular pathways mediating UVR effects in the skin, we performed comprehensive transcriptomic analyses to identify shared and distinctive molecular responses to UVR between human keratinocytes and melanocytes. Keratinocytes and melanocytes were irradiated with varying doses of UVB (10, 20 and 30 mJ/cm2) then analysed by RNA-Seq at different time points post-UVB radiation (4, 24 and 72 h). Under basal conditions, keratinocytes and melanocytes expressed similar number of genes, although they each expressed a distinctive subset of genes pertaining to their specific cellular identity. Upon UVB radiation, keratinocytes displayed a clear pattern of time- and dose-dependent changes in gene expression that was different from melanocytes. The early UVB-responsive gene set (4 h post-UVR) differed significantly from delayed UVB-responsive gene sets (24 and 72 h). We also identified multiple novel UVB signature genes including PRSS23, SERPINH1, LCE3D and CNFN, which were conserved between melanocyte and keratinocyte lines from different individuals. Taken together, our findings elucidated both common and distinctive molecular features between melanocytes and keratinocytes and uncovered novel UVB signature genes that might be utilized to predict UVB photobiological effects on the skin. PMID:27119462

  19. Polarized Integrin Mediates Human Keratinocyte Adhesion to Basal Lamina

    NASA Astrophysics Data System (ADS)

    de Luca, Michele; Tamura, Richard N.; Kajiji, Shama; Bondanza, Sergio; Rossino, Paola; Cancedda, Ranieri; Carlo Marchisio, Pier; Quaranta, Vito

    1990-09-01

    Epithelial cell interactions with matrices are critical to tissue organization. Indirect immunofluorescence and immunoprecipitations of cell lysates prepared from stratified cultures of human epidermal cells showed that the major integrins expressed by keratinocytes are α_Eβ_4 (also called α_6β_4) and α_2β_1/α_3β_1. The α_Eβ_4 integrin is localized at the surface of basal cells in contact with the basement membrane, whereas α_2β_1/ α_3β_1 integrins are absent from the basal surface and are localized only on the lateral surface of basal and spinous keratinocytes. Anti-β_4 antibodies potently inhibited keratinocyte adhesion to matrigel or purified laminin, whereas anti-β_1 antibodies were ineffective. Only anti-β_4 antibodies were able to detach established keratinocyte colonies. These data suggest that α_Eβ_4 mediates keratinocyte adhesion to basal lamina, whereas the β_1 subfamily is involved in cell-cell adhesion of keratinocytes.

  20. Induction of Apoptosis in Human Oral Keratinocyte by Doxorubicin.

    PubMed

    Sakagami, Hiroshi; Okudaira, Noriyuki; Masuda, Yoshiko; Amano, Osamu; Yokose, Satoshi; Kanda, Yumiko; Suguro, Madoka; Natori, Takenori; Oizumi, Hiroshi; Oizumi, Takaaki

    2017-03-01

    We have previously reported that doxorubicin (DXR) showed much higher cytotoxicity against human oral squamous cell carcinoma cell lines compared to normal human mesenchymal normal oral cells (gingival fibroblast, periodontal ligament fibroblast, pulp cell), yielding high tumor-specificity. However, we unexpectedly found that doxorubicin showed potent cytotoxicity against human normal oral keratinocytes and primary gingival epithelial cells. In the present study, we investigated the reproducibility, underlining mechanisms and generality of this unexpected finding. Viable cell number was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method, fine cell structure by transmission electron microscopy and apoptosis induction by western blot analysis. Doxorubicin induced keratinocyte toxicity, regardless of cell density and concentration of FBS in the culture medium. Doxorubicin induced apoptosis (characterized by the loss of cell surface microvilli, chromatin condensation, nuclear fragmentation and caspase-3 activation) in keratinocytes. A total of 11 anticancer drugs showed similar keratinocyte toxicity. Alkaline extract of the leaves of Sasa senanensis Rehder partially alleviated the DXR-induced keratinocyte cytotoxicity by promoting cell growth. The present study suggested that oral keratinocyte toxicity is a novel adverse effect of most anticancer agents. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  1. The UV (Ribotoxic) Stress Response of Human Keratinocytes Involves the Unexpected Uncoupling of the Ras-Extracellular Signal-Regulated Kinase Signaling Cascade from the Activated Epidermal Growth Factor Receptor

    PubMed Central

    Iordanov, Mihail S.; Choi, Remy J.; Ryabinina, Olga P.; Dinh, Thanh-Hoai; Bright, Robert K.; Magun, Bruce E.

    2002-01-01

    In mammals, UVB radiation is of biological relevance primarily for the cells of the epidermis. We report here the existence of a UVB response that is specific for proliferating human epidermal keratinocytes. Unlike other cell types that also display a UVB response, keratinocytes respond to UVB irradiation with a transient but potent downregulation of the Ras-extracellular signal-regulated kinase (ERK) signaling cascade. The downregulation of ERK precedes a profound decrease in the steady-state levels of cyclin D1, a mediator of the proliferative action of ERK. Keratinocytes exhibit high constitutive activity of the Ras-ERK signaling cascade even in culture medium lacking supplemental growth factors. The increased activity of Ras and phosphorylation of ERK in these cells are maintained by the autocrine production of secreted molecules that activate the epidermal growth factor receptor (EGFR). Irradiation of keratinocytes increases the phosphorylation of EGFR on tyrosine residues Y845, Y992, Y1045, Y1068, Y1086, Y1148, and Y1173 above the basal levels and leads to the increased recruitment of the adaptor proteins Grb2 and ShcA and of a p55 form of the regulatory subunit of the phosphatidylinositide 3-kinase to the UVB-activated EGFR. Paradoxically, however, UVB causes, at the same time, the inactivation of Ras and a subsequent dephosphorylation of ERK. By contrast, the signaling pathway leading from the activated EGFR to the phosphorylation of PKB/Akt1 is potentiated by UVB. The UVB response of keratinocytes appeared to be a manifestation of the more general ribotoxic stress response inasmuch as the transduction of the UVB-generated inhibitory signal to Ras and ERK required the presence of active ribosomes at the time of irradiation. PMID:12101233

  2. The lysosomal cysteine protease cathepsin L regulates keratinocyte proliferation by control of growth factor recycling.

    PubMed

    Reinheckel, Thomas; Hagemann, Sascha; Dollwet-Mack, Susanne; Martinez, Elke; Lohmüller, Tobias; Zlatkovic, Gordana; Tobin, Desmond J; Maas-Szabowski, Nicole; Peters, Christoph

    2005-08-01

    Mice deficient for cathepsin L (CTSL) show epidermal hyperplasia due to a hyperproliferation of basal keratinocytes. Here we show that the critical function of CTSL in the skin is keratinocyte specific. This is revealed by transgenic re-expression of CTSL in the keratinocytes of ctsl-/- mice, resulting in a rescue of the ctsl-/- skin phenotype. Cultivation of primary mouse keratinocytes with fibroblast- and keratinocyte-conditioned media, as well as heterologous organotypic co-cultures of mouse fibroblasts and human keratinocytes, showed that the altered keratinocyte proliferation is caused primarily by CTSL-deficiency in keratinocytes. In the absence of EGF, wild type and CTSL-knockout keratinocytes proliferate with the same rates, while in presence of EGF, ctsl-/- keratinocytes showed enhanced proliferation compared with controls. Internalization and degradation of radioactively labeled EGF was identical in both ctsl-/- and ctsl+/+ keratinocytes. However, ctsl-/- keratinocytes recycled more EGF to the cell surface, where it is bound to the EGF-receptor, which is also more abundant in ctsl-/- cells. We conclude that the hyperproliferation of keratinocytes in CTSL-knockout mice is caused by an enhanced recycling of growth factors and growth factor receptors from the endosomes to the keratinocyte plasma membrane, which result in sustained growth stimulation.

  3. 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

  4. 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.

  5. Transcriptional profiling of epidermal keratinocytes: comparison of genes expressed in skin, cultured keratinocytes, and reconstituted epidermis, using large DNA microarrays.

    PubMed

    Gazel, Alix; Ramphal, Patricia; Rosdy, Martin; De Wever, Bart; Tornier, Carine; Hosein, Nadia; Lee, Brian; Tomic-Canic, Marjana; Blumenberg, Miroslav

    2003-12-01

    Epidermal keratinocytes are complex cells that create a unique three-dimensional (3-D) structure, differentiate through a multistage process, and respond to extracellular stimuli from nearby cells. Consequently, keratinocytes express many genes, i.e., have a relatively large "transcriptome." To determine which of the expressed genes are innate to keratinocytes, which are specific for the differentiation and 3-D architecture, and which are induced by other cell types, we compared the transcriptomes of skin from human subjects, differentiating 3-D reconstituted epidermis, cultured keratinocytes, and nonkeratinocyte cell types. Using large oligonucleotide microarrays, we analyzed five or more replicates of each, which yielded statistically consistent data and allowed identification of the differentially expressed genes. Epidermal keratinocytes, unlike other cells, express many proteases and protease inhibitors and genes that protect from UV light. Skin specifically expresses a higher number of receptors, secreted proteins, and transcription factors, perhaps influenced by the presence of nonkeratinocyte cell types. Surprisingly, mitochondrial proteins were significantly suppressed in skin, suggesting a low metabolic rate. Three-dimensional samples, skin and reconstituted epidermis, are similar to each other, expressing epidermal differentiation markers. Cultured keratinocytes express many cell-cycle and DNA replication genes, as well as integrins and extracellular matrix proteins. These results define innate, architecture-specific, and cell-type-regulated genes in epidermis.

  6. Ryanodine receptors are expressed in epidermal keratinocytes and associated with keratinocyte differentiation and epidermal permeability barrier homeostasis.

    PubMed

    Denda, Sumiko; Kumamoto, Junichi; Takei, Kentaro; Tsutsumi, Moe; Aoki, Hirofumi; Denda, Mitsuhiro

    2012-01-01

    Ryanodine receptors (RyRs) have an important role as calcium channels in the regulation of intracellular calcium levels in the nervous system and muscle. In the present study, we investigated the expression of RyR in human epidermis. Immunohistochemical studies and reverse transcription-PCR indicated the expression of RyR type 1, 2, and 3 proteins in epidermal keratinocytes. The expression level of each RyR subtype was higher in differentiating keratinocytes than in proliferative cells. We also demonstrated the functional expression of RyR by calcium imaging. In cultured human keratinocytes, application of the RyR agonist 4-chloro-m-cresol (CMC) induced elevation of the intracellular calcium concentration, and co-application of the RyR antagonist 1,1'-diheptyl-4,4'-bipyridinium dibromide (DHBP) blocked the elevation. Application of CMC accelerated keratinocyte differentiation in vitro. On the other hand, topical application of CMC after tape-stripping of hairless mouse skin delayed barrier recovery, whereas application of an RyR antagonist, dantrolene or DHBP, accelerated the barrier recovery. These results suggest that RyR expressed in epidermal keratinocytes is associated with both differentiation of keratinocytes and epidermal barrier homeostasis.

  7. Expression and modulation of IL-1 alpha in murine keratinocytes

    SciTech Connect

    Ansel, J.C.; Luger, T.A.; Lowry, D.; Perry, P.; Roop, D.R.; Mountz, J.D.

    1988-04-01

    Murine and human keratinocytes produce an IL-1-like factor that appears to be similar if not identical to monocyte-derived IL-1. IL-1 may be an important mediator in cutaneous inflammatory responses, however, little is currently known concerning factors that may modulate IL-1 expression in keratinocytes. To address this issue we examined the effect of LPS, UV, and the cell differentiation state on murine keratinocyte IL-1 mRNA expression. Our results indicated that as with the murine P388D1 monocyte cell line, PAM 212 keratinocytes constitutively express abundant amounts of IL-1 alpha mRNA. On exposure to LPS (100 micrograms/ml) for 8 h there was more than 10 times the increase in PAM 212 IL-1 alpha mRNA which was accompanied by a sixfold increase in supernatant IL-1 activity. Similarly UV irradiation had a significant effect on keratinocyte IL-1 alpha expression. High dose UV (300 mJ/cm2) inhibited PAM 212 IL-1 alpha expression at 4, 8, 24, 48 h post-UV whereas a lower dose of UV (100 mJ/cm2) inhibited UV at 4 and 8 h post-UV, but induced IL-1 expression at 24 and 48 h post-UV. The expression of IL-1 alpha varied with the differentiation state of the keratinocytes. Freshly removed newborn murine keratinocytes were found to constitutively express IL-1 alpha mRNA. Keratinocytes grown in low (Ca2+) tissue culture media (0.05 mM) for 6 days, functionally and phenotypically become undifferentiated and express increased quantities of IL-1 alpha mRNA, whereas cells grown in high (Ca2+) media (1.2 mM) for 6 days become terminally differentiated and IL-1 expression ceased. Keratinocytes cultured for 3 days in low (Ca2+) conditions expressed an intermediate level of IL-1 alpha. In contrast, little or no IL-1 beta mRNA was detected in either the PAM 212 cells or newborn murine keratinocytes.

  8. AMPK regulation of the growth of cultured human keratinocytes

    SciTech Connect

    Saha, Asish K. . E-mail: aksaha@bu.edu; Persons, Kelly; Safer, Joshua D.; Luo Zhijun; Holick, Michael F.; Ruderman, Neil B.

    2006-10-20

    AMP kinase (AMPK) is a fuel sensing enzyme that responds to cellular energy depletion by increasing processes that generate ATP and inhibiting others that require ATP but are not acutely necessary for survival. In the present study, we examined the relationship between AMPK activation and the growth (proliferation) of cultured human keratinocytes and assessed whether the inhibition of keratinocyte growth by vitamin D involves AMPK activation. In addition, we explored whether the inhibition of keratinocyte proliferation as they approach confluence could be AMPK-related. Keratinocytes were incubated for 12 h with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside (AICAR). At concentrations of 10{sup -4} and 10{sup -3} M, AICAR inhibited keratinocyte growth by 50% and 95%, respectively, based on measurements of thymidine incorporation into DNA. It also increased AMPK and acetyl CoA carboxylase phosphorylation (P-AMPK and P-ACC) and decreased the concentration of malonyl CoA confirming that AMPK activation had occurred. Incubation with the thiazolidinedione, troglitazone (10{sup -6} M) caused similar alterations in P-AMPK, P-ACC, and cell growth. In contrast, the well known inhibition of keratinocyte growth by 1,25-dihydroxyvitamin D{sub 3} (10{sup -7} and 10{sup -6} M) was not associated with changes in P-AMPK or P-ACC. Like most cells, the growth of keratinocytes diminished as they approached confluence. Thus, it was of note that we found a progressive increase in P-AMPK (1.5- to 2-fold, p < 0.05) as keratinocytes grown in control medium went from 25% to 100% confluence. In conclusion, the data are consistent with the hypothesis that activation of AMPK acts as a signal to diminish the proliferation of cultured keratinocytes as they approach confluence. They also suggest that AMPK activators, such as AICAR and troglitazone, inhibit keratinocyte growth and that the inhibition of cell growth by 1,25-dihydroxyvitamin D{sub 3} is AMPK-independent.

  9. Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin.

    PubMed Central

    Clarke, J F; Young, P W; Yonezawa, K; Kasuga, M; Holman, G D

    1994-01-01

    Wortmannin is a potent and reversible inhibitor of insulin-stimulated PtdIns 3-kinase activity in 3T3-L1 cells (IC50 = 2.6 +/- 0.8 nM). Wortmannin inhibits the PtdIns 3-kinase activity which is precipitated with antibodies against insulin receptor substrate 1 and against the alpha-p85 subunit of PtdIns 3-kinase. These observations suggest that wortmannin inhibits at the p110 catalytic subunit of PtdIns 3-kinase. Insulin stimulation of glucose transport in permeabilized 3T3-L1 cells is also inhibited by wortmannin (IC50 = 6.4 +/- 1.4 nM). Wortmannin did not inhibit basal glucose transport activity. The close similarity of the IC50 values for wortmannin inhibition of insulin-stimulated PtdIns 3-kinase and glucose transport activities suggests that the PtdIns 3-kinase is a key intermediate in insulin signalling of glucose-transport stimulation. The wortmannin inhibitory effect on transport is associated with a reduction in the cell-surface, but not the total cellular, levels of both GLUT1 and GLUT4 glucose transporter isoforms that are accessible to the cell-impermeant photolabel, ATB-BMPA. These photolabelling results suggest that the glucose transporter translocation process is dependent upon PtdIns 3-kinase activity. The stimulatory effect of guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) on glucose transport activity in permeabilized cells is only partially blocked by concentrations of wortmannin that completely inhibit the stimulatory effect of insulin. The residual stimulatory effect of GTP gamma S that occurs in the presence of wortmannin suggests that at least part of the GTP gamma S effect is mediated at a signalling site that is downstream of the site at which wortmannin inhibits the insulin stimulation of PtdIns 3-kinase and glucose transport activities. PMID:8010944

  10. Anti-inflammatory activity of compounds isolated from Astragalus sinicus L. in cytokine-induced keratinocytes and skin

    PubMed Central

    Kim, Byung-Hak; Oh, Ikhoon; Kim, Jung-Ho; Jeon, Ju-eun; Jeon, Byeongwook; Shin, Jongheon; Kim, Tae-Yoon

    2014-01-01

    Inflammation is a part of the complex biological responses of a tissue to injury that protect the organ by removing injurious stimuli and initiating the healing process, and is considered as a mechanism of innate immunity. To identify biologically active compounds against pathogenic inflammatory and immune responses, we fractionated water, aqueous methanol and n-hexane layers from nine kinds of leguminosae and examined anti-inflammatory activity of the fractions in human keratinocytes and mouse skin. Among the fractions, rf3 and rf4, isolated from the aqueous methanol layer of Astragalus sinicus L., exhibited the strongest reactive oxygen species (ROS)-scavenging and anti-inflammatory activities as measured by inhibition of the intracellular ROS production, nuclear factor-kappaB (NF-κB), janus kinase (JAK)/signal transducer and activator of transcription (STAT), and phosphatidylinositol 3-kinase/Akt signaling in cytokine-stimulated human keratinocytes, as well as by effects on T-cell differentiation in mouse CD4+ T cells. In addition, topical application of rf3 and rf4 suppressed the progression of psoriasis-like dermatitis and expression of pro-inflammatory mediators in interleukin (IL)-23-injected mouse ears. Our results suggest that Astragalus sinicus L. may ameliorate chronic inflammatory skin diseases due to its antioxidant and anti-inflammatory activities via regulation of the intracellular ROS production, NF-κB, JAK/STAT and PI3/Akt signaling cascades as well as immune responses, and these results are the first report that Astragalus sinicus L. exhibits pharmacological activity. PMID:24651533

  11. Anti-inflammatory activity of compounds isolated from Astragalus sinicus L. in cytokine-induced keratinocytes and skin.

    PubMed

    Kim, Byung-Hak; Oh, Ikhoon; Kim, Jung-Ho; Jeon, Ju-Eun; Jeon, Byeongwook; Shin, Jongheon; Kim, Tae-Yoon

    2014-03-21

    Inflammation is a part of the complex biological responses of a tissue to injury that protect the organ by removing injurious stimuli and initiating the healing process, and is considered as a mechanism of innate immunity. To identify biologically active compounds against pathogenic inflammatory and immune responses, we fractionated water, aqueous methanol and n-hexane layers from nine kinds of leguminosae and examined anti-inflammatory activity of the fractions in human keratinocytes and mouse skin. Among the fractions, rf3 and rf4, isolated from the aqueous methanol layer of Astragalus sinicus L., exhibited the strongest reactive oxygen species (ROS)-scavenging and anti-inflammatory activities as measured by inhibition of the intracellular ROS production, nuclear factor-kappaB (NF-κB), janus kinase (JAK)/signal transducer and activator of transcription (STAT), and phosphatidylinositol 3-kinase/Akt signaling in cytokine-stimulated human keratinocytes, as well as by effects on T-cell differentiation in mouse CD4(+) T cells. In addition, topical application of rf3 and rf4 suppressed the progression of psoriasis-like dermatitis and expression of pro-inflammatory mediators in interleukin (IL)-23-injected mouse ears. Our results suggest that Astragalus sinicus L. may ameliorate chronic inflammatory skin diseases due to its antioxidant and anti-inflammatory activities via regulation of the intracellular ROS production, NF-κB, JAK/STAT and PI3/Akt signaling cascades as well as immune responses, and these results are the first report that Astragalus sinicus L. exhibits pharmacological activity.

  12. 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

  13. 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.

  14. Human Keratinocytes Radioprotection with Mentha Longifolia

    NASA Astrophysics Data System (ADS)

    Rizzo, Angela Maria; Berselli, P.; Zava, S.; Negroni, M.; Corsetto, P.; Montorfano, G.; Bertolotti, A.; Ranza, E.; Ottolenghi, A.; Berra, B.

    Antioxidants are suggested to act as radioprotectors, and dietary supplements based on antiox-idants have been proposed for astronauts involved in long-term space missions. Plant extracts with antioxidant properties may be used in dietetic supplements for astronauts; in fact recent nutritional guidelines suggest that "fruits and vegetables may become as important on space-going vessels as limes were on the sea-going vessels of old". Mint presents a large variety of biological properties, such as antiallergenic, antibacterial, anti-inflammatory, antitumor, an-tiviral, gastrointestinal protective, hepatoprotective, chemopreventive activities, most of which are attributable to its antioxidant activity. The aim of the present study is to evaluate the antioxidant properties and protective bio-efficacy of a phenol enriched Mentha longifolia ex-tract on gamma rays stressed human keratinocytes (NCTC2544). We assessed first the in vitro antioxidant activity (ABTS and DPPH), and then evaluated different stress markers in order to investigate various oxidative stress targets: cell viability (MTT); retained proliferating ca-pability (CA); DNA damage (histone H2AX) and protein damage (HSP70 induction). Results indicate that this Mint extract has a higher antioxidant activity respect to fresh extracts, that could be responsible of its really interesting radio-protective effects.

  15. Arachidonic acid metabolism in cultured mouse keratinocytes

    SciTech Connect

    Kondoh, H.; Sato, Y.; Kanoh, H.

    1985-07-01

    The authors attempted to characterize the general features of arachidonate metabolism in cultured mouse keratinocytes. The cells labeled with (/sup 3/H)arachidonate were stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), ionophore A23187, and fetal bovine serum (FBS). Common to the three substances, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine almost equally served as sources of arachidonate liberated by the action of phospholipase A2. The stimulation of phospholipase A2 action was observed in the order of A23187 greater than FBS greater than TPA. When stimulated by TPA or A23187, the radioactivity released into the extracellular medium was mostly found in prostaglandin (PG) E2. Formation of other PGs and hydroxyeicosatetraenoate (HETE) was extremely limited. In the case of stimulation by FBS, however, the released radioactivity was mainly associated with non-converted arachidonate. FBS also inhibited the TPA- and A23187-induced conversion of arachidonate to PGE2. Phospholipid degradation induced by the three stimulators was similarly dependent on extracellular Ca/sup 2 +/. The stimulation by FBS and A23187 was suppressed by calmodulin antagonists, though the effect of A23187 was much more sensitive to the antagonists when compared to that of FBS. The authors observed more than additive effects of the three stimulators when tested together.

  16. 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

  17. FHL-2 suppresses VEGF-induced phosphatidylinositol 3-kinase/Akt activation via interaction with sphingosine kinase-1.

    PubMed

    Hayashi, Hiroki; Nakagami, Hironori; Takami, Yoichi; Koriyama, Hiroshi; Mori, Masaki; Tamai, Katsuto; Sun, Jianxin; Nagao, Kaori; Morishita, Ryuichi; Kaneda, Yasufumi

    2009-06-01

    In the functional screening of a human heart cDNA library to identify a novel antiangiogenic factor, the prime candidate gene was "four-and-a-half LIM only protein-2" (FHL-2). The goal of this study is to clear the mechanism of antiangiogenic signaling of FHL-2 in endothelial cells (ECs). Overexpressed FHL-2 strongly inhibited vascular endothelial growth factor (VEGF)-induced EC migration. In the angiogenic signaling, we focused on sphingosine kinase-1 (SK1), which produces sphingosine-1-phosphate (S1P), a bioactive sphingolipid, as a potent angiogenic mediator in ECs. Immunoprecipitation and immunostaining analysis showed that FHL-2 might bind to SK1. Importantly, overexpression of FHL-2 in ECs inhibited VEGF-induced SK1 activity, phosphatidylinositol 3-kinase activity, and phosphorylation of Akt and eNOS. In contrast, overexpression of FHL-2 had no effect on S1P-induced Akt phosphorylation. Interestingly, VEGF stimulation decreased the binding of FHL-2 and SK1. Depletion of FHL-2 by siRNA increased EC migration accompanied with SK1 and Akt activation, and increased the expression of VEGF receptor-2 which further enhanced VEGF signaling. Furthermore, injection of FHL-2 mRNA into Xenopus embryos resulted in inhibition of vascular network development, assessed by in situ hybridization with endothelial markers. FHL-2 may regulate phosphatidylinositol 3-kinase/Akt via direct suppression of the SK1-S1P pathway in ECs.

  18. 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.

  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. 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

  1. Tec kinase signaling in T cells is regulated by phosphatidylinositol 3-kinase and the Tec pleckstrin homology domain.

    PubMed

    Yang, W C; Ching, K A; Tsoukas, C D; Berg, L J

    2001-01-01

    Tec, the prototypical member of the Tec family of tyrosine kinases, is abundantly expressed in T cells and other hemopoietic cell types. Although the functions of Itk and Txk have recently been investigated, little is known about the role of Tec in T cells. Using antisense oligonucleotide treatment to deplete Tec protein from primary T cells, we demonstrate that Tec plays a role in TCR signaling leading to IL-2 gene induction. Interestingly, Tec kinases are the only known family of tyrosine kinases containing a pleckstrin homology (PH) domain. Using several PH domain mutants overexpressed in Jurkat T cells, we show that the Tec PH domain is required for Tec-mediated IL-2 gene induction and TCR-mediated Tec tyrosine phosphorylation. Furthermore, we show that Tec colocalizes with the TCR after TCR cross-linking, and that both the Tec PH and Src homology (SH) 2 domains play a role in this association. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, abolishes Tec-mediated IL-2 gene induction and Tec tyrosine phosphorylation, and partially suppresses Tec colocalization with the activated TCR. Thus, our data implicate the Tec kinase PH domain and phosphatidylinositol 3-kinase in Tec signaling downstream of the TCR.

  2. 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.

  3. Stabilization of Quercetin Paradoxically Reduces Its Proapoptotic Effect on UVB-Irradiated Human Keratinocytes

    PubMed Central

    Olson, Erik R.; Melton, Tania; Dong, Zigang; Bowden, G. Tim

    2008-01-01

    UVB light promotes survival of initiated keratinocytes, in part, by the direct activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Novel chemopreventative agents targeting UVB-induced signaling pathways are needed to reduce the incidence of nonmelanoma skin cancer. Quercetin (Qu) is a dietary flavonoid and a known inhibitor of PI3K. We determined that Qu degrades rapidly when diluted in DMEM and incubated under normal cell culture conditions. Degradation was delayed by supplementing the medium with 1 mmol/L ascorbic acid (AA), and as expected, stabilization actually increased the effectiveness of Qu as a PI3K inhibitor because basal and UVB-induced Akt phosphorylation were reduced compared with Qu treatment in the absence of AA. Although AA stabilization increased Qu-induced apoptosis in mock-irradiated HaCaT cells, consistent with it acting as a PI3K inhibitor (13.4% Annexin V–positive cells for AA-stabilized Qu versus 6.3% for Qu), AA stabilization of Qu actually reduced the ability of the compound to induce apoptosis of UVB-irradiated HaCaTs (29.7% of Qu-treated cells versus 15.5% of AA + Qu–treated cells). Similar trends were seen in the analysis of caspase-3 and poly(ADP-ribose) polymerase cleavage. Qu is known to oxidize to form reactive products, and we found that dihydroethidium is oxidized by Qu regardless of whether or not it was stabilized. Although redox cycling occurs even in the presence of AA, stabilization reduces the accumulation of reactive Qu products that contribute to the proapoptotic effect of the compound, and thus reduces the ability of the compound to induce apoptosis of UVB-irradiated HaCaT cells. PMID:19138980

  4. Kinetics of growth and differentiation of cultured human epidermal keratinocytes

    SciTech Connect

    Albers, K.M.

    1985-01-01

    A study was made of the interrelationship between replication and differentiation in cultures of human epidermal keratinocytes. Measures of both parameters were made using newly developed methods to quantify the rate at which keratinocytes replicate and the rate at which they withdraw from the cell cycle. Keratinocyte replication was measured by determining the cell doubling time, labeling index, and cell cycle duration. Cell cycle length was measured using a double label assay that determines the length of time between two successive phases of DNA synthesis. The first DNA synthesis phase was marked by labeling keratinocytes with /sup 14/C-thymidine. At the next round of DNA synthesis, cells were labeled with bromodeoxyuridine, a heavy analog of thymidine. The cell cycle length is given by the time required for the /sup 14/C-labeled DNA to become double labeled. To measure keratinocyte differentiation, the rate at which cells withdraw from the cell cycle was determined. To measure withdrawal, the percentage of cells labeled by a pulse of /sup 14/C-thymidine that failed to undergo a second cycle of DNA synthesis, as measured by bromodeoxyuridine incorporation, was determined. Cells which failed to undergo a second cycle of synthesis were considered to have differentiated and withdrawn from the cell cycle.

  5. Chemosensory Information Processing between Keratinocytes and Trigeminal Neurons

    PubMed Central

    Sondersorg, Anna Christina; Busse, Daniela; Kyereme, Jessica; Rothermel, Markus; Neufang, Gitta; Gisselmann, Günter; Hatt, Hanns; Conrad, Heike

    2014-01-01

    Trigeminal fibers terminate within the facial mucosa and skin and transmit tactile, proprioceptive, chemical, and nociceptive sensations. Trigeminal sensations can arise from the direct stimulation of intraepithelial free nerve endings or indirectly through information transmission from adjacent cells at the peripheral innervation area. For mechanical and thermal cues, communication processes between skin cells and somatosensory neurons have already been suggested. High concentrations of most odors typically provoke trigeminal sensations in vivo but surprisingly fail to activate trigeminal neuron monocultures. This fact favors the hypothesis that epithelial cells may participate in chemodetection and subsequently transmit signals to neighboring trigeminal fibers. Keratinocytes, the major cell type of the epidermis, express various receptors that enable reactions to multiple environmental stimuli. Here, using a co-culture approach, we show for the first time that exposure to the odorant chemicals induces a chemical communication between human HaCaT keratinocytes and mouse trigeminal neurons. Moreover, a supernatant analysis of stimulated keratinocytes and subsequent blocking experiments with pyrodoxalphosphate-6-azophenyl-2′,4′-disulfonate revealed that ATP serves as the mediating transmitter molecule released from skin cells after odor stimulation. We show that the ATP release resulting from Javanol® stimulation of keratinocytes was mediated by pannexins. Consequently, keratinocytes act as chemosensors linking the environment and the trigeminal system via ATP signaling. PMID:24790106

  6. Proliferating cell nuclear antigen/cyclin in cultured human keratinocytes.

    PubMed

    Okada, N; Miyagawa, S; Steinberg, M L; Yoshikawa, K

    1990-09-01

    Expression of proliferating cell nuclear antigen (PCNA)/cyclin in cultured human keratinocytes was studied using an antibody from an SLE patient as the reagent. By indirect immunofluorescence staining, SV40-transformed human keratinocytes expressed PCNA/cyclin in 40-45% of the cells as a nulcear granular fluorescence. After synchronization of these cells, their nuclear distribution pattern during the S phase was sequential and showed a clear correlation with DNA synthesis. Primary cultured keratinocytes grown in high Ca+ medium expressed PCNA/cyclin in 10-15% of the cells with a similar staining pattern. These positively stained cells were confined to the basal and immediate suprabasal layers of the stratified culture sheet. The keratinocytes disaggregated by trypsin were separated according to cell size through a screen of Nitex monofilament cloth. The cells smaller than 15 microns in diameter synthesized abundant PCNA/cyclin, while the larger cells expressed very low levels. These results indicate that the expression of PCNA/cyclin correlates with DNA synthesis in cultured keratinocytes, but is not associated with their differentiation process.

  7. Polymeric membranes modulate human keratinocyte differentiation in specific epidermal layers.

    PubMed

    Salerno, Simona; Morelli, Sabrina; Giordano, Francesca; Gordano, Amalia; Bartolo, Loredana De

    2016-10-01

    In vitro models of human bioengineered skin substitutes are an alternative to animal experimentation for testing the effects and toxicity of drugs, cosmetics and pollutants. For the first time specific and distinct human epidermal strata were engineered by using membranes and keratinocytes. To this purpose, biodegradable membranes of chitosan (CHT), polycaprolactone (PCL) and a polymeric blend of CHT-PCL were prepared by phase-inversion technique and characterized in order to evaluate their morphological, physico-chemical and mechanical properties. The capability of membranes to modulate keratinocyte differentiation inducing specific interactions in epidermal membrane systems was investigated. The overall results demonstrated that the membrane properties strongly influence the cell morpho-functional behaviour of human keratinocytes, modulating their terminal differentiation, with the creation of specific epidermal strata or a fully proliferative epidermal multilayer system. In particular, human keratinocytes adhered on CHT and CHT-PCL membranes, forming the structure of the epidermal top layers, such as the corneum and granulosum strata, characterized by withdrawal or reduction from the cell cycle and cell proliferation. On the PCL membrane, keratinocytes developed an epidermal basal lamina, with high proliferating cells that stratified and migrated over time to form a complete differentiating epidermal multilayer system.

  8. Wound re-epithelialization: modulating keratinocyte migration in wound healing.

    PubMed

    Raja; Sivamani, K; Garcia, Miki Shirakawa; Isseroff, R Rivkah

    2007-05-01

    An essential feature of a healed wound is the restoration of an intact epidermal barrier through wound epithelialization, also known as re-epithelialization. The directed migration of keratinocytes is critical to wound epithelialization and defects in this function are associated with the clinical phenotype of chronic non-healing wounds. A complex balance of signaling factors and surface proteins are expressed and regulated in a temporospatial manner that promote keratinocyte motility and survival to activate wound re-epithelialization. The majority of this review focuses on the mechanisms that regulate keratinocyte migration in the re-epithelialization process. This includes a review of cell attachments via desmosomes, hemidesmosomes, and integrins, the expression of keratins, the role of growth factors, cytokines and chemokines, eicosanoids, oxygen tension, antimicrobial peptides, and matrix metalloproteinases. Also reviewed are recently emerging novel mediators of keratinocyte motility including the role of electric fields, and signaling via the acetylcholine and beta-adrenergic receptors. These multiple regulators impact the ability of keratinocytes to migrate from the wound edge or other epidermal reservoirs to efficiently re-epithelialize a breach in the integrity of the epidermis. New discoveries will continue to uncover the elegant network of events that result in restoration of epidermal integrity and complete the wound repair process.

  9. Dynamic Transcriptional and Epigenetic Regulation of Human Epidermal Keratinocyte Differentiation

    PubMed Central

    Cavazza, Alessia; Miccio, Annarita; Romano, Oriana; Petiti, Luca; Malagoli Tagliazucchi, Guidantonio; Peano, Clelia; Severgnini, Marco; Rizzi, Ermanno; De Bellis, Gianluca; Bicciato, Silvio; Mavilio, Fulvio

    2016-01-01

    Summary Human skin is maintained by the differentiation and maturation of interfollicular stem and progenitors cells. We used DeepCAGE, genome-wide profiling of histone modifications and retroviral integration analysis, to map transcripts, promoters, enhancers, and super-enhancers (SEs) in prospectively isolated keratinocytes and transit-amplifying progenitors, and retrospectively defined keratinocyte stem cells. We show that >95% of the active promoters are in common and differentially regulated in progenitors and differentiated keratinocytes, while approximately half of the enhancers and SEs are stage specific and account for most of the epigenetic changes occurring during differentiation. Transcription factor (TF) motif identification and correlation with TF binding site maps allowed the identification of TF circuitries acting on enhancers and SEs during differentiation. Overall, our study provides a broad, genome-wide description of chromatin dynamics and differential enhancer and promoter usage during epithelial differentiation, and describes a novel approach to identify active regulatory elements in rare stem cell populations. PMID:27050947

  10. Optimal differentiation of in vitro keratinocytes requires multifactorial external control.

    PubMed

    Borowiec, Anne-Sophie; Delcourt, Philippe; Dewailly, Etienne; Bidaux, Gabriel

    2013-01-01

    For almost 30 years, keratinocyte differentiation has been studied in numerous cell models including keratinocyte primary culture with various supplemented culture media. In this respect, it has become quite difficult to draw comparisons between studies using such a variety of culture conditions. Serum-free condition with low calcium has been used to culture basal proliferating cells, though differentiation is induced by various procedures. These latter include the addition of calcium at mM concentration and a concomitant addition of serum and calcium. Lowering the incubation temperature of cells has also been reported to induce a premature differentiation of keratinocytes in organotypic skin culture. This effect of temperature on keratinocyte differentiation has been poorly depicted, although average human skin temperature has been shown to be about 32 °C. However, studying differentiation and quantifying shifts in the differentiation rate of a cell population implies to precisely know i) the proportion of differentiated cells in the whole population, and ii) to which extent and to which level of expression, the induction of a gene or a protein might be considered as a marker of differentiation. This lack has rarely been taken into consideration and has surely led to over-interpretations of single protein induction and to consequent extrapolations to real differentiation processes. By means of paralleled analyses with immunocytofluorescence, flow cytometry, and with multiple differentiation markers quantify by qPCR and western-blot, we studied the paradoxical connection between calcium, serum, multilayer culture and incubation temperature on the differentiation of in vitro keratinocytes. Conversely to previous reports, we have shown that calcium switch is indeed a potent model for inducing calcium-dependent genes, but is not an efficient procedure when one wishes to assess the keratinocyte differentiation rate. Moreover, we have demonstrated that a synergic

  11. Hyaluronan Does Not Regulate Human Epidermal Keratinocyte Proliferation and Differentiation.

    PubMed

    Malaisse, Jérémy; Pendaries, Valérie; Hontoir, Fanny; De Glas, Valérie; Van Vlaender, Daniel; Simon, Michel; Lambert de Rouvroit, Catherine; Poumay, Yves; Flamion, Bruno

    2016-03-18

    Hyaluronan (HA) is synthesized by three HA synthases (HAS1, HAS2, and HAS3) and secreted in the extracellular matrix. In human skin, large amounts of HA are found in the dermis. HA is also synthesized by keratinocytes in the epidermis, although its epidermal functions are not clearly identified yet. To investigate HA functions, we studied the effects of HA depletion on human keratinocyte physiology within in vitro reconstructed human epidermis. Inhibition of HA synthesis with 4-methylumbelliferone (4MU) did not modify the expression profile of the epidermal differentiation markers involucrin, keratin 10, and filaggrin during tissue reconstruction. In contrast, when keratinocytes were incubated with 4MU, cell proliferation was decreased. In an attempt to rescue the proliferation function, HA samples of various mean molecular masses were added to keratinocyte cultures treated with 4MU. These samples were unable to rescue the initial proliferation rate. Furthermore, treatments with HA-specific hyaluronidase, although removing almost all HA from keratinocyte cultures, did not alter the differentiation or proliferation processes. The differences between 4MU and hyaluronidase effects did not result from differences in intracellular HA, sulfated glycosaminoglycan concentration, apoptosis, or levels of HA receptors, all of which remained unchanged. Similarly, knockdown of UDP-glucose 6-dehydrogenase (UGDH) using lentiviral shRNA effectively decreased HA production but did not affect proliferation rate. Overall, these data suggest that HA levels in the human epidermis are not directly correlated with keratinocyte proliferation and differentiation and that incubation of cells with 4MU cannot equate with HA removal. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Twist1 regulates keratinocyte proliferation and skin tumor promotion.

    PubMed

    Srivastava, Jaya; Rho, Okkyung; Youssef, Ronnie M; DiGiovanni, John

    2016-05-01

    In the present study, we evaluated the effect of deleting Twist1 on keratinocyte proliferation and on skin tumor development using the two-stage chemical carcinogenesis model. BK5.Cre × Twist1(flox/flox) mice, which have a keratinocyte-specific Twist1 knockout (Twist1 KO), developed significantly reduced numbers of papilloma (70% reduction) and squamous cell carcinoma (75% reduction) as well as delayed tumor latency compared to wild-type (WT) mice. Interestingly, knockdown of Twist1 in primary keratinocytes impeded cell cycle progression at the G1/S transition that coincided with reduced levels of the cell cycle proteins c-Myc, Cyclin E1, and E2F1 and increased levels of p53 and p21. Furthermore, ChIP analyses revealed that Twist1 bound to the promoter regions of Cyclin E1, E2F1, and c-Myc at the canonical E-box binding motif suggesting a direct transcriptional regulation. Further analyses of Twist1 KO mice revealed a significant reduction in the number of label-retaining cells as well as the number of α6-integrin(+) /CD34(+) cells in the hair follicles of untreated mice compared to WT mice. These mice also exhibited significantly reduced epidermal proliferation in response to TPA treatment that again correlated with reduced levels of cell cycle regulators and increased levels of p53 and p21. Finally, Twist1 deficiency in keratinocytes led to an upregulation of p53 via its stabilization and nuclear localization, which is responsible for the increased expression of p21 in these cells. Collectively, these findings indicate that Twist1 has a novel role in epithelial carcinogenesis by regulating proliferation of keratinocytes, including keratinocyte stem cells during tumor promotion. © 2015 Wiley Periodicals, Inc.

  13. Hyaluronan Does Not Regulate Human Epidermal Keratinocyte Proliferation and Differentiation*

    PubMed Central

    Malaisse, Jérémy; Pendaries, Valérie; Hontoir, Fanny; De Glas, Valérie; Van Vlaender, Daniel; Simon, Michel; Lambert de Rouvroit, Catherine; Poumay, Yves; Flamion, Bruno

    2016-01-01

    Hyaluronan (HA) is synthesized by three HA synthases (HAS1, HAS2, and HAS3) and secreted in the extracellular matrix. In human skin, large amounts of HA are found in the dermis. HA is also synthesized by keratinocytes in the epidermis, although its epidermal functions are not clearly identified yet. To investigate HA functions, we studied the effects of HA depletion on human keratinocyte physiology within in vitro reconstructed human epidermis. Inhibition of HA synthesis with 4-methylumbelliferone (4MU) did not modify the expression profile of the epidermal differentiation markers involucrin, keratin 10, and filaggrin during tissue reconstruction. In contrast, when keratinocytes were incubated with 4MU, cell proliferation was decreased. In an attempt to rescue the proliferation function, HA samples of various mean molecular masses were added to keratinocyte cultures treated with 4MU. These samples were unable to rescue the initial proliferation rate. Furthermore, treatments with HA-specific hyaluronidase, although removing almost all HA from keratinocyte cultures, did not alter the differentiation or proliferation processes. The differences between 4MU and hyaluronidase effects did not result from differences in intracellular HA, sulfated glycosaminoglycan concentration, apoptosis, or levels of HA receptors, all of which remained unchanged. Similarly, knockdown of UDP-glucose 6-dehydrogenase (UGDH) using lentiviral shRNA effectively decreased HA production but did not affect proliferation rate. Overall, these data suggest that HA levels in the human epidermis are not directly correlated with keratinocyte proliferation and differentiation and that incubation of cells with 4MU cannot equate with HA removal. PMID:26627828

  14. Analysis of Global Sumoylation Changes Occurring during Keratinocyte Differentiation

    PubMed Central

    Heaton, Phillip R.; Santos, Andres; Rosas-Acosta, Germán; Wilson, Van G.

    2012-01-01

    Sumoylation is a highly dynamic process that plays a role in a multitude of processes ranging from cell cycle progression to mRNA processing and cancer. A previous study from our lab demonstrated that SUMO plays an important role in keratinocyte differentiation. Here we present a new method of tracking the sumoylation state of proteins by creating a stably transfected HaCaT keratinocyte cell line expressing an inducible SNAP-SUMO3 protein. The SNAP-tag allows covalent fluorescent labeling that is denaturation resistant. When combined with two-dimensional gel electrophoresis, the SNAP-tag technology provides direct visualization of sumoylated targets and can be used to follow temporal changes in the global cohort of sumoylated proteins during dynamic processes such as differentiation. HaCaT keratinocyte cells expressing SNAP-SUMO3 displayed normal morphological and biochemical features that are consistent with typical keratinocyte differentiation. SNAP-SUMO3 also localized normally in these cells with a predominantly nuclear signal and some minor cytoplasmic staining, consistent with previous reports for untagged SUMO2/3. During keratinocyte differentiation the total number of proteins modified by SNAP-SUMO3 was highest in basal cells, decreased abruptly after induction of differentiation, and slowly rebounded beginning between 48 and 72 hours as differentiation progressed. However, within this overall trend the pattern of change for individual sumoylated proteins was highly variable with both increases and decreases in amount over time. From these results we conclude that sumoylation of proteins during keratinocyte differentiation is a complex process which likely reflects and contributes to the biochemical changes that drive differentiation. PMID:22291911

  15. The Role of Focal Adhesion Kinase in Keratinocyte Fibrogenic Gene Expression

    PubMed Central

    Januszyk, Michael; Kwon, Sun Hyung; Wong, Victor W.; Padmanabhan, Jagannath; Maan, Zeshaan N.; Whittam, Alexander J.; Major, Melanie R.; Gurtner, Geoffrey C.

    2017-01-01

    Abnormal skin scarring causes functional impairment, psychological stress, and high socioeconomic cost. Evidence shows that altered mechanotransduction pathways have been linked to both inflammation and fibrosis, and that focal adhesion kinase (FAK) is a key mediator of these processes. We investigated the importance of keratinocyte FAK at the single cell level in key fibrogenic pathways critical for scar formation. Keratinocytes were isolated from wildtype and keratinocyte-specific FAK-deleted mice, cultured, and sorted into single cells. Keratinocytes were evaluated using a microfluidic-based platform for high-resolution transcriptional analysis. Partitive clustering, gene enrichment analysis, and network modeling were applied to characterize the significance of FAK on regulating keratinocyte subpopulations and fibrogenic pathways important for scar formation. Considerable transcriptional heterogeneity was observed within the keratinocyte populations. FAK-deleted keratinocytes demonstrated increased expression of genes integral to mechanotransduction and extracellular matrix production, including Igtbl, Mmpla, and Col4a1. Transcriptional activities upon FAK deletion were not identical across all single keratinocytes, resulting in higher frequency of a minor subpopulation characterized by a matrix-remodeling profile compared to wildtype keratinocyte population. The importance of keratinocyte FAK signaling gene expression was revealed. A minor subpopulation of keratinocytes characterized by a matrix-modulating profile may be a keratinocyte subset important for mechanotransduction and scar formation. PMID:28880199

  16. 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.

  17. Identification of a Potent Phosphoinositide 3-Kinase Pan Inhibitor Displaying a Strategic Carboxylic Acid Group and Development of Its Prodrugs.

    PubMed

    Pirali, Tracey; Ciraolo, Elisa; Aprile, Silvio; Massarotti, Alberto; Berndt, Alex; Griglio, Alessia; Serafini, Marta; Mercalli, Valentina; Landoni, Clarissa; Campa, Carlo Cosimo; Margaria, Jean Piero; Silva, Rangel L; Grosa, Giorgio; Sorba, Giovanni; Williams, Roger; Hirsch, Emilio; Tron, Gian Cesare

    2017-09-21

    Activation of the phosphoinositide 3-kinase (PI3K) pathway is a key signaling event in cancer, inflammation, and other proliferative diseases. PI3K inhibitors are already approved for some specific clinical indications, but their systemic on-target toxicity limits their larger use. In particular, whereas toxicity is tolerable in acute treatment of life-threatening diseases, this is less acceptable in chronic conditions. In the past, the strategy to overcome this drawback was to block selected isoforms mainly expressed in leukocytes, but redundancy within the PI3K family members challenges the effectiveness of this approach. On the other hand, decreasing exposure to selected target cells represents a so-far unexplored alternative to circumvent systemic toxicity. In this manuscript, we describe the generation of a library of triazolylquinolones and the development of the first prodrug pan-PI3K inhibitor. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. 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.

  19. Novel domains in NADPH oxidase subunits, sorting nexins, and PtdIns 3-kinases: binding partners of SH3 domains?

    PubMed Central

    Ponting, C. P.

    1996-01-01

    Two SH3 domain-containing cytosolic components of the NADPH oxidase, p47phox and p40phox, are shown by analyses of their sequences to contain single copies of a novel class of domain, the PX (phox) domain. Homologous domains are demonstrated to be present in the Cpk class of phosphatidylinositol 3-kinase, S. cerevisiae Bem1p, and S. pombe Scd2, and a large family of human sorting nexin 1 (SNX1) homologues. The majority of these domains contains a polyproline motif, typical of SH3 domain-binding proteins. Two further findings are reported. A third NADPH oxidase subunit, p67phox, is shown to contain four tetratricopeptide repeats (TPRs) within its N-terminal RaclGTP-binding region, and a 28 residue motif in p40phox is demonstrated to be present in protein kinase C isoforms iota/lambda and zeta, and in three ZZ domain-containing proteins. PMID:8931154

  20. 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

  1. 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

  2. Clinical spectrum and features of activated phosphoinositide 3-kinase δ syndrome: A large patient cohort study.

    PubMed

    Coulter, Tanya I; Chandra, Anita; Bacon, Chris M; Babar, Judith; Curtis, James; Screaton, Nick; Goodlad, John R; Farmer, George; Steele, Cathal Laurence; Leahy, Timothy Ronan; Doffinger, Rainer; Baxendale, Helen; Bernatoniene, Jolanta; Edgar, J David M; Longhurst, Hilary J; Ehl, Stephan; Speckmann, Carsten; Grimbacher, Bodo; Sediva, Anna; Milota, Tomas; Faust, Saul N; Williams, Anthony P; Hayman, Grant; Kucuk, Zeynep Yesim; Hague, Rosie; French, Paul; Brooker, Richard; Forsyth, Peter; Herriot, Richard; Cancrini, Caterina; Palma, Paolo; Ariganello, Paola; Conlon, Niall; Feighery, Conleth; Gavin, Patrick J; Jones, Alison; Imai, Kohsuke; Ibrahim, Mohammad A A; Markelj, Gašper; Abinun, Mario; Rieux-Laucat, Frédéric; Latour, Sylvain; Pellier, Isabelle; Fischer, Alain; Touzot, Fabien; Casanova, Jean-Laurent; Durandy, Anne; Burns, Siobhan O; Savic, Sinisa; Kumararatne, D S; Moshous, Despina; Kracker, Sven; Vanhaesebroeck, Bart; Okkenhaug, Klaus; Picard, Capucine; Nejentsev, Sergey; Condliffe, Alison M; Cant, Andrew James

    2017-02-01

    Activated phosphoinositide 3-kinase δ syndrome (APDS) is a recently described combined immunodeficiency resulting from gain-of-function mutations in PIK3CD, the gene encoding the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ). We sought to review the clinical, immunologic, histopathologic, and radiologic features of APDS in a large genetically defined international cohort. We applied a clinical questionnaire and performed review of medical notes, radiology, histopathology, and laboratory investigations of 53 patients with APDS. Recurrent sinopulmonary infections (98%) and nonneoplastic lymphoproliferation (75%) were common, often from childhood. Other significant complications included herpesvirus infections (49%), autoinflammatory disease (34%), and lymphoma (13%). Unexpectedly, neurodevelopmental delay occurred in 19% of the cohort, suggesting a role for PI3Kδ in the central nervous system; consistent with this, PI3Kδ is broadly expressed in the developing murine central nervous system. Thoracic imaging revealed high rates of mosaic attenuation (90%) and bronchiectasis (60%). Increased IgM levels (78%), IgG deficiency (43%), and CD4 lymphopenia (84%) were significant immunologic features. No immunologic marker reliably predicted clinical severity, which ranged from asymptomatic to death in early childhood. The majority of patients received immunoglobulin replacement and antibiotic prophylaxis, and 5 patients underwent hematopoietic stem cell transplantation. Five patients died from complications of APDS. APDS is a combined immunodeficiency with multiple clinical manifestations, many with incomplete penetrance and others with variable expressivity. The severity of complications in some patients supports consideration of hematopoietic stem cell transplantation for severe childhood disease. Clinical trials of selective PI3Kδ inhibitors offer new prospects for APDS treatment. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights

  3. A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1

    PubMed Central

    Ozes, Osman Nidai; Akca, Hakan; Mayo, Lindsey D.; Gustin, Jason A.; Maehama, Tomohiko; Dixon, Jack E.; Donner, David B.

    2001-01-01

    Tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) by the insulin receptor permits this docking protein to interact with signaling proteins that promote insulin action. Serine phosphorylation uncouples IRS-1 from the insulin receptor, thereby inhibiting its tyrosine phosphorylation and insulin signaling. For this reason, there is great interest in identifying serine/threonine kinases for which IRS-1 is a substrate. Tumor necrosis factor (TNF) inhibited insulin-promoted tyrosine phosphorylation of IRS-1 and activated the Akt/protein kinase B serine-threonine kinase, a downstream target for phosphatidylinositol 3-kinase (PI 3-kinase). The effect of TNF on insulin-promoted tyrosine phosphorylation of IRS-1 was blocked by inhibition of PI 3-kinase and the PTEN tumor suppessor, which dephosphorylates the lipids that mediate PI 3-kinase functions, whereas constitutively active Akt impaired insulin-promoted IRS-1 tyrosine phosphorylation. Conversely, TNF inhibition of IRS-1 tyrosine phosphorylation was blocked by kinase dead Akt. Inhibition of IRS-1 tyrosine phosphorylation by TNF was blocked by rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a downstream target of Akt. mTOR induced the serine phosphorylation of IRS-1 (Ser-636/639), and such phosphorylation was inhibited by rapamycin. These results suggest that TNF impairs insulin signaling through IRS-1 by activation of a PI 3-kinase/Akt/mTOR pathway, which is antagonized by PTEN. PMID:11287630

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. Effect of nifedipine on the expression of keratinocyte growth factor and its receptor in cocultured/monocultured fibroblasts and keratinocytes.

    PubMed

    Di, C-P; Sun, Y; Zhao, L; Li, L; Ding, C; Xu, Y; Fan, Y

    2013-12-01

    Keratinocyte growth factor (KGF) and its receptor (KGFR) are involved in hyperplastic diseases. This study explored the effect of intercellular communication on KGF and KGFR in cocultured/monocultured gingival fibroblasts and keratinocytes following treatment with nifedipine. Human gingival fibroblasts and keratinocytes were monocultured and cocultured, respectively. MTT was used to investigate the effects of nifedipine on the proliferation of gingival fibroblasts and keratinocytes. Monoculture and coculture systems were treated with different concentrations (0, 0.2 or 20 μg/mL) of nifedipine, and the expression of KGF and KGFR mRNAs was examined by RT-PCR, whilst the secretion of KGF and the expression of KGFR on the membrane were analyzed using ELISA and flow cytometry, respectively. Nifedipine (0, 0.2 and 20 μg/mL) had no influence on cell proliferation within 3 d. KGF and KGFR mRNAs were up-regulated, but only in the cocultures. In coculture, the secretion of KGF was significantly increased by nifedipine, while it was only significantly up-regulated by 20 μg/mL of nifedipine in monoculture. Moreover, the level of KGFR protein in the membrane was significantly increased by 20 μg/mL of nifedipine in monocultures, while it was significantly down-regulated by 20 μg/mL of nifedipine in cocultures. The expression of KGF and KGFR are influenced by the interplay of gingival keratinocytes and fibroblasts. Epithelial keratinocytes and mesenchymal fibroblasts may interplay to dynamically regulate gene expression, which may have an effect on the gingival condition following treatment with nifedipine. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation

    NASA Technical Reports Server (NTRS)

    Leong, J.; Hughes-Fulford, M.; Rakhlin, N.; Habib, A.; Maclouf, J.; Goldyne, M. E.

    1996-01-01

    Epidermal expression of the two isoforms of the prostaglandin H-generating cyclooxygenase (COX-1 and COX-2) was evaluated both by immunohistochemistry performed on human and mouse skin biopsy sections and by Western blotting of protein extracts from cultured human neonatal foreskin keratinocytes. In normal human skin, COX-1 immunostaining is observed throughout the epidermis whereas COX-2 immunostaining increases in the more differentiated, suprabasilar keratinocytes. Basal cell carcinomas express little if any COX-1 or COX-2 immunostaining whereas both isozymes are strongly expressed in squamous cell carcinomas deriving from a more differentiated layer of the epidermis. In human keratinocyte cultures, raising the extracellular calcium concentration, a recognized stimulus for keratinocyte differentiation, leads to an increased expression of both COX-2 protein and mRNA; expression of COX-1 protein, however, shows no significant alteration in response to calcium. Because of a recent report that failed to show COX-2 in normal mouse epidermis, we also looked for COX-1 and COX-2 immunostaining in sections of normal and acetone-treated mouse skin. In agreement with a previous report, some COX-1, but no COX-2, immunostaining is seen in normal murine epidermis. However, following acetone treatment, there is a marked increase in COX-1 expression as well as the appearance of significant COX-2 immunostaining in the basal layer. These data suggest that in human epidermis as well as in human keratinocyte cultures, the expression of COX-2 occurs as a part of normal keratinocyte differentiation whereas in murine epidermis, its constitutive expression is absent, but inducible as previously published.

  10. Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation

    NASA Technical Reports Server (NTRS)

    Leong, J.; Hughes-Fulford, M.; Rakhlin, N.; Habib, A.; Maclouf, J.; Goldyne, M. E.

    1996-01-01

    Epidermal expression of the two isoforms of the prostaglandin H-generating cyclooxygenase (COX-1 and COX-2) was evaluated both by immunohistochemistry performed on human and mouse skin biopsy sections and by Western blotting of protein extracts from cultured human neonatal foreskin keratinocytes. In normal human skin, COX-1 immunostaining is observed throughout the epidermis whereas COX-2 immunostaining increases in the more differentiated, suprabasilar keratinocytes. Basal cell carcinomas express little if any COX-1 or COX-2 immunostaining whereas both isozymes are strongly expressed in squamous cell carcinomas deriving from a more differentiated layer of the epidermis. In human keratinocyte cultures, raising the extracellular calcium concentration, a recognized stimulus for keratinocyte differentiation, leads to an increased expression of both COX-2 protein and mRNA; expression of COX-1 protein, however, shows no significant alteration in response to calcium. Because of a recent report that failed to show COX-2 in normal mouse epidermis, we also looked for COX-1 and COX-2 immunostaining in sections of normal and acetone-treated mouse skin. In agreement with a previous report, some COX-1, but no COX-2, immunostaining is seen in normal murine epidermis. However, following acetone treatment, there is a marked increase in COX-1 expression as well as the appearance of significant COX-2 immunostaining in the basal layer. These data suggest that in human epidermis as well as in human keratinocyte cultures, the expression of COX-2 occurs as a part of normal keratinocyte differentiation whereas in murine epidermis, its constitutive expression is absent, but inducible as previously published.

  11. Identification of an epidermal keratinocyte AMPA glutamate receptor involved in dermatopathies associated with sensory abnormalities

    PubMed Central

    Cabañero, David; Irie, Takeshi; Celorrio, Marta; Trousdale, Christopher; Owens, David M.; Virley, David; Albrecht, Phillip J.; Caterina, Michael J.; Rice, Frank L.; Morón, Jose A.

    2016-01-01

    Introduction Epidermal keratinocytes are increasingly recognized as active participants in the sensory transduction of itch and pain, processes known to involve primary afferent glutamatergic neurons. However the role of keratinocyte glutamate signaling in sensory functioning is not fully understood. Here, we present the observation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid type glutamate receptors (AMPAR) in epidermal keratinocytes. Methods Immunohistochemical and in situ hybridization analyses were conducted to assess the expression of AMPAR subunits in epidermal keratinocytes in mouse and human skin samples, and in organotypic cultures of human keratinocytes. In addition, RTPCR further confirmed the expression of GluA4-containing AMPAR in epidermal keratinocytes. Results We found prominent immunolabeling (IL) for the GluA4 subunit of AMPAR in keratinocytes of glabrous and hairy skin of mouse epidermis, as well as in human epidermal keratinocytes. RTPCR confirmed Gria4 transcript expression in epidermal mouse keratinocytes. In addition, expression of GRIA4 mRNA was confirmed in epidermal human keratinocytes by in situ hybridization. Immunohistochemical studies conducted in human skin biopsies from patients with atopic dermatitis (AD) and postherpetic neuralgia (PHN) demonstrate that keratinocyte expression of GluA4 can be altered under pathological conditions. Moreover, a decrease of GluA4 expression was observed in organotypic cultures of human keratinocytes after direct application of algogenic agents. Conclusions We provide evidence that GluA4-containing AMPAR are expressed in epidermal keratinocytes, that human pruritic and painful dermatopathologies have alterations in the keratinocyte expression levels of GluA4-containing AMPAR, and that itch and pain producing substances can directly regulate their production in keratinocytes. PMID:28210712

  12. Co-culture of healthy human keratinocytes and T-cells promotes keratinocyte chemokine production and RORγt-positive IL-17 producing T-cell populations.

    PubMed

    Peters, Jorieke H; Tjabringa, Geuranne S; Fasse, Esther; de Oliveira, Vivian L; Schalkwijk, Joost; Koenen, Hans J P M; Joosten, Irma

    2013-01-01

    Both keratinocytes and T-cells are crucial players in cutaneous immune responses. We hypothesized that direct interactions between keratinocytes and T-cell subsets could shape the nature or strength of the local immune response. We investigated direct interactions between keratinocytes and T-cell subsets, focused on keratinocyte chemokine production and T-cell phenotype and cytokine production. A newly developed in vitro serum free co-culture model using primary keratinocytes and T-cells subsets from healthy human donors was used. Keratinocyte chemokine production was analyzed with luminex, T-cell phenotype and cytokine production were analyzed with flow cytometry. Our data show that upon co-culture with CD4(pos) or CD8(pos) T-cells primary human keratinocytes increased production of functionally active chemokines CCL2, CCL20 and CXCL10 and that regulatory T-cells did not regulate keratinocyte chemokine production. Next to that, we found that keratinocytes skewed CD4(pos) and CD8(pos) T-cell populations toward an IL-17(pos) CCR6(pos) RORγt(pos) phenotype in a cell-cell contact independent manner, and that Treg were able to decrease the absolute number of IL-17 producing T-cells in keratinocyte/T-cell co-cultures. Correspondingly, freshly isolated skin-derived T-cell populations contained relatively high percentages of IL-17(pos) cells. We provide evidence that keratinocyte/T-cell communication may regulate leukocyte influx in the skin, and that keratinocytes enrich T-cell populations for Th17/Tc17 cells. Accumulation of Th17/Tc17 cells, but not chemokine production, appears under the control of regulatory T-cells. Dysregulation of these processes may well contribute to the pathophysiology of inflammatory skin diseases. Copyright © 2012. Published by Elsevier Ireland Ltd.

  13. Molecular cloning, cDNA sequence, and chromosomal localization of the human phosphatidylinositol 3-kinase p110{alpha} (PIK3CA) gene

    SciTech Connect

    Volinia, S.; Hiles, I.; Waterfield, M.D.

    1994-12-01

    Phosphatidylinositol (PI) 3-kinase is a heterodimeric enzyme comprising a 110-kDa catalytic subunit and an 85-kDa regulatory subunit that binds to tyrosine phosphopeptide sites linked directly or indirectly to receptors serving diverse signal functions. Knowledge of the structure and function of PI 3-kinase was greatly advanced by the purification, cDNA cloning, and subsequent expression of the bovine enzyme. Here the cloning of the cDNA for the human p110{alpha}subunit of PI 3-kinase (PIK3CA), encoding a protein 99% identical to the bovine p110, and of its gene in YAC is described. The chromosomal localization of the gene for PIK3CA is shown to be at 3q21-qter as determined using somatic cell hybrids. In situ hybridization performed using Alu-PCR from the YAC DNA located the gene in 3q26.3. 30 refs., 3 figs., 1 tab.

  14. 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

  15. Paraquat-induced Oxidative Stress Represses Phosphatidylinositol 3-Kinase Activities Leading to Impaired Glucose Uptake in 3T3-L1 Adipocytes*

    PubMed Central

    Shibata, Michihiro; Hakuno, Fumihiko; Yamanaka, Daisuke; Okajima, Hiroshi; Fukushima, Toshiaki; Hasegawa, Takashi; Ogata, Tomomi; Toyoshima, Yuka; Chida, Kazuhiro; Kimura, Kumi; Sakoda, Hideyuki; Takenaka, Asako; Asano, Tomoichiro; Takahashi, Shin-Ichiro

    2010-01-01

    Accumulated evidence indicates that oxidative stress causes and/or promotes insulin resistance; however, the mechanism by which this occurs is not fully understood. This study was undertaken to elucidate the molecular mechanism by which oxidative stress induced by paraquat impairs insulin-dependent glucose uptake in 3T3-L1 adipocytes. We confirmed that paraquat-induced oxidative stress decreased glucose transporter 4 (GLUT4) translocation to the cell surface, resulting in repression of insulin-dependent 2-deoxyglucose uptake. Under these conditions, oxidative stress did not affect insulin-dependent tyrosine phosphorylation of insulin receptor, insulin receptor substrate (IRS)-1 and -2, or binding of the phosphatidylinositol 3′-OH kinase (PI 3-kinase) p85 regulatory subunit or p110α catalytic subunit to each IRS. In contrast, we found that oxidative stress induced by paraquat inhibited activities of PI 3-kinase bound to IRSs and also inhibited phosphorylation of Akt, the downstream serine/threonine kinase that has been shown to play an essential role in insulin-dependent translocation of GLUT4 to the plasma membrane. Overexpression of active form Akt (myr-Akt) restored inhibition of insulin-dependent glucose uptake by paraquat, indicating that paraquat-induced oxidative stress inhibits insulin signals upstream of Akt. Paraquat treatment with and without insulin treatment decreased the activity of class Ia PI 3-kinases p110α and p110β that are mainly expressed in 3T3-L1 adipocytes. However, paraquat treatment did not repress the activity of the PI 3-kinase p110α mutated at Cys90 in the p85 binding region. These results indicate that the PI 3-kinase p110 is a possible primary target of paraquat-induced oxidative stress to reduce the PI 3-kinase activity and impaired glucose uptake in 3T3-L1 adipocytes. PMID:20430890

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

    PubMed

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

    2000-02-24

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

  17. 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.

  18. Phosphatidylinositol 3-kinase binding to polyoma virus middle tumor antigen mediates elevation of glucose transport by increasing translocation of the GLUT1 transporter.

    PubMed Central

    Young, A T; Dahl, J; Hausdorff, S F; Bauer, P H; Birnbaum, M J; Benjamin, T L

    1995-01-01

    Elevation in the rate of glucose transport in polyoma virus-infected mouse fibroblasts was dependent upon phosphatidylinositol 3-kinase (PI 3-kinase; EC 2.7.1.137) binding to complexes of middle tumor antigen (middle T) and pp60c-src. Wild-type polyoma virus infection led to a 3-fold increase in the rate of 2-deoxyglucose (2DG) uptake, whereas a weakly transforming polyoma virus mutant that encodes a middle T capable of activating pp60c-src but unable to promote binding of PI 3-kinase induced little or no change in the rate of 2DG transport. Another transformation-defective mutant encoding a middle T that retains functional binding of both pp60c-src and PI 3-kinase but is incapable of binding Shc (a protein involved in activation of Ras) induced 2DG transport to wild-type levels. Wortmannin (< or = 100 nM), a known inhibitor of PI 3-kinase, blocked elevation of glucose transport in wild-type virus-infected cells. In contrast to serum stimulation, which led to increased levels of glucose transporter 1 (GLUT1) RNA and protein, wild-type virus infection induced no significant change in levels of either GLUT1 RNA or protein. Nevertheless, virus-infected cells did show increases in GLUT1 protein in plasma membranes. These results point to a posttranslational mechanism in the elevation of glucose transport by polyoma virus middle T involving activation of PI 3-kinase and translocation of GLUT1. Images Fig. 1 Fig. 3 Fig. 5 PMID:8524814

  19. Role of keratin 24 in human epidermal keratinocytes

    PubMed Central

    Min, Min; Chen, Xi-Bei; Wang, Ping; Landeck, Lilla; Chen, Jia-Qi; Li, Wei; Cai, Sui-Qing; Zheng, Min; Man, Xiao-Yong

    2017-01-01

    Keratin 24 (K24) is a new kind of keratin genes, which encodes a novel keratin protein, K24 that bears high similarity to the type I keratins and displays a unique expression profile. However, the role of K24 is incompletely understood. In our study, we investigated the localization of K24 within the epidermis and possible functions. Keratin 24 was found to be modestly overexpressed in senescent keratinocytes and was mainly restricted to the upper stratum spinosum of epidermis. The protein was required for terminal differentiation upon CaCl2-induced differentiation. In vitro results showed that increased K24 in keratinocytes dramatically changed the differentiation of primary keratinocytes. It also inhibited cell survival by G1/S phase cell cycle arrest and induced senescence, autophagy and apoptosis of keratinocytes. In addition, K24 activated PKCδ signal pathway involving in cellular survival. In summary, K24 may be suggested as a potential differentiation marker and anti-proliferative factor in the epidermis. PMID:28362807

  20. The Galvanotactic Migration of Keratinocytes is Enhanced by Hypoxic Preconditioning

    PubMed Central

    Guo, Xiaowei; Jiang, Xupin; Ren, Xi; Sun, Huanbo; Zhang, Dongxia; Zhang, Qiong; Zhang, Jiaping; Huang, Yuesheng

    2015-01-01

    The endogenous electric field (EF)-directed migration of keratinocytes (galvanotaxis) into wounds is an essential step in wound re-epithelialization. Hypoxia, which occurs immediately after injury, acts as an early stimulus to initiate the healing process; however, the mechanisms for this effect, remain elusive. We show here that the galvanotactic migration of keratinocytes was enhanced by hypoxia preconditioning as a result of the increased directionality rather than the increased motility of keratinocytes. This enhancement was both oxygen tension- and preconditioning time-dependent, with the maximum effects achieved using 2% O2 preconditioning for 6 hours. Hypoxic preconditioning (2% O2, 6 hours) decreased the threshold voltage of galvanotaxis to < 25 mV/mm, whereas this value was between 25 and 50 mV/mm in the normal culture control. In a scratch-wound monolayer assay in which the applied EF was in the default healing direction, hypoxic preconditioning accelerated healing by 1.38-fold compared with the control conditions. Scavenging of the induced ROS by N-acetylcysteine (NAC) abolished the enhanced galvanotaxis and the accelerated healing by hypoxic preconditioning. Our data demonstrate a novel and unsuspected role of hypoxia in supporting keratinocyte galvanotaxis. Enhancing the galvanotactic response of cells might therefore be a clinically attractive approach to induce improved wound healing. PMID:25988491

  1. Keratinocytes in the treatment of severe burn injury: an update.

    PubMed

    Lootens, Liesbeth; Brusselaers, Nele; Beele, Hilde; Monstrey, Stan

    2013-02-01

    Burns are among the most life-threatening physical injuries, in which fast wound closure is crucial. The surgical burn care has evolved considerably throughout the past decennia resulting in a shift of therapeutic goals. Therapies aiming to provide coverage of the burn have been replaced by treatments that have both functional as aesthetic outcomes. The standard in treating severe burns is still early excision followed by skin grafting. The use of cultured keratinocytes to cover extensive burn wounds appeared very promising at first, but the technique still has several limitations of which the long time to culture, the major costs, the risk of infection and the need for an adequate dermal layer limit clinical application. The introduction of dermal substitutes, composite grafts, tissue engineering based on stem cell application have been advocated. The aim of this review is to assess the use of cultured keratinocytes in terms of technical aspects, clinical application, limitations and future perspectives. Cultured keratinocytes are expected to keep playing a role in wound healing, especially in the field of chronic wounds. In severe burns, despite its limitations, keratinocytes can be beneficial if implemented as one of the elements in a broader wound management. © 2012 The Authors. International Wound Journal © 2012 Blackwell Publishing Ltd and Medicalhelplines.com Inc.

  2. FOXO1 expression in keratinocytes promotes connective tissue healing

    PubMed Central

    Zhang, Chenying; Lim, Jason; Liu, Jian; Ponugoti, Bhaskar; Alsadun, Sarah; Tian, Chen; Vafa, Rameen; Graves, Dana T.

    2017-01-01

    Wound healing is complex and highly orchestrated. It is well appreciated that leukocytes, particularly macrophages, are essential for inducing the formation of new connective tissue, which requires the generation of signals that stimulate mesenchymal stem cells (MSC), myofibroblasts and fibroblasts. A key role for keratinocytes in this complex process has yet to be established. To this end, we investigated possible involvement of keratinocytes in connective tissue healing. By lineage-specific deletion of the forkhead box-O 1 (FOXO1) transcription factor, we demonstrate for the first time that keratinocytes regulate proliferation of fibroblasts and MSCs, formation of myofibroblasts and production of collagen matrix in wound healing. This stimulation is mediated by a FOXO1 induced TGFβ1/CTGF axis. The results provide direct evidence that epithelial cells play a key role in stimulating connective tissue healing through a FOXO1-dependent mechanism. Thus, FOXO1 and keratinocytes may be an important therapeutic target where healing is deficient or compromised by a fibrotic outcome. PMID:28220813

  3. Canine distemper virus infection: proliferation of canine footpad keratinocytes.

    PubMed

    Gröne, A; Engelhardt, P; Zurbriggen, A

    2003-09-01

    The proliferation of footpad keratinocytes of canine distemper virus (CDV)-infected dogs was investigated. Footpads of 19 dogs inoculated experimentally with a virulent distemper strain (A75/17) and of two noninoculated control dogs were collected at necropsy. Dogs were divided into four groups according to results of the postmortem examination: dogs with severe distemper (group 1), dogs with mild distemper (group 2), inoculated dogs without distemper (group 3) and noninoculated dogs (group 4). There was no distinct difference of epidermal thickness among the four groups. Infection of the footpad epidermis with CDV was demonstrated using immunohistochemistry for viral nucleoprotein and in situ hybridization for nucleoprotein messenger ribonucleic acid (mRNA). Only group 1 dogs had viral antigen and mRNA in the footpad epidermis with the same distribution. Footpad epidermis of group 1 dogs had more mitotic figures in the basal layer, and significantly more basal keratinocytes were positive for the proliferation markers Ki-67 and proliferating cell nuclear antigen. Double-staining for Ki-67 and viral nucleoprotein identified rare double-labeled basal keratinocytes. These findings suggest that the presence of CDV particles in the footpad epidermis is associated with keratinocyte proliferation.

  4. Recovery and Cultivation of Keratinocytes From Shipped Mouse Skin.

    PubMed

    Yang, Hsin-Ya; La, Thi Dinh; Gurenko, Zhanna; Steenhuis, Pieter; Liu, Wei; Isseroff, R Rivkah

    2015-02-01

    Murine keratinocyte culture from neonatal skin is an important tool for studying the functional role of specific genes in epithelial biology. However, when the transgenic animal is only available in a geographically distant local, obtaining viable keratinocytes can be problematic. A method for transferring the isolated murine skin from collaborating labs could decrease the cost of shipping live animals, and would allow the efficient use of the tissues from the transgenic animals. Here we optimized shipping conditions and characterized the cells retrieved and cultured from mouse skin shipped for 48 h at 0 °C. The cultured keratinocytes from the control, non-shipped skin and the 2-day shipped skin were 43.6 +/- 7.8% viable, doubled every 2 days, and expressed comparable amounts of heat shock proteins and CD29/integrin beta-1. However, under the same shipping conditions, the 3-day shipped tissue failed to establish colonies in the culture. Therefore, this 2-day shipping technique allows the transfer mouse skin from distant locations with recovery of viable, propagatable keratinocytes, facilitating long-distance collaborations.

  5. Desmoglein 3-Dependent Signaling Regulates Keratinocyte Migration and Wound Healing.

    PubMed

    Rötzer, Vera; Hartlieb, Eva; Winkler, Julia; Walter, Elias; Schlipp, Angela; Sardy, Miklós; Spindler, Volker; Waschke, Jens

    2016-01-01

    The desmosomal transmembrane adhesion molecules desmoglein 3 (Dsg3) and desmocollin 3 (Dsc3) are required for strong keratinocyte cohesion. Recently, we have shown that Dsg3 associates with p38 mitogen-activated protein kinase (p38MAPK) and suppresses its activity. Here, we further investigated the role of Dsg3-dependent control of p38MAPK function. Dsg3-deficient mice display recurrent spontaneously healing skin erosions. In lesional and perilesional biopsies, p38MAPK activation was detectable compared with control animals. This led us to speculate that Dsg3 regulates wound repair in a p38MAPK-dependent manner. Indeed, scratch-wounded keratinocyte monolayers exhibited p38MAPK activation and loss of Dsg3 in cells lining the wound edge. Human keratinocytes after silencing of Dsg3 as well as primary cells isolated from Dsg3 knockout animals exhibited accelerated migration, which was further corroborated in an ex vivo skin outgrowth assay. Importantly, migration was efficiently blocked by inhibition of p38MAPK, indicating that p38MAPK mediates the effects observed upon loss of Dsg3. In line with this, we show that levels of active p38MAPK associated with Dsc3 are increased in Dsg3-deficient cells. These data indicate that Dsg3 controls a switch from an adhesive to a migratory keratinocyte phenotype via p38MAPK inhibition. Thus, loss of Dsg3 adhesion may foster wound closure by allowing p38MAPK-dependent migration.

  6. Oxidative damage in keratinocytes exposed to cigarette smoke and aldehydes.

    PubMed

    Avezov, Katia; Reznick, Abraham Z; Aizenbud, Dror

    2014-06-01

    Cigarette smoke (CS) is a significant environmental source of human exposure to chemically active saturated (acetaldehyde) and α,β-unsaturated aldehydes (acrolein) inducing protein carbonylation and dysfunction. The exposure of oral tissues to environmental hazards is immense, especially in smokers. The objectives of the current study were to examine the effect of aldehydes originating from CS on intracellular proteins of oral keratinocytes and to observe the antioxidant response in these cells. Intracellular protein carbonyl modification under CS, acrolein and acetaldehyde exposure in the HaCaT keratinocyte cell line, representing oral keratinocytes was examined by Western blot. Possible intracellular enzymatic dysfunction under the above conditions was examined by lactate dehydrogenase (LDH) activity assay. Oxidative stress response was investigated, by DCF (2,7-dichlorodihydrofluorescein) assay and GSH (glutathione) oxidation. Intracellular protein carbonyls increased 5.2 times after CS exposure and 2.7 times after exposure to 1 μmol of acrolein. DCF assay revealed an increase of fluorescence intensity 3.2 and 3.1 times after CS and acrolein exposure, respectively. CS caused a 72.5% decrease in intracellular GSH levels compared to controls. Activity of intracellular LDH was preserved. α,β-Unsaturated aldehydes from CS are capable of intracellular protein carbonylation and have a role in intracellular oxidative stress elevation in keratinocytes, probably due to the reduction in GSH levels.

  7. Keratinocytes can modulate and directly initiate nociceptive responses

    PubMed Central

    Baumbauer, Kyle M; DeBerry, Jennifer J; Adelman, Peter C; Miller, Richard H; Hachisuka, Junichi; Lee, Kuan Hsien; Ross, Sarah E; Koerber, H Richard; Davis, Brian M; Albers, Kathryn M

    2015-01-01

    How thermal, mechanical and chemical stimuli applied to the skin are transduced into signals transmitted by peripheral neurons to the CNS is an area of intense study. Several studies indicate that transduction mechanisms are intrinsic to cutaneous neurons and that epidermal keratinocytes only modulate this transduction. Using mice expressing channelrhodopsin (ChR2) in keratinocytes we show that blue light activation of the epidermis alone can produce action potentials (APs) in multiple types of cutaneous sensory neurons including SA1, A-HTMR, CM, CH, CMC, CMH and CMHC fiber types. In loss of function studies, yellow light stimulation of keratinocytes that express halorhodopsin reduced AP generation in response to naturalistic stimuli. These findings support the idea that intrinsic sensory transduction mechanisms in epidermal keratinocytes can directly elicit AP firing in nociceptive as well as tactile sensory afferents and suggest a significantly expanded role for the epidermis in sensory processing. DOI: http://dx.doi.org/10.7554/eLife.09674.001 PMID:26329459

  8. The peanut lectin-binding glycoproteins of human epidermal keratinocytes

    SciTech Connect

    Morrison, A.I. ); Keeble, S.; Watt, F.M. )

    1988-08-01

    The peanut lectin (PNA) is known to bind more strongly to keratinocytes that are undergoing terminal differentiation than to proliferating keratinocytes. In order to investigate the significance of this change in cell-surface carbohydrate authors have identified the PNA-binding glycoproteins of cultured human keratinocytes and antibodies against them. Two heavily glycosylated bands of 110 and 250 kDa were resolved by PAGE of ({sup 14}C)galactose- or ({sup 14}C)mannose- and ({sup 14}C)glucosamine-labeled cell extracts eluted with galactose from PNA affinity columns. The higher molecular weight band was also detected on PNA blots of unlabeled cell extracts transferred to nitrocellulose. Both bands were sensitive to pronase digestion, but only the 250-kDa band was digested with trypsin. A rabbit antiserum that we prepared (anti-PNA-gp) immunoprecipitated both bands from cell extracts. In contrast to PNA, anti-PNA-gp bound equally to proliferating and terminally differentiating cells, indicating that some epitope(s) of the PNA-binding glycoproteins is present on the cell surface prior to terminal differentiation. When keratinocytes grown as a monolayer in low-calcium medium were switched to medium containing 2 mM calcium ions in order to induce desmosome formation and stratification, there was a dramatic redistribution of the PNA-binding glycoproteins, which became concentrated at the boundaries between cells. This may suggest a role for the glycoproteins in cell-cell interactions during stratification.

  9. Cell shape controls terminal differentiation of human epidermal keratinocytes.

    PubMed Central

    Watt, F M; Jordan, P W; O'Neill, C H

    1988-01-01

    Cultures of human epidermal keratinocytes provide a useful experimental model with which to study the factors that regulate cell proliferation and terminal differentiation. One situation that is known to trigger premature terminal differentiation is suspension culture, when keratinocytes are deprived of substratum and intercellular contact. We have now investigated whether area of substratum contact, and hence cell shape, can regulate terminal differentiation. Keratinocytes were grown on circular adhesive islands that prevented cell-cell contact. By varying island area we could vary cell shape from fully spread to almost spherical. We found that when substratum contact was restricted, DNA synthesis was inhibited and expression of involucrin, a marker of terminal differentiation, was stimulated. Inhibition of proliferation was not a sufficient stimulus for involucrin synthesis in fully spread cells. When DNA synthesis and involucrin expression were plotted against contact area, classic dose-response curves were obtained. Thus cell shape acts as a signal for the terminal differentiation of keratinocytes in culture. Images PMID:2456572

  10. Gamma Interferon Reduces the Synthesis of Fibronectin by Human Keratinocytes

    DTIC Science & Technology

    1988-10-06

    mmm mm m m m m mmm• m 13. Wikner NE, Dixit VM, Frazier WA, Clark RAF: Human keratinocytes synthesize and secrete the extracellular matrix protein...keratinoctye-derived lymphocyte inhibitory factor. J Investig Dermatol 90:592A, 1988 18. Wikner NE, Baskin JB, Nielsen LD, McPherson JM, Clark RAF

  11. Human keratinocytes synthesize and secrete the extracellular matrix protein, thrombospondin.

    PubMed

    Wikner, N E; Dixit, V M; Frazier, W A; Clark, R A

    1987-02-01

    Thrombospondin (TSP) a glycoprotein originally identified as the endogenous lectin of platelets, is also synthesized by fibroblasts, endothelial cells, pneumocytes, smooth muscle cells, and macrophages. Thrombospondin is subdivided into functional domains which bind specifically to heparin, fibronectin, collagen, and to specific cellular receptors. It is found within the basement membranes of kidney, lung, smooth muscle, and skin. Thus TSP may serve as an important link between cells and matrices. Thrombospondin also has been reported at the epidermal-dermal junction. We wished to determine whether human keratinocytes synthesize and secrete TSP. Pure human keratinocytes were grown in defined medium without fibroblast feeder layers. Immunofluorescent staining with either rabbit polyclonal or mouse monoclonal antibodies to human platelet TSP yielded specific granular staining within the cytoplasm of keratinocytes. Culture media and cellular lysates were harvested from cultures metabolically labeled with [35S]methionine. Trichloroacetic acid precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography revealed a major labeled band comigrating with purified platelet TSP in both the media and the cellular lysates. Immunoprecipitation with either the polyclonal or the monoclonal anti-TSP antibodies followed by SDS-PAGE and autoradiography identified this band as TSP. Thus keratinocytes in culture synthesize and secrete TSP. Thrombospondin may play an important role in epidermal interactions with extracellular matrix.

  12. Prediction of drug-induced liver injury using keratinocytes.

    PubMed

    Hirashima, Rika; Itoh, Tomoo; Tukey, Robert H; Fujiwara, Ryoichi

    2017-01-31

    Drug-induced liver injury (DILI) is one of the most common adverse drug reactions. DILI is often accompanied by skin reactions, including rash and pruritus. However, it is still unknown whether DILI-associated genes such as S100 calcium-binding protein A and interleukin (IL)-1β are involved in drug-induced skin toxicity. In the present study, most of the tested hepatotoxic drugs such as pioglitazone and diclofenac induced DILI-associated genes in human and mouse keratinocytes. Keratinocytes of mice at higher risk for DILI exhibited an increased IL-1β basal expression. They also showed a higher inducibility of IL-1β when treated by pioglitazone. Mice at higher risk for DILI showed even higher sums of DILI-associated gene basal expression levels and induction rates in keratinocytes. Our data suggest that DILI-associated genes might be involved in the onset and progression of drug-induced skin toxicity. Furthermore, we might be able to identify individuals at higher risk of developing DILI less invasively by examining gene expression patterns in keratinocytes. Copyright © 2017 John Wiley & Sons, Ltd.

  13. Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector.

    PubMed

    Nanba, Daisuke; Matsushita, Natsuki; Toki, Fujio; Higashiyama, Shigeki

    2013-10-18

    The development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment. In this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry. Polybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene. This study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine.

  14. Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector

    PubMed Central

    2013-01-01

    Introduction The development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment. Methods In this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry. Results Polybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene. Conclusions This study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine. PMID:24406242

  15. Novel method for proliferation of oral keratinocyte stem cells.

    PubMed

    Calenic, B; Paun, I A; van Staden, R I; Didilescu, A; Petre, A; Dinescu, M; Greabu, M

    2014-12-01

    Stem cell-based tissue engineering offers clear advantages over conventional normal cell approaches. Owing to their specific characteristics, oral keratinocyte stem cells represent an attractive solution for therapeutic applications. However, when cultured in vitro, these cells lose their unique properties, acquiring a limited capacity for self-renewal, and differentiate rapidly into normal functional keratinocytes. The main aim of the present study was to develop an in-vitro method for the expansion of oral keratinocyte stem cells using a biomaterial approach. Oral keratinocyte stem cells were isolated based on the identification of two surface markers - integrin α6β4 and CD71 - using a magnetic method. The cells were cultured on specific substrates formed from blends of polymers: poly(lactide-co-glycolide) (PLGA); poly(lactide-co-glycolide) + polyurethane (PLGA + PU); and poly(lactide-co-glycolide) + extracellular matrix (PLGA + ECM). The polymers were deposited using a laser-based technique - matrix-assisted pulsed laser evaporation. The cells were analyzed for cell size, cell proliferation, colony-forming efficiency, cell adhesion markers (such as E-cadherin and beta 1 integrin), keratinocyte stem cells and differentiation markers. The methods included ELISAs, immunofluorescence and atomic force microscopy imaging. After 14 d in culture, cells seeded on PLGA + PU stained positive for p63, cd44H, cytokeratin 19 and integrin α6β4 and negative for involucrin, cytokeratin 14 and cytokeratin 10. The levels of adhesion molecules were significantly increased in cells grown on PLGA + PU: at 14 d the E-cadherin levels were 5.4 ± 0.2 ng/mL (for cells grown on PLGA + PU) vs. 4.1 ± 0.4 ng/mL (for cells grown on control medium) (n = 5, p < 0.05 Bonferroni). Oral keratinocyte stem cells grown on PLGA + PU had the highest colony-forming efficiency and proliferation rate, together with the smallest cell size, compared with cells grown on

  16. Vimentin is necessary for colony growth of human diploid keratinocytes.

    PubMed

    Castro-Muñozledo, Federico; Velez-DelValle, Cristina; Marsch-Moreno, Meytha; Hernández-Quintero, Miriam; Kuri-Harcuch, Walid

    2015-01-01

    The role of vimentin (Vim) in diploid epithelial cells is not well known. To understand its biological function, we cultured human epidermal keratinocytes under conditions that support migration, proliferation, stratification and terminal differentiation. We identified a keratinocyte subpopulation that shows a p63(+)/α5β1(bright) phenotype and displays Vim intermediate filaments (IFs) besides their keratin IF network. These cells were mainly located at the proliferative/migratory rim of the growing colonies; but also, they were scarce and scattered or formed small groups of basal cells in confluent stratified epithelia. Stimulation of cells with EGF and wounding experiments in confluent arrested epithelia increased the number of Vim(+) keratinocytes in an extent higher to the expected for a cell population doubling. BrdU labeling demonstrated that most of the proliferative cells located at the migratory border of the colony have Vim, in contrast with proliferative cells located at the basal layer at the center of big colonies which lacked of Vim IFs, suggesting that Vim expression was not solely linked to proliferation. Therefore, we silenced Vim mRNA in the cultured keratinocytes and observed an inhibition of colony growth. Such results, together with long-term cultivation assays which showed that Vim might be associated to pattern formation in cultured epithelia, suggest that Vim expression is essential for a highly motile phenotype, which is necessary for keratinocyte colony growth and possibly for development and wound healing. Vim(+)/p63(+)/α5β1(bright) epithelial cells may play a significant physiological role in embryonic morphogenetic movements; wound healing and other pathologies such as carcinomas and hyperproliferative diseases.

  17. Partial loss of epithelial phenotype in kindlin-1-deficient keratinocytes.

    PubMed

    Qu, Haiyan; Wen, Tingting; Pesch, Monika; Aumailley, Monique

    2012-04-01

    Kindlin-1 is an adaptor protein that is expressed by most epithelial cells and has been implicated in integrin bidirectional signaling. Mutations in the gene encoding kindlin-1 are associated with Kindler syndrome, a recessively inherited disorder that is characterized by fragile skin. Functionally, a loss of kindlin-1 impairs the adhesion of basal keratinocytes to the extracellular matrix both in vivo and in vitro. In this study, we show that the phenotype of mutant keratinocytes deficient in kindlin-1 is characterized by the modification of the cortical actin network and increased plasticity of the plasma membrane. At the molecular level, expression of several proteins associated with an epithelial phenotype, such as α6β4 integrin, collagen XVII, E-cadherin, and desmoglein-3, is strongly reduced, whereas, surprisingly, laminin 332 is synthesized in larger amounts than in control keratinocytes. In contrast, mesenchymal markers such as vimentin and fibronectin are increased in keratinocytes lacking kindlin-1. The switch in cell plasticity and protein expression was confirmed by siRNA-mediated down-regulation of kindlin-1 in HaCaT epithelial cells. Furthermore, there was up-regulation of matrix metalloproteinases and pro-inflammatory cytokines in kindlin-1-deficient keratinocytes. These results provide new insights into the pathogenic mechanisms that take place in Kindler syndrome. Moreover, the constellation of molecular defects associated with the loss of kindlin-1 may explain the higher incidence of skin cancer observed in patients affected with this disorder. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  18. Inhibition of the phosphoinositide 3-kinase pathway for the treatment of patients with metastatic metaplastic breast cancer.

    PubMed

    Moulder, S; Helgason, T; Janku, F; Wheler, J; Moroney, J; Booser, D; Albarracin, C; Morrow, P K; Atkins, J; Koenig, K; Gilcrease, M; Kurzrock, R

    2015-07-01

    Mesenchymal/metaplastic breast cancers (MpBCs) are often triple-negative (TNBC), and chemo-refractory, and can harbor phosphoinositide 3-kinase (PI3kinase) alterations; thus, therapy with mTor inhibitors may demonstrate activity. Patients with mesenchymal/MpBC treated with temsirolimus-based regimens were evaluated. Mutational analyses [polymerase chain reaction (PCR)-based DNA sequencing method, mass spectrometric detection (Sequenom MassARRAY), or next-generation sequencing] as well as loss of phosphatase and tensin homolog (PTEN) (immunohistochemistry) were performed (archived tissue when available). Twenty-three patients (one of whom was on two separate trials) were treated using temsirolimus-containing regimens: temsirolimus alone (n = 1 patient) or combined with the following: liposomal doxorubicin and bevacizumab (DAT, n = 18); liposomal doxorubicin (DT, n = 1); paclitaxel and bevacizumab (TAT, n = 2); paclitaxel (TT, n = 1); carboplatin and bevacizumab (CAT, n = 1). Response rate [complete response (CR) + partial response (PR)] was 25% across all regimens; 32% in the anthracycline-based regimens [DAT and DT (CR = 2, PR = 4; N = 19)]. An additional two patients achieved stable disease (SD) ≥6 months [total SD ≥6 months/CR/PR = 8 (33%)]. Molecular aberrations in the PI3K pathway were common: PIK3CA mutation = 6/15 (40%), PTEN mutation = 3/11 (27%), and PTEN loss = 2/11 (18%). A point mutation in the NF2 gene (K159fs*16; NF2 alterations can activate mTor) was found in one patient who attained CR (3+ years). Of the eight patients who achieved SD ≥6 months/CR/PR, all 4 patients with available tissue had a molecular aberration that activate the PIK3CA/Akt/mTOR axis: PIK3CA mutation = 2; PTEN loss = 1; NF2 aberration = 1. DAT has activity in MpBCs including complete CRs. Molecular aberrations that can activate the PI3 K/Akt/mTOR axis are common in MpBC. © The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical

  19. Role of Class III phosphoinositide 3-kinase in the brain development: possible involvement in specific learning disorders.

    PubMed

    Inaguma, Yutaka; Matsumoto, Ayumi; Noda, Mariko; Tabata, Hidenori; Maeda, Akihiko; Goto, Masahide; Usui, Daisuke; Jimbo, Eriko F; Kikkawa, Kiyoshi; Ohtsuki, Mamitaro; Momoi, Mariko Y; Osaka, Hitoshi; Yamagata, Takanori; Nagata, Koh-Ichi

    2016-10-01

    Class III phosphoinositide 3-kinase (PIK3C3 or mammalian vacuolar protein sorting 34 homolog, Vps34) regulates vesicular trafficking, autophagy, and nutrient sensing. Recently, we reported that PIK3C3 is expressed in mouse cerebral cortex throughout the developmental process, especially at early embryonic stage. We thus examined the role of PIK3C3 in the development of the mouse cerebral cortex. Acute silencing of PIK3C3 with in utero electroporation method caused positional defects of excitatory neurons during corticogenesis. Time-lapse imaging revealed that the abnormal positioning was at least partially because of the reduced migration velocity. When PIK3C3 was silenced in cortical neurons in one hemisphere, axon extension to the contralateral hemisphere was also delayed. These aberrant phenotypes were rescued by RNAi-resistant PIK3C3. Notably, knockdown of PIK3C3 did not affect the cell cycle of neuronal progenitors and stem cells at the ventricular zone. Taken together, PIK3C3 was thought to play a crucial role in corticogenesis through the regulation of excitatory neuron migration and axon extension. Meanwhile, when we performed comparative genomic hybridization on a patient with specific learning disorders, a 107 Kb-deletion was identified on 18q12.3 (nt. 39554147-39661206) that encompasses exons 5-23 of PIK3C3. Notably, the above aberrant migration and axon growth phenotypes were not rescued by the disease-related truncation mutant (172 amino acids) lacking the C-terminal kinase domain. Thus, functional defects of PIK3C3 might impair corticogenesis and relate to the pathophysiology of specific learning disorders and other neurodevelopmental disorders. Acute knockdown of Class III phosphoinositide 3-kinase (PIK3C3) evokes migration defects of excitatory neurons during corticogenesis. PIK3C3-knockdown also disrupts axon outgrowth, but not progenitor proliferation in vivo. Involvement of PIK3C3 in neurodevelopmental disorders might be an interesting future

  20. 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.

  1. 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

  2. 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

  3. PI3 Kinase Disease

    MedlinePlus

    ... NIAID Scientists Discover Rare Genetic Susceptibility to Common Cold , June 12, 2017 NIAID Research Aids Discovery of ... Duke University Boston University Zoonotic Influenza Vaccine Seed Viruses Bioinformatics Resource Centers PATRIC Resources Bioinformatics Resource Centers ...

  4. Overexpression of a rat kinase-deficient phosphoinositide 3-kinase, Vps34p, inhibits cathepsin D maturation.

    PubMed Central

    Row, P E; Reaves, B J; Domin, J; Luzio, J P; Davidson, H W

    2001-01-01

    Lipid kinases and their phosphorylated products are important regulators of many cellular processes, including intracellular membrane traffic. The best example of this is provided by the class III phosphoinositide 3-kinase (PI-3K), Vps34p, which is required for correct targeting of newly synthesized carboxypeptidase Y to the yeast vacuole. A probable mammalian Vps34p orthologue has been previously identified, but its function in the trafficking of lysosomal enzymes has not been resolved. To investigate the possible role(s) of mammalian Vps34p in protein targeting to lysosomes, we have cloned the rat orthologue and overexpressed a kinase-deficient mutant in HeLa cells. Expression of the mutant protein inhibited both maturation of procathepsin D and basal secretion of the precursor. In contrast wortmannin, which also inhibited maturation, caused hypersecretion of the precursor. We propose that mammalian Vps34p plays a direct role in targeting lysosomal enzyme precursors to the endocytic pathway in an analogous fashion to its role in the fusion of early endocytic vesicles with endosomes. We further suggest that inhibition of a wortmannin-sensitive enzyme, other than mammalian Vps34p, is responsible for the failure to recycle unoccupied mannose 6-phosphate receptors to the trans-Golgi network, and consequent hypersecretion of lysosomal enzyme precursors observed in the presence of this drug. PMID:11171063

  5. 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

  6. p85α subunit of class IA PI-3 kinase is crucial for macrophage growth and migration

    PubMed Central

    Munugalavadla, Veerendra; Borneo, Jovencio; Ingram, David A.; Kapur, Reuben

    2005-01-01

    Macrophages play an essential role in defending against invading pathogens by migrating to the sites of infection, removing apoptotic cells, and secreting inflammatory cytokines. The molecular mechanisms whereby macrophages regulate these processes are poorly understood. Using bone marrow–derived macrophages (BMMs) deficient in the expression of p85α-subunit of class IA phosphatidylinositol 3 (PI-3) kinase, we demonstrate 50% reduction in proliferation in response to macrophage–colony-stimulating factor (M-CSF) as well as granulocyte macrophage–colony-stimulating factor (GM-CSF) compared with wild-type controls. Furthermore, p85α–/– BMMs demonstrate a significant reduction in migration in a wound-healing assay compared with wild-type controls. The reduction in migration due to p85α deficiency in BMMs is associated with reduced adhesion and directed migration on fibronectin and vascular cell adhesion molecule-1. In addition, deficiency of p85α in BMMs also results in defective phagocytosis of sheep red blood cells. Biochemically, loss of p85α in BMMs results in reduced activation of Akt and Rac, but not Erk (extracellular signal-related kinase) mitogen-activated protein (MAP) kinase. Taken together, our results provide genetic evidence for the importance of p85α in regulating both actin- and growth-based functions in macrophages, and provide a potential therapeutic target for the treatment of diseases involving macrophages, including inflammation. PMID:15769893

  7. 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.

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

    PubMed

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

    2012-07-01

    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.

  9. 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

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

    PubMed Central

    Zhu, Weiguo; Oxford, Gerry S.

    2009-01-01

    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 coexpressing 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 wildtype or Y785F (PLC signal deficient) mutant human trkA reconstituted NGF sensitization. In contrast, TRPV1 coexpressed 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 p85α (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. PMID:17324588

  11. 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

  12. 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

  13. Cationic liposomes suppress intracellular calcium ion concentration increase via inhibition of PI3 kinase pathway in mast cells.

    PubMed

    Inoh, Yoshikazu; Haneda, Aki; Tadokoro, Satoshi; Yokawa, Satoru; Furuno, Tadahide

    2017-09-28

    Cationic liposomes are commonly used as vectors to effectively introduce foreign genes (antisense DNA, plasmid DNA, siRNA, etc.) into target cells. Cationic liposomes are also known to affect cellular immunocompetences such as the mast cell function in allergic reactions. In particular, we previously showed that the cationic liposomes bound to the mast cell surface suppress the degranulation induced by cross-linking of high affinity IgE receptors in a time- and dose-dependent manner. This suppression is mediated by impairment of the sustained level of intracellular Ca(2+) concentration ([Ca(2+)]i) via inhibition of store-operated Ca(2+) entry (SOCE). Here we study the mechanism underlying an impaired [Ca(2+)]i increase by cationic liposomes in mast cells. We show that cationic liposomes inhibit the phosphorylation of Akt and PI3 kinases but not Syk and LAT. As a consequence, SOCE is suppressed but Ca(2+) release from endoplasmic reticulum (ER) is not. Cationic liposomes inhibit the formation of STIM1 puncta, which is essential to SOCE by interacting with Orai1 following the Ca(2+) concentration decrease in the ER. These data suggest that cationic liposomes suppress SOCE by inhibiting the phosphorylation of PI3 and Akt kinases in mast cells. Copyright © 2017. Published by Elsevier B.V.

  14. The phosphatidylinositol 3-kinase/Akt/mTOR signaling network as a therapeutic target in acute myelogenous leukemia patients

    PubMed Central

    Martelli, Alberto M.; Evangelisti, Camilla; Chiarini, Francesca; McCubrey, James A.

    2010-01-01

    The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling axis plays a central role in cell proliferation, growth, and survival under physiological conditions. However, aberrant PI3K/Akt/mTOR signaling has been implicated in many human cancers, including acute myelogenous leukemia (AML). Therefore, the PI3K/Akt/mTOR network is considered as a validated target for innovative cancer therapy. The limit of acceptable toxicity for standard polychemotherapy has been reached in AML. Novel therapeutic strategies are therefore needed. This review highlights how the PI3K/Akt/mTOR signaling axis is constitutively active in AML patients, where it affects survival, proliferation, and drug-resistance of leukemic cells including leukemic stem cells. Effective targeting of this pathway with small molecule kinase inhibitors, employed alone or in combination with other drugs, could result in the suppression of leukemic cell growth. Furthermore, targeting the PI3K/Akt/mTOR signaling network with small pharmacological inhibitors, employed either alone or in combinations with other drugs, may result in less toxic and more efficacious treatment of AML patients. Efforts to exploit pharmacological inhibitors of the PI3K/Akt/mTOR cascade which show efficacy and safety in the clinical setting are now underway. PMID:20671809

  15. 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

  16. 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

  17. 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.

  18. 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

  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. 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.

  1. Specific roles of the p110alpha isoform of phosphatidylinsositol 3-kinase in hepatic insulin signaling and metabolic regulation.

    PubMed

    Sopasakis, Victoria Rotter; Liu, Pixu; Suzuki, Ryo; Kondo, Tatsuya; Winnay, Jonathon; Tran, Thien T; Asano, Tomoichiro; Smyth, Graham; Sajan, Mini P; Farese, Robert V; Kahn, C Ronald; Zhao, Jean J

    2010-03-03

    The class I(A) phosphatidylinsositol 3-kinases (PI3Ks) form a critical node in the insulin metabolic pathway; however, the precise roles of the different isoforms of this enzyme remain elusive. Using tissue-specific gene inactivation, we demonstrate that p110alpha catalytic subunit of PI3K is a key mediator of insulin metabolic actions in the liver. Thus, deletion of p110alpha in liver results in markedly blunted insulin signaling with decreased generation of PIP(3) and loss of insulin activation of Akt, defects that could not be rescued by overexpression of p110beta. As a result, mice with hepatic knockout of p110alpha display reduced insulin sensitivity, impaired glucose tolerance, and increased gluconeogenesis, hypolipidemia, and hyperleptinemia. The diabetic syndrome induced by loss of p110alpha in liver did not respond to metformin treatment. Together, these data indicate that the p110alpha isoform of PI3K plays a fundamental role in insulin signaling and control of hepatic glucose and lipid metabolism. 2010 Elsevier Inc. All rights reserved.

  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. 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.

  4. Co-amplification of phosphoinositide 3-kinase enhancer A and cyclin-dependent kinase 4 triggers glioblastoma progression.

    PubMed

    Qi, Q; Kang, S S; Zhang, S; Pham, C; Fu, H; Brat, D J; Ye, K

    2017-08-10

    Glioblastoma (GBM) is the most common primary brain tumor and has a dismal prognosis. Amplification of chromosome 12q13-q15 (Cyclin-dependent kinase 4 (CDK4) amplicon) is frequently observed in numerous human cancers including GBM. Phosphoinositide 3-kinase enhancer (PIKE) is a group of GTP-binding proteins that belong to the subgroup of centaurin GTPase family, encoded by CENTG1 located in CDK4 amplicon. However, the pathological significance of CDK4 amplicon in GBM formation remains incompletely understood. In the current study, we show that co-expression of PIKE-A and CDK4 in TP53/PTEN double knockout GBM mouse model additively shortens the latency of glioma onset and survival compared to overexpression of these genes alone. Consequently, p-mTOR, p-Akt and p-ERK pathways are highly upregulated in the brain tumors, in alignment with their oncogenic activities by CDK4 and PIKE-A stably transfected in GBM cell lines. Hence, our findings support that PIKE amplification or overexpression coordinately acts with CDK4 to drive GBM tumorigenesis.

  5. 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.

  6. 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

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

    PubMed Central

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

    2012-01-01

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

  8. The insulin sensitizing effect of homoisoflavone-enriched fraction in Liriope platyphylla Wang et Tang via PI3-kinase pathway.

    PubMed

    Choi, Soo Bong; Wha, Jun Dong; Park, Sunmin

    2004-10-15

    In the present study, we screened candidates for enhancing insulin action, using glucose uptake as an indicator, from Liriope platyphylla Wang et Tang (LPWT) extract, Liliaceae, in 3T3-L1 adipocytes. The mechanism of insulin sensitizing action in the fractions was also investigated. LPWT extract with 70% MeOH was sequentially separated with Diaion HP-20 and silica gel column chromatography. The 9:1 fraction from silica gel column chromatography increased glucose uptake with 1 ng/mL up to glucose uptake with 50 ng/mL insulin. The 9:1 fraction, determined as homoisoflavone-enriched fraction, worked as an insulin sensitizer. It increased insulin stimulated glucose uptake in 3T3-L1 adipocytes, insulin responsive cells, through increased glucose transporter 4 (GLUT4) contents in the plasma membrane. GLUT4 translocation was increased through insulin receptor substrate 1 (IRS1)-PI3 kinase-Akt signaling mechanism. Thus, homoisoflavone-enriched fraction in LPWT extract played an important role as an insulin sensitizer in adipocytes.

  9. Phosphatidylinositol 3-kinase and calcium-activated transcription pathways are required for VLDL-induced smooth muscle cell proliferation.

    PubMed

    Lipskaia, Larissa; Pourci, Marie-Luce; Deloménie, Claudine; Combettes, Laurent; Goudounèche, Dominique; Paul, Jean-Louis; Capiod, Thierry; Lompré, Anne-Marie

    2003-05-30

    Little is known regarding the molecular mechanisms of atherogenicity of triglyceride-rich lipoproteins such as very low-density lipoproteins (VLDLs). We examined the effect of VLDL on proliferation of rat aortic smooth muscle cells, intracellular Ca2+ handling, and activity of cAMP-responsive element binding protein (CREB) and nuclear factor of activated T cells (NFAT) transcription factors. VLDL, isolated from human serum, dose- and time-dependently promoted proliferation. After 4 hours of exposure to VLDL (0.15 g/L proteins), the caffeine-induced Ca2+ release was inhibited and the IP3-sensitive Ca2+ release induced by ATP (10 micromol/L) was markedly prolonged. In quiescent cells, CREB was phosphorylated (pCREB) and NFAT was present in the cytosol, whereas in cells exposed to VLDL for 4 to 24 hours, pCREB disappeared and NFAT was translocated to the nucleus. VLDL-induced NFAT translocation and proliferation were blocked by cyclosporin A and LY294002 involving calcineurin and phosphatidylinositol 3-kinase (PI3K) pathways. Indeed, VLDLs rapidly phosphorylate protein kinase B and glycogen synthase kinase-3beta in a PI3K-dependent way. These results provide the first evidence that VLDLs induce smooth muscle cell proliferation by activating the PI3K pathway and nuclear NFAT translocation. Blockade of the Ca2+-induced Ca2+ release mechanism and dephosphorylation of pCREB contribute but were not sufficient to induce a proliferating phenotype.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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.

  18. Functional genomics reveals that tumors with activating phosphoinositide 3-kinase mutations are dependent on accelerated protein turnover.

    PubMed

    Davoli, Teresa; Mengwasser, Kristen E; Duan, Jingjing; Chen, Ting; Christensen, Camilla; Wooten, Eric C; Anselmo, Anthony N; Li, Mamie Z; Wong, Kwok-Kin; Kahle, Kristopher T; Elledge, Stephen J

    2016-12-15

    Activating mutations in the phosphoinositide 3-kinase (PI3K) signaling pathway are frequently identified in cancer. To identify pathways that support PI3K oncogenesis, we performed a genome-wide RNAi screen in isogenic cell lines harboring wild-type or mutant PIK3CA to search for PI3K synthetic-lethal (SL) genes. A combined analysis of these results with a meta-analysis of two other large-scale RNAi screening data sets in PI3K mutant cancer cell lines converged on ribosomal protein translation and proteasomal protein degradation as critical nononcogene dependencies for PI3K-driven tumors. Genetic or pharmacologic inhibition of either pathway alone, but not together, selectively killed PI3K mutant tumor cells in an mTOR-dependent manner. The expression of ribosomal and proteasomal components was significantly up-regulated in primary human colorectal tumors harboring PI3K pathway activation. Importantly, a PI3K SL gene signature containing the top hits of the SL genes identified in our meta-analysis robustly predicted overall patient survival in colorectal cancer, especially among patients with tumors with an activated PI3K pathway. These results suggest that disruption of protein turnover homeostasis via ribosome or proteasome inhibition may be a novel treatment strategy for PI3K mutant human tumors. © 2016 Davoli et al.; Published by Cold Spring Harbor Laboratory Press.

  19. 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

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

    PubMed Central

    Wieman, Heather L.; Wofford, Jessica A.

    2007-01-01

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

  1. 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.

  2. 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.

  3. 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.

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

    PubMed Central

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

    2010-01-01

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

  5. 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.

  6. 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.

  7. 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

  8. Acute wounding alters the beta2-adrenergic signaling and catecholamine synthetic pathways in keratinocytes.

    PubMed

    Sivamani, Raja K; Shi, Biao; Griffiths, Elizabeth; Vu, Shirley M; Lev-Tov, Hadar A; Dahle, Sara; Chigbrow, Marianne; La, Thi Dinh; Mashburn, Chelcy; Peavy, Thomas R; Rivkah Isseroff, R

    2014-08-01

    Keratinocyte migration is critical for wound re-epithelialization. Previous studies showed that epinephrine activates the beta2-adrenergic receptor (B2AR), impairing keratinocyte migration. Here, we investigated the keratinocyte catecholamine synthetic pathway in response to acute trauma. Cultured keratinocytes were scratch wounded and expression levels of the B2AR and catecholamine synthetic enzymes tyrosine hydroxylase and phenylethanolamine-N-methyltransferase were assayed. The binding affinity of the B2AR was measured. Wounding downregulated B2AR, tyrosine hydroxylase, and phenylethanolamine-N-methyltransferase expression, but pre-exposure to timolol, a beta-adrenergic receptor antagonist, delayed this effect. In wounded keratinocytes, B2AR-binding affinity remained depressed even after its expression returned to prewounding levels. Keratinocyte-derived norepinephrine increased after wounding. Norepinephrine impaired keratinocyte migration; this effect was abrogated with B2AR-selective antagonist ICI-118,551 but not with B1AR-selective antagonist bisoprolol. Finally, for clinical relevance, we determined that norepinephrine was present in freshly wounded skin, thus providing a potential mechanism for impaired healing by local B2AR activation in wound-edge keratinocytes. Taken together, the data show that keratinocytes modulate catecholamine synthetic enzymes and release norepinephrine after scratch wounding. Norepinephrine appears to be a stress-related mediator that impairs keratinocyte migration through activation of the B2AR. Future therapeutic strategies evaluating modulation of norepinephrine-related effects in the wound are warranted.

  9. 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.

  10. Genetic ablation of cyclooxygenase-2 in keratinocytes produces a cell-autonomous defect in tumor formation.

    PubMed

    Lao, Huei-Chen; Akunda, Jacqueline K; Chun, Kyung-Soo; Flake, Gordon P; Yuspa, Stuart H; Langenbach, Robert

    2012-11-01

    Using a mouse skin tumor model, we reported previously that cyclooxygenase-2 (COX-2) deficiency reduced papilloma formation. However, this model did not differentiate between the effects of systemic COX-2-deficiency and keratinocyte-specific COX-2 deficiency on tumor formation. To determine whether keratinocyte-specific COX-2 deficiency reduced papilloma formation, v-H-ras-transformed COX-2+/+ and COX-2-/- keratinocytes were grafted onto nude mice and tumor development was compared. Transformed COX-2+/+ and COX-2-/- keratinocytes expressed similar levels of H-ras, epidermal growth factor receptor and phospho-extracellular signal-regulated kinase 1/2 in vitro; and COX-2-deficiency did not reduce uninfected or v-H-ras infected keratinocyte replication. In contrast, tumors arising from grafted transformed COX-2+/+ and COX-2-/- keratinocytes expressed similar levels of H-ras, but COX-2 deficiency reduced phospho-extracellular signal-regulated kinase 1/2 and epidermal growth factor receptor levels 50-60% and tumor volume by 80% at 3 weeks. Two factors appeared to account for the reduced papilloma size. First, papillomas derived from COX-2-/- keratinocytes showed about 70% decreased proliferation, as measured by bromodeoxyuridine incorporation, compared with papillomas derived from COX-2+/+ keratinocytes. Second, keratin 1 immunostaining of papillomas indicated that COX-2-/- keratinocytes prematurely initiated terminal differentiation. Differences in the levels of apoptosis and vascularization did not appear to be contributing factors as their levels were similar in tumors derived from COX-2-/- and COX-2+/+ keratinocytes. Overall, the data are in agreement with our previous observations that decreased papilloma number and size on COX-2-/- mice resulted from reduced keratinocyte proliferation and accelerated keratinocyte differentiation. Furthermore, the data indicate that deficiency/inhibition of COX-2 in the initiated keratinocyte is an important determinant of

  11. Genetic ablation of cyclooxygenase-2 in keratinocytes produces a cell-autonomous defect in tumor formation

    PubMed Central

    Langenbach, Robert

    2012-01-01

    Using a mouse skin tumor model, we reported previously that cyclooxygenase-2 (COX-2) deficiency reduced papilloma formation. However, this model did not differentiate between the effects of systemic COX-2-deficiency and keratinocyte-specific COX-2 deficiency on tumor formation. To determine whether keratinocyte-specific COX-2 deficiency reduced papilloma formation, v-H-ras-transformed COX-2+/+ and COX-2−/− keratinocytes were grafted onto nude mice and tumor development was compared. Transformed COX-2+/+ and COX-2−/− keratinocytes expressed similar levels of H-ras, epidermal growth factor receptor and phospho-extracellular signal-regulated kinase1/2 in vitro; and COX-2-deficiency did not reduce uninfected or v-H-ras infected keratinocyte replication. In contrast, tumors arising from grafted transformed COX-2+/+ and COX-2−/− keratinocytes expressed similar levels of H-ras, but COX-2 deficiency reduced phospho-extracellular signal-regulated kinase 1/2 and epidermal growth factor receptor levels 50–60% and tumor volume by 80% at 3 weeks. Two factors appeared to account for the reduced papilloma size. First, papillomas derived from COX-2−/− keratinocytes showed about 70% decreased proliferation, as measured by bromodeoxyuridine incorporation, compared with papillomas derived from COX-2+/+ keratinocytes. Second, keratin 1 immunostaining of papillomas indicated that COX-2−/− keratinocytes prematurely initiated terminal differentiation. Differences in the levels of apoptosis and vascularization did not appear to be contributing factors as their levels were similar in tumors derived from COX-2−/− and COX-2+/+ keratinocytes. Overall, the data are in agreement with our previous observations that decreased papilloma number and size on COX-2−/− mice resulted from reduced keratinocyte proliferation and accelerated keratinocyte differentiation. Furthermore, the data indicate that deficiency/inhibition of COX-2 in the initiated keratinocyte is an

  12. Identification of glucocorticoid-induced TNF receptor-related protein ligand on keratinocytes: ligation by GITR induces keratinocyte chemokine production and augments T-cell proliferation.

    PubMed

    Byrne, Aideen M; Goleva, Elena; Leung, Donald Y M

    2009-12-01

    Glucocorticoid-induced tumor necrosis factor (TNF) receptor-related protein ligand (GITRL) is a recently described co-stimulatory molecule expressed by antigen-presenting cells (APCs). Activated keratinocytes are known to engage intraepithelial T cells through co-stimulatory molecules. This study investigated the expression and function of GITRL in resting keratinocytes. We showed by immunofluorescence and flow cytometry that keratinocytes from Balb/C and C57Bl/6 mice, as well as PAM 212 murine cell line keratinocytes and human epidermal keratinocytes (HEK), express cell-surface GITRL. Stimulation of murine skin biopsies and HEK with GITR fusion protein (GITR: Fc FP) resulted in mRNA induction for chemoattractants: cutaneous T-cell-attracting chemokine (CTACK), thymus and activation-regulated chemokine (TARC), IL-8, monocyte chemoattractant protein-1 (MCP-1), and murine beta-defensin 3 (MBD3). Immunofluorescent studies on mouse biopsies treated with GITR: Fc FP confirmed corresponding TARC and MCP-1 protein production by keratinocytes. Chemokine induction was shown to be NF-kappaB-mediated. T-cell proliferation was enhanced by the addition of keratinocytes. This was reversed by pretreatment with an anti-GITRL antibody. We conclude that keratinocytes express GITRL, and that through this important co-stimulatory molecule, they have the potential to influence T-cell numbers in the skin through chemokine production and through a direct cell-cell effect on T-cell proliferation.

  13. CCN2 is transiently expressed by keratinocytes during re-epithelialization and regulates keratinocyte migration in vitro by the ras-MEK-ERK signaling pathway.

    PubMed

    Kiwanuka, Elizabeth; Hackl, Florian; Caterson, Edward J; Nowinski, Daniel; Junker, Johan P E; Gerdin, Bengt; Eriksson, Elof

    2013-12-01

    CCN2 (previously known as connective tissue growth factor) is a multifunctional matricellular protein that has numerous effects on cell life and cell interactions with the connective tissue. Although the importance of CCN2 for the fibrotic process in wound healing has been well studied, the involvement of CCN2 in keratinocyte function has not yet been explored. Therefore, the aim of the present study was to investigate the role of CCN2 in the epidermis during wound healing. Immunohistochemistry was done on sections from full-thickness porcine wounds. The effect of CCN2 on the migration of cultured human keratinocytes exposed to scratch wounds, the effect on phosphorylation of extracellular signal-related kinases (ERK), and the effect of adding inhibitors to the ERK/mitogen-activated protein kinase pathway to human keratinocytes were studied. The CCN2 protein was transiently expressed in vivo at the leading keratinocyte edge during re-epithelialization of full-thickness porcine wounds. In vitro, exogenous addition of CCN2 to human keratinocyte cultures regulated keratinocyte migration and resulted in phosphorylation of ERK. The addition of inhibitors of ERK/mitogen-activated protein kinase counteracted the effect of CCN2 on migration. CCN2 was transiently expressed at the leading keratinocyte edge in vivo. The biologic importance of this was supported in vitro, because CCN2 regulated human keratinocyte migration through activation of the Ras-mitogen-activated protein kinase kinase-ERK signal transduction pathway. Published by Elsevier Inc.

  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. 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

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

    PubMed

    Yu, Seon-Mi; Kim, Song Ja

    2016-06-01

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

  17. Disruption of GLUT1 glucose carrier trafficking in L6E9 and Sol8 myoblasts by the phosphatidylinositol 3-kinase inhibitor wortmannin.

    PubMed Central

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

    1995-01-01

    In this study we have used wortmannin, a highly specific inhibitor of phosphatidylinositol (PI) 3-kinase, to assess the role of this enzyme on GLUT1 glucose carrier distribution and glucose transport activity in myoblasts from two skeletal-muscle cell lines, L6E9 and Sol8. As detected in L6E9 cells, myoblasts exhibited basal and insulin-stimulated PI 3-kinase activities. Incubation of intact myoblasts with wortmannin resulted in a marked inhibition of both basal and insulin-stimulated PI 3-kinase activities. L6E9 and Sol8 myoblasts showed basal and insulin-stimulated glucose transport activities, both of them inhibited by wortmannin in a dose-dependent manner (IC50 approximately 10-20 nM). Concomitantly, immunofluorescence analysis revealed that 1 h treatment with wortmannin led to a dramatic intracellular accumulation of GLUT1 carriers (the main glucose transporter expressed in L6E9 and Sol8 myoblasts) in both cell systems. The effect of wortmannin on GLUT1 cellular redistribution was independent of the presence of insulin. The cellular distribution of two structural plasma-membrane components such as beta 1-integrin or the alpha 1 subunit of the Na(+)-K(+)-ATPase were unaffected by wortmannin in both the absence and the presence of insulin. As a whole, our results indicate that PI 3-kinase is necessary to basal and insulin-stimulated glucose transport in L6E9 and Sol8 myoblasts. Moreover, immunofluorescence assays suggest that in both cellular models there is a constitutive GLUT 1 trafficking pathway (independent of insulin) that involves PI 3-kinase and which, when blocked, locks GLUT1 in a perinuclear compartment. Images Figure 1 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8526858

  18. Transcriptional responses of human epidermal keratinocytes to Oncostatin-M.

    PubMed

    Finelt, Nika; Gazel, Alix; Gorelick, Steven; Blumenberg, Miroslav

    2005-08-21

    Oncostatin-M (OsM) plays an important role in inflammatory and oncogenic processes in skin, including psoriasis and Kaposi sarcoma. However, the molecular responses to OsM in keratinocytes have not been explored in depth. Here we show the results of transcriptional profiling in OsM-treated primary human epidermal keratinocytes, using high-density DNA microarrays. We find that OsM strongly and specifically affects the expression of many genes, in particular those involved with innate immunity, angiogenesis, adhesion, motility, tissue remodeling, cell cycle and transcription. The timing of the responses to OsM comprises two waves, early at 1h, and late at 48 h, with much fewer genes regulated in the intervening time points. Secreted cytokines and growth factors and their receptors, as well as nuclear transcription factors, are primary targets of OsM regulation, and these, in turn, effect the secondary changes.

  19. Control of keratinocyte proliferation and differentiation by p63.

    PubMed

    Truong, Amy B; Khavari, Paul A

    2007-02-01

    The p53 family member p63 has been implicated in both the development and maintenance of stratified epithelial tissues, including the epidermis. Increasing data support p63 function in the regenerative capacity of basal keratinocytes by maintaining cell proliferation. Recent studies further suggest this regulation relies on inhibition of p53 activity. In addition, p63 appears to exert separate control over epidermal differentiation, which may involve control of such key signaling molecules as IKKalpha and Notch. While studies over the past decade have greatly expanded our knowledge of p63 function, much remains to be understood regarding how p63 regulates epidermal homeostasis. Future efforts to identify and validate direct p63 target genes as well as to understand the expression and function of individual p63 isoforms will be important to further define how p63 functions in the control of keratinocyte proliferation and differentiation.

  20. Complementation assays adapted for DNA repair-deficient keratinocytes.

    PubMed

    Fréchet, Mathilde; Bergoglio, Valérie; Chevallier-Lagente, Odile; Sarasin, Alain; Magnaldo, Thierry

    2006-01-01

    Genetic alterations affecting nucleotide excision repair, the most versatile DNA-repair mechanism responsible for removal of bulky DNA adducts including ultraviolet (UV) light-induced DNA lesions, may result in the rare, recessively inherited autosomal syndromes xeroderma pigmentosum (XP), Cockayne syndrome (CS), or trichothiodystrophy (TTD). Classical approaches such as somatic cell fusions or microinjection assays have formalized the genetic complexity of these related but clinically distinct syndromes, and contributed to the determination of seven, five, and three complementation groups for XP, CS, and TTD, respectively. XP patients are highly susceptible to photoinduced cutaneous cancers of epidermal origin. To better study the responses to UV irradiation of XP keratinocytes, and to objectively determine the extent to which cutaneous gene therapy may be realized, we set up experimental procedures adapted to ex vivo genetic complementation of keratinocytes from XP patients. We provide here detailed rationales and procedures for these approaches.

  1. Inhibition of JNK promotes differentiation of epidermal keratinocytes.

    PubMed

    Gazel, Alix; Banno, Tomohiro; Walsh, Rebecca; Blumenberg, Miroslav

    2006-07-21

    In inflamed tissue, normal signal transduction pathways are altered by extracellular signals. For example, the JNK pathway is activated in psoriatic skin, which makes it an attractive target for treatment. To define comprehensively the JNK-regulated genes in human epidermal keratinocytes, we compared the transcriptional profiles of control and JNK inhibitor-treated keratinocytes, using DNA microarrays. We identified the differentially expressed genes 1, 4, 24, and 48 h after the treatment with SP600125. Surprisingly, the inhibition of JNK in keratinocyte cultures in vitro induces virtually all aspects of epidermal differentiation in vivo: transcription of cornification markers, inhibition of motility, withdrawal from the cell cycle, stratification, and even production of cornified envelopes. The inhibition of JNK also induces the production of enzymes of lipid and steroid metabolism, proteins of the diacylglycerol and inositol phosphate pathways, mitochondrial proteins, histones, and DNA repair enzymes, which have not been associated with differentiation previously. Simultaneously, basal cell markers, including integrins, hemidesmosome and extracellular matrix components, are suppressed. Promoter analysis of regulated genes finds that the binding sites for the forkhead family of transcription factors are over-represented in the SP600125-induced genes and c-Fos sites in the suppressed genes. The JNK-induced proliferation appears to be secondary to inhibition of differentiation. The results indicate that the inhibition of JNK in epidermal keratinocytes is sufficient to initiate their differentiation program and suggest that augmenting JNK activity could be used to delay cornification and enhance wound healing, whereas attenuating it could be a differentiation therapy-based approach for treating psoriasis.

  2. Hypoxia induces an undifferentiated phenotype of oral keratinocytes in vitro.

    PubMed

    Kato, Hiroko; Izumi, Kenji; Uenoyama, Atsushi; Shiomi, Aki; Kuo, Shiuhyang; Feinberg, Stephen E

    2014-01-01

    The aim of this study was to determine the effects of hypoxia on the proliferating potential and phenotype of primary human oral keratinocytes cultured at ambient oxygen tension (20%) or at different levels of hypoxia (2 and 0.5% O2). The effects of oxygen tensions on cellular metabolic activity, cell proliferation, clonogenicity and proliferation heterogeneity were measured. Cell cycle profiles were analyzed by a fluorescent-activated cell sorter, and p21(WAF1/CIP1) expression in the G0/G1 phase was also concomitantly quantitated. The expression levels of cell cycle regulatory proteins were examined by immunoblotting, and the cellular senescence was assessed by senescence-associated β-galactosidase staining. Basal and suprabasal keratinocyte phenotypes were determined by the expression levels of 14-3-3σ, p75(NTR) and α6 integrin. Despite having a lower metabolism, the proliferation rate and clonogenic potential were remarkably enhanced in hypoxic cells. The significantly higher percentage of cells in the G0/G1 phase under hypoxia and the expression patterns of cell cycle regulatory proteins in hypoxic cells were indicative of a state of cell cycle arrest in hypoxia. Furthermore, a decrease in the expression of p21(WAF1/CIP1) and p16(INK4A) and fewer β-galactosidase-positive cells suggested a quiescent phenotype rather than a senescent one in hypoxic cells. Compared with normoxic cells, the differential expression patterns of keratinocyte phenotypic markers suggest that hypoxic cells that generate minimal reactive oxygen species, suppress the mammalian target of rapamycin activity and express hypoxia-inducible factor-1α favor a basal cell phenotype. Thus, regardless of the predisposition to the state of cell cycle arrest, hypoxic conditions can maintain oral keratinocytes in vitro in an undifferentiated and quiescent state.

  3. Melanoma cells influence the differentiation pattern of human epidermal keratinocytes.

    PubMed

    Kodet, Ondřej; Lacina, Lukáš; Krejčí, Eliška; Dvořánková, Barbora; Grim, Miloš; Štork, Jiří; Kodetová, Daniela; Vlček, Čestmír; Šáchová, Jana; Kolář, Michal; Strnad, Hynek; Smetana, Karel

    2015-01-05

    Nodular melanoma is one of the most life threatening tumors with still poor therapeutic outcome. Similarly to other tumors, permissive microenvironment is essential for melanoma progression. Features of this microenvironment are arising from molecular crosstalk between the melanoma cells (MC) and the surrounding cell populations in the context of skin tissue. Here, we study the effect of melanoma cells on human primary keratinocytes (HPK). Presence of MC is as an important modulator of the tumor microenvironment and we compare it to the effect of nonmalignant lowly differentiated cells also originating from neural crest (NCSC). Comparative morphometrical and immunohistochemical analysis of epidermis surrounding nodular melanoma (n = 100) was performed. Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively. Differentially expressed candidate genes were verified by RT-qPCR. Biological activity of candidate proteins was assessed on cultured HPK. Epidermis surrounding nodular melanoma exhibits hyperplastic features in 90% of cases. This hyperplastic region exhibits aberrant suprabasal expression of keratin 14 accompanied by loss of keratin 10. We observe that MC and NCSC are able to increase expression of keratins 8, 14, 19, and vimentin in the co-cultured HPK. This in vitro finding partially correlates with pseudoepitheliomatous hyperplasia observed in melanoma biopsies. We provide evidence of FGF-2, CXCL-1, IL-8, and VEGF-A participation in the activity of melanoma cells on keratinocytes. We conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro. This interaction further highlights the role of intercellular interactions in melanoma. The reciprocal role of activated keratinocytes on biology of melanoma cells shall be verified in the future.

  4. Climbazole increases expression of cornified envelope proteins in primary keratinocytes.

    PubMed

    Pople, J E; Moore, A E; Talbot, D C S; Barrett, K E; Jones, D A; Lim, F L

    2014-10-01

    Dandruff is a troubling consumer problem characterized by flaking and pruritus of the scalp and is considered a multifactorial condition with sebum, individual susceptibility and the fungus Malassezia all thought to play a part. The condition is commonly treated with shampoo products containing antifungal ingredients such as zinc pyrithione and climbazole. It is hypothesized that these ingredients may be delivering additional scalp skin benefits besides their antifungal activity helping to relieve dandruff effectively. The objective of this study was to evaluate the anti-dandruff ingredient climbazole for potential skin benefits using genomics and in vitro assays. Microarray analysis was performed to profile gene expression changes in climbazole-treated primary human keratinocyte cells. Results were independently validated using qPCR and analysis of protein expression using ELISA and immunocytochemistry. Microarray analysis of climbazole-treated keratinocytes showed statistically significant expression changes in genes associated with the gene ontology groups encompassing epidermal differentiation, keratinization, cholesterol biosynthesis and immune response. Upregulated genes included a number encoding cornified envelope proteins such as group 3 late-cornified envelope proteins, LCE3 and group 2 small-proline-rich proteins, SPRR2. Protein analysis studies of climbazole-treated primary keratinocytes using ELISA and immunocytochemistry were able to demonstrate that the increase in gene transcripts translated into increased protein expression of these cornified envelope markers. Climbazole treatment of primary keratinocytes results in an upregulation in expression of a number of genes including those encoding proteins involved in cornified envelope formation with further studies demonstrating this did translate into increased protein expression. A climbazole-driven increase in cornified envelope proteins may improve the scalp skin barrier, which is known to be weaker

  5. A Model to Predict the Risk of Keratinocyte Carcinomas.

    PubMed

    Whiteman, David C; Thompson, Bridie S; Thrift, Aaron P; Hughes, Maria-Celia; Muranushi, Chiho; Neale, Rachel E; Green, Adele C; Olsen, Catherine M

    2016-06-01

    Basal cell and squamous cell carcinomas of the skin are the commonest cancers in humans, yet no validated tools exist to estimate future risks of developing keratinocyte carcinomas. To develop a prediction tool, we used baseline data from a prospective cohort study (n = 38,726) in Queensland, Australia, and used data linkage to capture all surgically excised keratinocyte carcinomas arising within the cohort. Predictive factors were identified through stepwise logistic regression models. In secondary analyses, we derived separate models within strata of prior skin cancer history, age, and sex. The primary model included terms for 10 items. Factors with the strongest effects were >20 prior skin cancers excised (odds ratio 8.57, 95% confidence interval [95% CI] 6.73-10.91), >50 skin lesions destroyed (odds ratio 3.37, 95% CI 2.85-3.99), age ≥ 70 years (odds ratio 3.47, 95% CI 2.53-4.77), and fair skin color (odds ratio 1.75, 95% CI 1.42-2.15). Discrimination in the validation dataset was high (area under the receiver operator characteristic curve 0.80, 95% CI 0.79-0.81) and the model appeared well calibrated. Among those reporting no prior history of skin cancer, a similar model with 10 factors predicted keratinocyte carcinoma events with reasonable discrimination (area under the receiver operator characteristic curve 0.72, 95% CI 0.70-0.75). Algorithms using self-reported patient data have high accuracy for predicting risks of keratinocyte carcinomas. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Retinoid-Responsive Transcriptional Changes in Epidermal Keratinocytes

    PubMed Central

    Krzyzanowska, Agata; Vouthounis, Constantinos; Blumenberg, Miroslav; Tomic-Canic, Marjana

    2015-01-01

    Retinoids (RA) have been used as therapeutic agents for numerous skin diseases, from psoriasis to acne and wrinkles. While RA is known to inhibit keratinocyte differentiation, the molecular effects of RA in epidermis have not been comprehensively defined. To identify the transcriptional targets of RA in primary human epidermal keratinocytes, we compared the transcriptional profiles of cells grown in the presence or absence of all-trans retinoic acid for 1, 4, 24, 48 and 72 hours, using large DNA microarrays. As expected, RA suppresses the protein markers of cornification; however the genes responsible for biosynthesis of epidermal lipids, long-chain fatty acids, cholesterol, and sphingolipids, are also suppressed. Importantly, the pathways of RA synthesis, esterification and metabolism are activated by RA; therefore, RA regulates its own bioavailability. Unexpectedly, RA regulates many genes associated with the cell cycle and programmed cell death. This led us to reveal novel effects of RA on keratinocyte proliferation and apoptosis. The response to RA is very fast: 315 genes were regulated already after 1 h. More than one-third of RA-regulated genes function in signal transduction and regulation of transcription. Using in silico analysis, we identified a set of over-represented transcription factor binding sites in the RA-regulated genes. Many psoriasis-related genes are regulated by RA, some induced, others suppressed. These results comprehensively document the transcriptional changes caused by RA in keratinocytes, add new insights into the molecular mechanism influenced by RA in the epidermis and demonstrate the hypothesis-generating power of DNA microarray analysis. PMID:19388012

  7. Keratinocytes at the uppermost layer of epidermis might act as sensors of atmospheric pressure change.

    PubMed

    Denda, Mitsuhiro

    2016-01-01

    It has long been suggested that climate, especially atmospheric pressure change, can cause health problems ranging from migraine to myocardial infarction. Here, I hypothesize that the sensory system of epidermal keratinocytes mediates the influence of atmospheric pressure change on the human physiological condition. We previously demonstrated that even subtle changes of atmospheric pressure (5-20 hPa) induce elevation of intracellular calcium level in cultured human keratinocytes (excitation of keratinocytes). It is also established that communication occurs between epidermal keratinocytes and peripheral nerve systems. Moreover, various neurotransmitters and hormones that influence multiple systems (nervous, cardiovascular, endocrine, and immune systems) are generated and released from epidermal keratinocytes in response to various external stimuli. Thus, I suggest that pathophysiological phenomena induced by atmospheric pressure changes might be triggered by epidermal keratinocytes.

  8. Protective effect of geranylgeranylacetone against radiation-induced delayed effects on human keratinocytes.

    PubMed

    Isoir, Muriel; Roque, Telma; Squiban, Claire; Milliat, Fabien; Mondon, Philippe; Mas-Chamberlin, Claire; Benderitter, Marc; Guipaud, Olivier; Tamarat, Radia

    2013-02-01

    Skin exposure to ionizing radiation affects the normal wound healing process. We investigated the beneficial effects of a pharmacological treatment with geranylgeranylacetone (GGA) on keratinocytes using in vitro scratch wound injury assay in nonirradiated and irradiated conditions. Irradiation affected the wound closure of keratinocytes 24 h after scratch injury, whereas re-epithelialization was markedly accelerated after GGA treatment when compared to nontreated keratinocytes. We demonstrated that GGA treatment increased migration of human epidermal keratinocytes and this migratory property was not related to RhoA signaling. Interestingly, Western blot analysis revealed that GGA treatment down-regulated caspase 3 active form expression and up-regulated the activated phenotype by inducing both keratin 6 (K6) expression and interleukin-1β (IL-1β) release without modification of the differentiate phenotype. Finally, the proteomic profiling was performed on keratinocytes, showing that global protein changes occurred after irradiation of keratinocytes treated or untreated with GGA.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. p70S6 kinase is a critical node that integrates HER-family and PI3 kinase signaling networks

    PubMed Central

    Axelrod, Mark J.; Gordon, Vicki; Mendez, Rolando E.; Leimgruber, Stephanie S.; Conaway, Mark R.; Sharlow, Elizabeth R.; Jameson, MarkJ.; Gioeli, Daniel G.; Weber, Michael J.

    2014-01-01

    Therapies targeting oncogenic drivers rapidly induce compensatory adaptive responses that blunt drug effectiveness, contributing to therapeutic resistance. Adaptive responses are characteristic of robust cell signaling networks, and thus there is increasing interest in drug combinations that co-target the driver and the adaptive response. An alternative approach to co-inhibiting oncogenic and adaptive targets is to identify a critical node where the activities of these targets converge. Nodes of convergence between signaling modules represent potential therapeutic vulnerabilities because their inhibition could result in collapse of the network, leading to enhanced cytotoxicity. In this report we demonstrate that p70S6 kinase (p70S6K) can function as a critical node linking HER-family and phosphoinositide-3-kinase (PI3K) pathway signaling. We used high-throughput combinatorial drug screening to identify adaptive survival responses to targeted therapies, and found that HER-family and PI3K represented compensatory signaling pathways. Co-targeting these pathways with drug combinations caused synergistic cytotoxicity in cases where inhibition of neither target was effective as a monotherapy. We utilized Reverse Phase Protein Arrays and determined that phosphorylation of ribosomal protein S6 was synergistically down-regulated upon HER-family and PI3K/mammalian target of rapamycin (mTOR) co-inhibition. Expression of constitutively active p70S6K protected against apoptosis induced by combined HER-family and PI3K/mTOR inhibition. Direct inhibition of p70S6K with small molecule inhibitors phenocopied HER-family and PI3K/mTOR co-inhibition. These data implicate p70S6K as a critical node in the HER-family/PI3K signaling network. The ability of direct inhibitors of p70S6K to phenocopy co-inhibition of two upstream signaling targets indicates that identification and targeting of critical nodes can overcome adaptive resistance to targeted therapies. PMID:24662264

  15. Signalling mechanisms mediated by the phosphoinositide 3-kinase/Akt cascade in synaptic plasticity and memory in the rat.

    PubMed

    Horwood, Jennifer M; Dufour, Franck; Laroche, Serge; Davis, Sabrina

    2006-06-01

    The phosphoinositide 3-kinase (PI3K)/Akt signalling cascade has classically been implicated in promoting cell survival but more recently has been shown to regulate a number of other cellular functions. In particular, studies have suggested that PI3K contributes to mechanisms associated with synaptic plasticity and memory processes but the function of this cascade in forms of synaptic plasticity, such as long-term potentiation, remains controversial and the PI3K substrates which mediate these effects are poorly understood. Here we report that the PI3K inhibitor LY294002 infused i.c.v. in vivo blocked maintenance of long-term potentiation induced in the dentate gyrus with a single tetanus to the perforant path but not with repeated tetani. This pattern of stimulation led to rapid and transient phosphorylation of the PI3K substrate Akt at Ser473 but not at Thr308. Functional readout of partial activation of Akt was demonstrated by an increase in phosphorylation of two downstream substrates, Forkhead (FKHR) and mammalian target of rapamycin (mTOR), in a delayed and prolonged manner at Akt-specific phosphorylation sites. LY294002 blocked phosphorylation of Akt and the prolonged phosphorylation of FKHR and mTOR but did not impair long-term potentiation-induced phosphorylation of extracellular receptor kinase. In addition, the same i.c.v. concentration of LY294002 impaired long-term consolidation of recognition memory but not short-term recognition memory or spatial learning and repeated training in the recognition memory task overcame the deficit in consolidation. These results suggest that activation of the PI3K/Akt pathway may contribute to the mechanisms of synaptic plasticity and memory consolidation by promoting cell survival via FKHR and protein synthesis via mTOR. Importantly, only partial activation of Akt at its Ser473 residue was necessary to mediate these effects.

  16. 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.

  17. 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.

  18. 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.

  19. Chronic Blockade of Phosphatidylinositol 3-Kinase in the Nucleus Tractus Solitarii Is Prohypertensive in the Spontaneously Hypertensive Rat

    PubMed Central

    Zubcevic, Jasenka; Waki, Hidefumi; Diez-Freire, Carlos; Gampel, Alexandra; Raizada, Mohan K.; Paton, Julian F.R.

    2009-01-01

    Phosphatidylinositol 3-kinase (PI3K) within brain stem neurons has been implicated in hypertension in the spontaneously hypertensive rat (SHR). Previously, we demonstrated elevated expression of PI3K subunits in rostral ventrolateral medulla and paraventricular nucleus of SHRs compared with Wistar-Kyoto rats. Here, we considered expression levels of PI3K in the nucleus tractus solitarii, a pivotal region in reflex regulation of arterial pressure, and determined its functional role for arterial pressure homeostasis in SHRs and Wistar-Kyoto rats. We found elevated mRNA levels of p110β and p110δ catalytic PI3K subunits in the nucleus tractus solitarii of adult (12 to 14 weeks old) SHRs relative to the age-matched Wistar-Kyoto rats (fold differences relative to β-actin: 1.7±0.2 versus 1.01±0.08 for p110β, n=6, P<0.05; 1.62±0.15 versus 1.02±0.1 for p110δ, n=6, P<0.05). After chronic blockade of PI3K signaling in the nucleus tractus solitarii by lentiviral-mediated expression of a mutant form of p85α, systolic pressure increased from 175±3 mm Hg to 191±6 mm Hg (P<0.01) in SHRs but not in Wistar-Kyoto rats. In addition, heart rate increased (from 331±6 to 342±6 bpm; P<0.05) and spontaneous baroreflex gain decreased (from 0.7±0.07 to 0.5±0.04 ms/mm Hg; P<0.001) in the SHRs. Thus, PI3K signaling in the nucleus tractus solitarii of SHR restrains arterial pressure in this animal model of neurogenic hypertension. PMID:19015400

  20. 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.

  1. Klebsiella pneumoniae Translocates across the Intestinal Epithelium via Rho GTPase- and Phosphatidylinositol 3-Kinase/Akt-Dependent Cell Invasion

    PubMed Central

    Hsu, Chun-Ru; Pan, Yi-Jiun; Liu, Ju-Yun; Chen, Chun-Tang; Lin, Tzu-Lung

    2014-01-01

    Klebsiella pneumoniae is an important pathogen that causes hospital-acquired septicemia and is associated with the recent emergence of community-acquired pyogenic liver abscess (PLA). Clinical typing suggests that K. pneumoniae infections originate from the gastrointestinal reservoir. However, the underlying mechanism remains unknown. Here, we have sought to determine how K. pneumoniae penetrates the intestinal barrier. We identified that bacteremia and PLA clinical isolates adhered to and invaded intestinal epithelial cells. Internalization of K. pneumoniae in three different human colonic cell lines was visualized by confocal microscopy and three-dimensional (3D) imaging. Using a Transwell system, we demonstrated that these K. pneumoniae isolates translocated across a polarized Caco-2 monolayer. No disruptions of transepithelial electrical resistance and altered distribution of tight junction protein ZO-1 or occludin were observed. Therefore, K. pneumoniae appeared to penetrate the intestinal epithelium via a transcellular pathway. Using specific inhibitors, we characterized the host signaling pathways involved. Inhibition by cytochalasin D and nocodazole suggested that actin and microtubule cytoskeleton were both important for K. pneumoniae invasion. A Rho inhibitor, ML141, LY294002, and an Akt1/2 inhibitor diminished K. pneumoniae invasion in a dose-dependent manner, indicating that Rho family GTPases and phosphatidylinositol 3-kinase (PI3K)/Akt signaling were required. By a mouse model of gastrointestinal colonization, in vivo invasion of K. pneumoniae into colonic epithelial cells was demonstrated. Our results present evidence to describe a possible mechanism of gastrointestinal translocation for K. pneumoniae. Cell invasion by manipulating host machinery provides a pathway for gut-colonized K. pneumoniae cells to penetrate the intestinal barrier and access extraintestinal locations to cause disease. PMID:25452552

  2. Klebsiella pneumoniae translocates across the intestinal epithelium via Rho GTPase- and phosphatidylinositol 3-kinase/Akt-dependent cell invasion.

    PubMed

    Hsu, Chun-Ru; Pan, Yi-Jiun; Liu, Ju-Yun; Chen, Chun-Tang; Lin, Tzu-Lung; Wang, Jin-Town

    2015-02-01

    Klebsiella pneumoniae is an important pathogen that causes hospital-acquired septicemia and is associated with the recent emergence of community-acquired pyogenic liver abscess (PLA). Clinical typing suggests that K. pneumoniae infections originate from the gastrointestinal reservoir. However, the underlying mechanism remains unknown. Here, we have sought to determine how K. pneumoniae penetrates the intestinal barrier. We identified that bacteremia and PLA clinical isolates adhered to and invaded intestinal epithelial cells. Internalization of K. pneumoniae in three different human colonic cell lines was visualized by confocal microscopy and three-dimensional (3D) imaging. Using a Transwell system, we demonstrated that these K. pneumoniae isolates translocated across a polarized Caco-2 monolayer. No disruptions of transepithelial electrical resistance and altered distribution of tight junction protein ZO-1 or occludin were observed. Therefore, K. pneumoniae appeared to penetrate the intestinal epithelium via a transcellular pathway. Using specific inhibitors, we characterized the host signaling pathways involved. Inhibition by cytochalasin D and nocodazole suggested that actin and microtubule cytoskeleton were both important for K. pneumoniae invasion. A Rho inhibitor, ML141, LY294002, and an Akt1/2 inhibitor diminished K. pneumoniae invasion in a dose-dependent manner, indicating that Rho family GTPases and phosphatidylinositol 3-kinase (PI3K)/Akt signaling were required. By a mouse model of gastrointestinal colonization, in vivo invasion of K. pneumoniae into colonic epithelial cells was demonstrated. Our results present evidence to describe a possible mechanism of gastrointestinal translocation for K. pneumoniae. Cell invasion by manipulating host machinery provides a pathway for gut-colonized K. pneumoniae cells to penetrate the intestinal barrier and access extraintestinal locations to cause disease. Copyright © 2015, American Society for Microbiology. All

  3. 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

  4. Early Activation of Phosphatidylinositol 3-Kinase after Ischemic Stroke Reduces Infarct Volume and Improves Long-Term Behavior.

    PubMed

    Kim, Young Seo; Yoo, Arum; Son, Jeong Woo; Kim, Hyun Young; Lee, Young-Jun; Hwang, Sejin; Lee, Kyu-Yong; Lee, Young Joo; Ayata, Cenk; Kim, Hyung-Hwan; Koh, Seong-Ho

    2017-09-01

    Phosphatidylinositol 3-kinases (PI3Ks) have recently been implicated in apoptosis and ischemic cell death. We tested the efficacy of early intervention with a peptide PI3K activator in focal cerebral ischemia. After determining the most effective dose (24 μg/kg) and time window (2 h after MCAO) of treatment, a total of 48 rats were subjected to middle cerebral artery occlusion (MCAO). Diffusion weighted MRI (DWI) was performed 1 h after MCAO and rats with lesion sizes within a predetermined range were randomized to either PI3K activator or vehicle treatment arms. Fluid attenuated inversion recovery (FLAIR) MRI, neurological function, western blots, and immunohistochemistry were blindly assessed. Initial DWI lesion volumes were nearly identical between two groups prior to treatment. However, FLAIR showed significantly smaller infarct volumes in the PI3K activator group compared with vehicle (146 ± 81 mm(3) and 211 ± 96 mm(3), p = 0.045) at 48 h. The PI3K activator group also had better neurological function for up to 2 weeks. In addition, PI3K activator decreased the number of TUNEL-positive cells in the peri-infarct region compared with the control group. Western blot and immunohistochemistry showed increased expression of phosphorylated Akt (Ser473) and GSK-3β (Ser9) and decreased expression of cleaved caspase-9 and caspase-3. Our results suggest a neuroprotective role of early activation of PI3K in ischemic stroke. The use of DWI in the randomization of experimental groups may reduce bias.

  5. 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.

  6. A novel macrolide/fluoroketolide, solithromycin (CEM-101), reverses corticosteroid insensitivity via phosphoinositide 3-kinase pathway inhibition

    PubMed Central

    Kobayashi, Y; Wada, H; Rossios, C; Takagi, D; Charron, C; Barnes, P J; Ito, K

    2013-01-01

    Background and Purpose Corticosteroid insensitivity is a major therapeutic problem for some inflammatory diseases including chronic obstructive pulmonary disease (COPD), and it is known to be induced by reduced histone deacetylase (HDAC)-2 activities via activation of the phosphoinositide 3-kinase (PI3K) pathway. The aim of this study is to evaluate effects of a novel macrolide/fluoroketolide, solithromycin (SOL, CEM-101), on corticosteroid sensitivity induced by oxidative stress. Experimental Approach Corticosteroid sensitivity was determined by IC50/EC50 of dexamethasone (Dex) on TNF-α-induced CXCL8 production in U937 monocytic cell line and peripheral blood mononuclear cells (PBMC) from COPD patients. Activities of HDAC and protein phosphatase 2A (PP2A) were measured by fluorescence-based assay in cells exposed to hydrogen peroxide (H2O2). We also investigated steroid insensitive airway neutrophilia in cigarette smoke exposed mice in vivo. Key Results SOL (10 μM) restored Dex sensitivity in PBMC from COPD patients, H2O2-treated U937 cells and phorbol 12-myristate 13-acetate-differentiated U937 cells. In addition, SOL restored HDAC activity with concomitant inhibition of Akt phosphorylation as surrogate marker of PI3K activation. The inhibition of Akt phosphorylation by SOL was due to increased PP2A phosphatase activity, which was reduced in COPD and oxidative stress model. Other known macrolides, such as eryhthromycin, clarithromycin and azithromycin, were significantly less effective in these responses. In cigarette smoke-exposed mice, SOL (100 mg kg−1, po) showed significant but weak inhibition of neutrophilia, whereas Dex (10 mg kg−1, p.o.) showed no such effect. However, a combination of SOL and Dex inhibited neutrophilia by over 50%. Conclusions and Implications SOL has potential as novel therapy for corticosteroid-insensitive diseases such as COPD. PMID:23758162

  7. 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.

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

    PubMed Central

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

    2009-01-01

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

  9. Liver Dysfunction and Phosphatidylinositol-3-Kinase Signalling in Early Sepsis: Experimental Studies in Rodent Models of Peritonitis

    PubMed Central

    Westermann, Martin; Lambeck, Sandro; Lupp, Amelie; Rudiger, Alain; Dyson, Alex; Carré, Jane E.; Kortgen, Andreas; Krafft, Christoph; Popp, Jürgen; Sponholz, Christoph; Fuhrmann, Valentin; Hilger, Ingrid; Claus, Ralf A.; Riedemann, Niels C.; Wetzker, Reinhard; Singer, Mervyn; Trauner, Michael; Bauer, Michael

    2012-01-01

    Background Hepatic dysfunction and jaundice are traditionally viewed as late features of sepsis and portend poor outcomes. We hypothesized that changes in liver function occur early in the onset of sepsis, yet pass undetected by standard laboratory tests. Methods and Findings In a long-term rat model of faecal peritonitis, biotransformation and hepatobiliary transport were impaired, depending on subsequent disease severity, as early as 6 h after peritoneal contamination. Phosphatidylinositol-3-kinase (PI3K) signalling was simultaneously induced at this time point. At 15 h there was hepatocellular accumulation of bilirubin, bile acids, and xenobiotics, with disturbed bile acid conjugation and drug metabolism. Cholestasis was preceded by disruption of the bile acid and organic anion transport machinery at the canalicular pole. Inhibitors of PI3K partially prevented cytokine-induced loss of villi in cultured HepG2 cells. Notably, mice lacking the PI3Kγ gene were protected against cholestasis and impaired bile acid conjugation. This was partially confirmed by an increase in plasma bile acids (e.g., chenodeoxycholic acid [CDCA] and taurodeoxycholic acid [TDCA]) observed in 48 patients on the day severe sepsis was diagnosed; unlike bilirubin (area under the receiver-operating curve: 0.59), these bile acids predicted 28-d mortality with high sensitivity and specificity (area under the receiver-operating curve: CDCA: 0.77; TDCA: 0.72; CDCA+TDCA: 0.87). Conclusions Liver dysfunction is an early and commonplace event in the rat model of sepsis studied here; PI3K signalling seems to play a crucial role. All aspects of hepatic biotransformation are affected, with severity relating to subsequent prognosis. Detected changes significantly precede conventional markers and are reflected by early alterations in plasma bile acids. These observations carry important implications for the diagnosis of liver dysfunction and pharmacotherapy in the critically ill. Further clinical work is

  10. Phosphatidylinositol 3-kinase-dependent signaling modulates taurochenodeoxycholic acid-induced liver injury and cholestasis in perfused rat livers.

    PubMed

    Rust, Christian; Bauchmuller, Kris; Fickert, Peter; Fuchsbichler, Andrea; Beuers, Ulrich

    2005-07-01

    Taurochenodeoxycholic acid (TCDCA), but not glycochenodeoxycholic acid (GCDCA), activates a phosphatidylinositol 3-kinase (PI3-K)-mediated survival pathway in vitro. Here, the effects of PI3-K inhibition on TCDCA- and GCDCA-induced hepatocellular injury, apoptosis, and bile secretion were examined in the intact liver. In isolated perfused rat livers, bile flow was determined gravimetrically. Hepatovenous lactate dehydrogenase and alanine aminotransferase efflux as markers of liver integrity and biliary secretion of 2,4-dinitrophenyl-S-glutathione (DNP-GS) were determined photometrically. Apoptosis was assessed by immunohistochemistry of active caspase-3 and cytokeratin 18 in liver tissue. Phosphorylation of protein kinase B (PKB/Akt) as a readout of PI3-K activity was determined by immunoblot analysis. Bile acid concentrations were determined by gas chromatography. TCDCA (25 muM) induced moderate liver injury by hepatocellular apoptosis and distinctly reduced bile flow and DNP-GS secretion. In contrast, GCDCA (25 muM) induced severe liver injury by extensive hepatocyte apoptosis. TCDCA strongly activated PI3-K, whereas GCDCA did not markedly affect PI3-K activity. Inhibition of PI3-K by 100 nM wortmannin enhanced TCDCA-induced liver injury and apoptosis and tended to aggravate the cholestatic effect of TCDCA. In contrast, wortmannin reduced GCDCA-induced liver injury and apoptosis. Bile acid uptake tended to be reduced by wortmannin. The cholestatic effect of GCDCA was aggravated by wortmannin. Inhibition of PI3-K markedly aggravated TCDCA-induced but not GCDCA-induced liver damage and hepatocyte apoptosis. Thus TCDCA appears to block its inherent toxicity by a PI3-K-dependent survival pathway in the intact liver.

  11. 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.

  12. 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.

  13. Phosphoinositide 3-kinase gamma mediates angiotensin II-induced stimulation of L-type calcium channels in vascular myocytes.

    PubMed

    Quignard, J F; Mironneau, J; Carricaburu, V; Fournier, B; Babich, A; Nurnberg, B; Mironneau, C; Macrez, N

    2001-08-31

    Previous results have shown that in rat portal vein myocytes the betagamma dimer of the G(13) protein transduces the angiotensin II-induced stimulation of calcium channels and increase in intracellular Ca(2+) concentration through activation of phosphoinositide 3-kinase (PI3K). In the present work we determined which class I PI3K isoforms were involved in this regulation.