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Sample records for 3-kinase pathway-dependent keratinocyte

  1. 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.

  2. 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

  3. Distinct roles for extracellular-signal-regulated protein kinase (ERK) mitogen-activated protein kinases and phosphatidylinositol 3-kinase in the regulation of Mcl-1 synthesis.

    PubMed Central

    Schubert, K M; Duronio, V

    2001-01-01

    Alterations in the expression of various Bcl-2 family members may act as one means by which a cell's survival may be regulated. The mechanism by which cytokines regulate expression of Bcl-2 family members was examined in the haemopoietic cell line TF-1. Cytokine-induced Mcl-1 protein expression was shown to be controlled through a pathway dependent upon phosphatidylinositol 3-kinase (PI 3-kinase). The cytokine-induced increase in mRNA transcription was not dependent upon PI 3-kinase, thus dissociating the immediate-early transcription factors responsible for Mcl-1 transcription from the PI 3-kinase signalling pathway. In contrast, Mcl-1 mRNA levels were dependent upon MEK [mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated protein kinase kinase] activation, suggesting a role for the Ras/MEK/MAPK pathway in Mcl-1 transcription. Activation of PI 3-kinase was shown to be necessary to stimulate Mcl-1 protein translation. This was not due to any effect on prolonging the half-life of the protein. Finally, the lipid second messenger ceramide was shown to cause a reduction in Mcl-1 protein translation, probably via its ability to inhibit protein kinase B activation, providing further clues regarding the death-inducing effect of this lipid. PMID:11368774

  4. Endothelial PI 3-kinase activity regulates lymphocyte diapedesis.

    PubMed

    Nakhaei-Nejad, Maryam; Hussain, Amer M; Zhang, Qiu-Xia; Murray, Allan G

    2007-12-01

    Lymphocyte recruitment to sites of inflammation involves a bidirectional series of cues between the endothelial cell (EC) and the leukocyte that culminate in lymphocyte migration into the tissue. Remodeling of the EC F-actin cytoskeleton has been observed after leukocyte adhesion, but the signals to the EC remain poorly defined. We studied the dependence of peripheral blood lymphocyte transendothelial migration (TEM) through an EC monolayer in vitro on EC phosphatidylinositol 3-kinase (PI 3-kinase) activity. Lymphocytes were perfused over cytokine-activated EC using a parallel-plate laminar flow chamber. Inhibition of EC PI 3-kinase activity using LY-294002 or wortmannin decreased lymphocyte TEM (48 +/- 6 or 34 +/- 7%, respectively, vs. control; mean +/- SE; P < 0.05). Similarly, EC knockdown of the p85alpha regulatory subunit of PI 3-kinase decreased lymphocyte transmigration. Treatment of EC with jasplakinolide to inhibit EC F-actin remodeling also decreased lymphocyte TEM to 24 +/- 10% vs. control (P < 0.05). EC PI 3-kinase inhibition did not change the strength of lymphocyte adhesion to the EC or formation of the EC "docking structure" after intercellular adhesion molecule-1 ligation, whereas this was inhibited by jasplakinolide treatment. A similar fraction of lymphocytes migrated on control or LY-294002-treated EC and localized to interendothelial junctions. However, lymphocytes failed to extend processes below the level of vascular endothelial (VE)-cadherin on LY-294002-treated EC. Together these observations indicate that EC PI 3-kinase activity and F-actin remodeling are required during lymphocyte diapedesis and identify a PI 3-kinase-dependent step following initial separation of the VE-cadherin barrier.

  5. 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

  6. The Role of Phosphoinositide 3-Kinase Signaling in Intestinal Inflammation

    PubMed Central

    Cahill, Catherine M.; Rogers, Jack T.; Walker, W. Allan

    2012-01-01

    The phosphatidylinositol 3-kinase signaling pathway plays a central role in regulating the host inflammatory response. The net effect can either be pro- or anti-inflammatory depending on the system and cellular context studied. This paper focuses on phosphatidylinositol 3-kinase signaling in innate and adaptive immune cells of the intestinal mucosa. The role of phosphatidylinositol 3-kinase signaling in mouse models of inflammatory bowel disease is also discussed. With the development of new isoform specific inhibitors, we are beginning to understand the specific role of this complex pathway, in particular the role of the γ isoform in intestinal inflammation. Continued research on this complex pathway will enhance our understanding of its role and provide rationale for the design of new approaches to intervention in chronic inflammatory conditions such as inflammatory bowel disease. PMID:22570785

  7. Beta Adrenergic Receptors in Keratinocytes

    PubMed Central

    Sivamani, Raja K.; Lam, Susanne T.; Isseroff, R. Rivkah

    2007-01-01

    Synopsis Beta2 adrenergic receptors were identified in keratinocytes more than 30 years ago, but their function in the epidermis continues to be elucidated. Abnormalities in their expression, signaling pathway, or in the generation of endogenous catecholamine agonists by keratinocytes have been implicated in the pathogenesis of cutaneous diseases such as atopic dermatitis, vitiligo and psoriasis. New studies also indicate that the beta2AR also modulates keratinocyte migration, and thus can function to regulate wound re-epithelialization. This review focuses on the function of these receptors in keratinocytes and their contribution to cutaneous physiology and disease. PMID:17903623

  8. 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

  9. PtdIns 3-Kinase Orchestrates Autophagosome Formation in Yeast

    PubMed Central

    Obara, Keisuke; Ohsumi, Yoshinori

    2011-01-01

    Eukaryotic cells can massively transport their own cytoplasmic contents into a lytic compartment, the vacuole/lysosome, for recycling through a conserved system called autophagy. The key process in autophagy is the sequestration of cytoplasmic contents within a double-membrane structure, the autophagosome. Autophagosome formation requires the elaborate cooperation of Atg (autophagy-related) proteins and lipid molecules. Phosphorylation of phosphatidylinositol (PtdIns) by a PtdIns 3-kinase, Vps34, is a key step in coordinating Atg proteins and lipid molecules. Vps34 forms two distinct protein complexes, only one of which is involved in generating autophagic membranes. Upon induction of autophagy, PtdIns(3)P, the enzymatic product of PtdIns 3-kinase, is massively transported into the lumen of the vacuole via autophagy. PtdIns(3)P is enriched on the inner membrane of the autophagosome. PtdIns(3)P recruits the Atg18−Atg2 complex and presumably other Atg proteins to autophagic membranes, thereby coordinating lipid molecules and Atg proteins. PMID:21490802

  10. 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

  11. Susi, a negative regulator of Drosophila PI3-kinase.

    PubMed

    Wittwer, Franz; Jaquenoud, Malika; Brogiolo, Walter; Zarske, Marcel; Wüstemann, Philipp; Fernandez, Rafael; Stocker, Hugo; Wymann, Matthias P; Hafen, Ernst

    2005-06-01

    The Phosphatidylinositol-3 kinase/Protein Kinase B (PI3K/PKB) signaling pathway controls growth, metabolism, and lifespan in animals, and deregulation of its activity is associated with diabetes and cancer in humans. Here, we describe Susi, a coiled-coil domain protein that acts as a negative regulator of insulin signaling in Drosophila. Whereas loss of Susi function increases body size, overexpression of Susi reduces growth. We provide genetic evidence that Susi negatively regulates dPI3K activity. Susi directly binds to dP60, the regulatory subunit of dPI3K. Since Susi has no overt similarity to known inhibitors of PI3K/PKB signaling, it defines a novel mechanism by which this signaling cascade is kept in check. The fact that Susi is expressed in a circadian rhythm, with highest levels during the night, suggests that Susi attenuates insulin signaling during the fasting period.

  12. 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.

  13. Interleukin-1 of cholesteatomatous keratinocytes.

    PubMed

    Kakiuchi, H; Kinoshita, K; Katoh, Y; Tabata, T

    1992-10-01

    Interleukin-1 (IL-1) has been thought to be one of the essential cytokines mainly produced by macrophages. It has recently been reported that epidermal keratinocytes produce IL-1, and attention is being paid to local immune reactions mediated with this cytokine. Interleukin-1 not only activates lymphocytes, but also acts as an osteoclast-activating factor. In this study, we used immunohistochemistry and immunoblotting on cholesteatomatous epithelium with anti-IL-1 alpha antibody and anti-IL-1 beta antibody. Next, the relationship of cholesteatomatous debris to the production of IL-1 by keratinocytes was evaluated. Highly concentrated IL-1 alpha was found in the cholesteatomatous epithelium, especially in the basal cell layer. The intensity of IL-1 beta staining was weaker than that of IL-1 alpha staining. In the immunoblotting study, the 31 kd band, an intracellular immature precursor molecule, was identified. The production of IL-1 alpha from keratinocytes was augmented to a greater degree by cholesteatomatous debris than by lipopolysaccharide or keratin. The keratinocytes did not produce IL-1 beta. These findings suggest that IL-1 alpha is derived from cholesteatomatous keratinocytes. Interleukin-1, mainly IL-1 alpha, from the stimulated cholesteatomatous keratinocytes may be an important factor in the markedly increased bone resorption observed in cholesteatoma.

  14. Class I PI 3-kinases: Function and evolution.

    PubMed

    Kriplani, Nisha; Hermida, Miguel A; Brown, Euan R; Leslie, Nicholas R

    2015-09-01

    In many human cell types, the class I phosphoinositide 3-kinases play key roles in the control of diverse cellular processes including growth, proliferation, survival and polarity. This is achieved through their activation by many cell surface receptors, leading to the synthesis of the phosphoinositide lipid signal, PIP3, which in turn influences the function of numerous direct PIP3-binding proteins. Here we review PI3K pathway biology and analyse the evolutionary distribution of its components and their functions. The broad phylogenetic distribution of class I PI3Ks in metazoa, amoebozoa and choannoflagellates, implies that these enzymes evolved in single celled organisms and were later co-opted into metazoan intercellular communication. A similar distribution is evident for the AKT and Cytohesin groups of downstream PIP3-binding proteins, with other effectors and pathway components appearing to evolve later. The genomic and functional phylogeny of regulatory systems such as the PI3K pathway provides a framework to improve our understanding of the mechanisms by which key cellular processes are controlled in humans.

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

    PubMed

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

    2010-04-01

    In vertebrates and some invertebrates, odorant molecules bind to G protein-coupled receptors 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 G protein-coupled receptor-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 PI3Kgamma and cloned a gene for a PI3K with amino acid homology with PI3Kbeta. The lobster olfactory PI3K co-immunoprecipitates with the G protein alpha and beta subunits, and an odorant-evoked increase in phosphatidylinositol (3,4,5)-trisphosphate can be detected in the signal transduction compartment of the ORNs. PI3Kgamma and beta 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.

  16. Phosphatidylinositol-3-kinase regulates PKCtheta activity in cytotoxic T cells.

    PubMed

    Puente, Lawrence G; Mireau, Laura R; Lysechko, Tara L; Ostergaard, Hanne L

    2005-06-01

    Protein kinase C (PKC) theta plays a crucial role in T cell activation. We, therefore, examined the regulation of PKCtheta activity in cytotoxic T lymphocytes (CTL). We demonstrated that PMA did not stimulate PKCtheta activation and phospholipase C inhibition did not block anti-CD3-stimulated PKCtheta activation in a CTL clone. This suggests that diacylglycerol is neither sufficient nor required for PKCtheta activation. Furthermore, PKCtheta was only activated in a CTL clone stimulated with plate-bound anti-CD3 but not soluble anti-CD3. However, PMA or cross-linked anti-CD3 stimulated phosphorylation of PKCtheta as measured by a migratory shift, suggesting that phosphorylation was not sufficient for activity. Phosphatidylinositol 3-kinase activity was required for anti-CD3, but not PMA, stimulated phosphorylation and for immobilized anti-CD3-triggered PKCtheta activity. A substantial fraction of PKCtheta was constitutively membrane associated and PMA or CD3 stimulation did not significantly increase membrane association. Our data indicate that phosphorylation of PKCtheta is not a suitable surrogate measurement for PKCtheta activity and that additional, yet to be defined steps, are required for the regulation of PKCtheta enzymatic activity in CTL.

  17. Human Keratinocytes Are Vanilloid Resistant

    PubMed Central

    Pecze, László; Szabó, Kornélia; Széll, Márta; Jósvay, Katalin; Kaszás, Krisztián; Kúsz, Erzsébet; Letoha, Tamás; Prorok, János; Koncz, István; Tóth, András; Kemény, Lajos; Vizler, Csaba; Oláh, Zoltán

    2008-01-01

    Background Use of capsaicin or resiniferatoxin (RTX) as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1) permanently and selectively removes nociceptive neurons by Ca2+-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects. Methods To address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies. Results Although both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca2+-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1–50 nM) of vanilloids. The TRPV1-mediated and non-receptor specific Ca2+-cytotoxity ([RTX]>15 µM) could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca2+-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes. Conclusion TRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar) doses of RTX. This additional safety information might be useful for planning future human clinical trials. PMID:18852901

  18. Inositol polyphosphate multikinase is a physiologic PI3-kinase that activates Akt/PKB.

    PubMed

    Maag, David; Maxwell, Micah J; Hardesty, Douglas A; Boucher, Katie L; Choudhari, Namrata; Hanno, Adam G; Ma, Jenny F; Snowman, Adele S; Pietropaoli, Joseph W; Xu, Risheng; Storm, Phillip B; Saiardi, Adolfo; Snyder, Solomon H; Resnick, Adam C

    2011-01-25

    The second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), formed by the p110 family of PI3-kinases, promotes cellular growth, proliferation, and survival, in large part by activating the protein kinase Akt/PKB. We show that inositol polyphosphate multikinase (IPMK) physiologically generates PIP(3) as well as water soluble inositol phosphates. IPMK deletion reduces growth factor-elicited Akt signaling and cell proliferation caused uniquely by loss of its PI3-kinase activity. Inhibition of p110 PI3-kinases by wortmannin prevents IPMK phosphorylation and activation. Thus, growth factor stimulation of Akt signaling involves PIP(3) generation through the sequential activations of the p110 PI3-kinases and IPMK. As inositol phosphates inhibit Akt signaling, IPMK appears to act as a molecular switch, inhibiting or stimulating Akt via its inositol phosphate kinase or PI3-kinase activities, respectively. Drugs regulating IPMK may have therapeutic relevance in influencing cell proliferation.

  19. DNA repair in cultured keratinocytes.

    PubMed

    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. We have examined 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. In addition, since portions of human skin are chronically exposed to sunlight, we have assessed the repair response to ultraviolet (UV) irradiation (254 nm) when the cells are conditioned by chronic low-level UV irradiation. The methods of Liu and Karasek were used to grow pure keratinocytes on collagen gels following their isolation from abdominal skin of newborns and adults at autopsy. Density labeling with 5-bromodeoxyuridine was used to resolve repair replication from the semiconservative mode. We found similar repair characteristics in human epidermal keratinocytes to those previously reported for cultured fibroblasts. However, the DNA repair response in basal cells was much greater than that in differentiated cells from the same skin preparation. Our 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.

  20. 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.

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

    PubMed Central

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

    2016-01-01

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

  2. 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.

  3. Phosphatidylinositol 3-kinase association with the osteoclast cytoskeleton, and its involvement in osteoclast attachment and spreading.

    PubMed

    Lakkakorpi, P T; Wesolowski, G; Zimolo, Z; Rodan, G A; Rodan, S B

    1997-12-15

    Osteoclast activation involves attachment to the mineralized bone matrix and reorganization of the cytoskeleton, leading to polarization of the cell. Signaling molecules, PI3-kinase, rho A, and pp60c-src, were shown to be essential for osteoclastic bone resorption. In this study we have focused on the involvement of these signaling molecules in the early event of osteoclast activation: attachment, spreading, and organization of the cytoskeleton. Highly purified osteoclasts were fractionated into Triton X-100-soluble or cytosolic and Triton X-100-insoluble or cytoskeletal fractions, and the distribution of above-mentioned signaling molecules between the two fractions was examined. PI3-kinase, rho A, and pp60c-src all showed translocation to the cytoskeletal fraction upon osteoclast attachment to plastic. However, PI3-kinase and rho A, but not pp60c-src, showed further translocation of 2.4- and 3.2-fold, respectively, upon attachment of osteoclasts to bone. PI3-kinase translocation to the cytoskeleton was inhibited by either cytochalasin B or colchicine. Furthermore, treatment of osteoclasts with the PI3-kinase inhibitor wortmannin decreased its translocation, suggesting that PI3-kinase activity was needed for its translocation. Moreover, wortmannin inhibited osteoclast attachment to both bone and plastic and caused drastic changes in osteoclast morphology resulting in rounding of the cells, disappearance of F-actin structures or podosomes, and appearance of punctate or vesicular structures inside the cells. Osteoblastic MB1.8 cells and IC-21 macrophages did not show additional translocation of PI3-kinase or rho A upon attachment to bone or changes in attachment or morphology in response to wortmannin. Finally, PI3-kinase coimmunoprecipitated with alpha v beta 3 integrin from osteoclasts. PMID:9434625

  4. Regulation of mRNA export by the PI3 kinase/AKT signal transduction pathway

    PubMed Central

    Quaresma, Alexandre Jose Christino; Sievert, Rachel; Nickerson, Jeffrey A.

    2013-01-01

    UAP56, ALY/REF, and NXF1 are mRNA export factors that sequentially bind at the 5′ end of a nuclear mRNA but are also reported to associate with the exon junction complex (EJC). To screen for signal transduction pathways regulating mRNA export complex assembly, we used fluorescence recovery after photobleaching to measure the binding of mRNA export and EJC core proteins in nuclear complexes. The fraction of UAP56, ALY/REF, and NXF1 tightly bound in complexes was reduced by drug inhibition of the phosphatidylinositide 3-kinase (PI3 kinase)/AKT pathway, as was the tightly bound fraction of the core EJC proteins eIF4A3, MAGOH, and Y14. Inhibition of the mTOR mTORC1 pathway decreased the tight binding of MAGOH. Inhibition of the PI3 kinase/AKT pathway increased the export of poly(A) RNA and of a subset of candidate mRNAs. A similar effect of PI3 kinase/AKT inhibition was observed for mRNAs from both intron-containing and intronless histone genes. However, the nuclear export of mRNAs coding for proteins targeted to the endoplasmic reticulum or to mitochondria was not affected by the PI3 kinase/AKT pathway. These results show that the active PI3 kinase/AKT pathway can regulate mRNA export and promote the nuclear retention of some mRNAs. PMID:23427269

  5. Membrane Ig cross-linking regulates phosphatidylinositol 3-kinase in B lymphocytes.

    PubMed

    Gold, M R; Chan, V W; Turck, C W; DeFranco, A L

    1992-04-01

    Cross-linking of the B cell AgR results in activation of mature B cells and tolerization of immature B cells. The initial signaling events stimulated by membrane immunoglobulin (mIg) cross-linking are tyrosine phosphorylation of a number of proteins. Among the targets of mIg-induced tyrosine phosphorylation are the tyrosine kinases encoded by the lyn, blk, fyn, and syk genes, the mIg-associated proteins MB-1 and Ig-beta, phospholipase C-gamma 1 and -gamma 2, as well as many unidentified proteins. In this report we show that mIg cross-linking also regulates phosphatidylinositol 3-kinase (PtdIns 3-kinase), an enzyme that phosphorylates inositol phospholipids and plays a key role in mediating the effects of tyrosine kinases on growth control in fibroblasts. Cross-linking mIg on B lymphocytes greatly increased the amount of PtdIns 3-kinase activity which could be immunoprecipitated with anti-phosphotyrosine (anti-tyr(P) antibodies. This response was observed after mIg cross-linking in mIgM- and mIgG-bearing B cell lines and after cross-linking either mIgM or mIgD in murine splenic B cells. Thus, regulation of PtdIns 3-kinase is a common feature of signaling by several different isotypes of mIg. This response was rapid and peaked 2 to 3 min after the addition of anti-Ig antibodies. The anti-Ig-stimulated increase in PtdIns 3-kinase activity associated with anti-Tyr(P) immunoprecipitates could reflect increased tyrosine phosphorylation of PtdIns 3-kinase, increased activity of the enzyme, or both. In favor of the first possibility, the tyrosine kinase inhibitor herbimycin A blocked the increase in ant-Tyr(P)-immunoprecipitated PtdIns 3-kinase activity as well as the anti-Ig-induced tyrosine phosphorylation. Moreover, this response was not secondary to phospholipase C activation but rather seemed to be a direct consequence of mIg-induced tyrosine phosphorylation. Activation of the phosphoinositide pathway by a transfected M1 muscarinic acetylcholine receptor expressed in

  6. Auxin-induced reactive oxygen species production requires the activation of phosphatidylinositol 3-kinase.

    PubMed

    Joo, Jung Hee; Yoo, Ho Jung; Hwang, Inhwan; Lee, June Seung; Nam, Kyoung Hee; Bae, Yun Soo

    2005-02-14

    We recently reported that production of reactive oxygen species (ROS) is essential for auxin-induced gravitropic signaling. Here, we investigated the role of phosphatidylinositol 3-kinase and its product, PtdIns(3)P, in auxin-mediated ROS production and the root gravitropic response. Pretreatment with LY294002, an inhibitor of PtdIns 3-kinase activity, blocked auxin-mediated ROS generation, and reduced the sensitivity of root tissue to gravistimulation. The amount of PtdIns(3)P increased in response to auxin, and this effect was abolished by pretreatment with LY294002. In addition, sequestration of PtdIns(3)P by transient expression of the endosome binding domain in protoplasts abrogated IAA-induced ROS accumulation. These results indicate that activation of PtdIns 3-kinase and its product PtdIns(3)P are required for auxin-induced production of ROS and root gravitropism. PMID:15710420

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

    PubMed

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

    2007-01-01

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

  8. Role of taurine accumulation in keratinocyte hydration.

    PubMed

    Janeke, Guido; Siefken, Wilfried; Carstensen, Stefanie; Springmann, Gunja; Bleck, Oliver; Steinhart, Hans; Höger, Peter; Wittern, Klaus-Peter; Wenck, Horst; Stäb, Franz; Sauermann, Gerhard; Schreiner, Volker; Doering, Thomas

    2003-08-01

    Epidermal keratinocytes are exposed to a low water concentration at the stratum corneum-stratum granulosum interface. When epithelial tissues are osmotically perturbed, cellular protection and cell volume regulation is mediated by accumulation of organic osmolytes such as taurine. Previous studies reported the presence of taurine in the epidermis of several animal species. Therefore, we analyzed human skin for the presence of the taurine transporter (TAUT) and studied the accumulation of taurine as one potential mechanism protecting epidermal keratinocytes from dehydration. According to our results, TAUT is expressed as a 69 kDa protein in human epidermis but not in the dermis. For the epidermis a gradient was evident with maximal levels of TAUT in the outermost granular keratinocyte layer and lower levels in the stratum spinosum. No TAUT was found in the basal layer or in the stratum corneum. Keratinocyte accumulation of taurine was induced by experimental induction of skin dryness via application of silica gel to human skin. Cultured human keratinocytes accumulated taurine in a concentration- and osmolarity-dependent manner. TAUT mRNA levels were increased after exposure of human keratinocytes to hyperosmotic culture medium, indicating osmosensitive TAUT mRNA expression as part of the adaptation of keratinocytes to hyperosmotic stress. Keratinocyte uptake of taurine was inhibited by beta-alanine but not by other osmolytes such as betaine, inositol, or sorbitol. Accumulation of taurine protected cultured human keratinocytes from both osmotically induced and ultraviolet-induced apoptosis. Our data indicate that taurine is an important epidermal osmolyte required to maintain keratinocyte hydration in a dry environment. PMID:12880428

  9. 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

  10. 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.

  11. 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. PMID:20333370

  12. Phosphatidylinositol-3 kinase activation induced upon Fc gamma RIIIA- ligand interaction

    PubMed Central

    1994-01-01

    Induced activation of protein tyrosine kinase(s) is a central event in signal transduction mediated via the low affinity receptor for IgG (Fc gamma RIIIA, CD16) in natural killer (NK) cells. Tyrosine phosphorylation may affect the function of several protein directly, or indirectly by inducing their association with other tyrosine phosphorylated proteins. Here, we report that Fc gamma RIII stimulation induces activation of phosphatidylinositol (PI)-3 kinase in NK cells. Phosphotyrosine immunoprecipitates from Fc gamma RIII-stimulated NK cells contain PI-kinase activity and PI-3 kinase can be directly precipitated from them. Conversely, a series of tyrosine-phosphorylated proteins is coprecipitated with PI-3 kinase from the stimulated, but not from control cells. Analogous results obtained using Jurkat T cells expressing transfected Fc gamma RIIIA alpha ligand binding chain in association with gamma 2 or zeta 2 homodimers indicate that both complexes transduce this effect, although the Fc gamma RIIIA-zeta 2 complexes do so with greater efficiency. Accumulation of phosphoinositide D3 phosphorylated products in stimulated cells confirms PI-3 kinase activation, indicating the participation of this enzyme in Fc gamma RIIIA-mediated signal transduction. PMID:8294866

  13. Beclin 1 Forms Two Distinct Phosphatidylinositol 3-Kinase Complexes with Mammalian Atg14 and UVRAG

    PubMed Central

    Itakura, Eisuke; Kishi, Chieko; Inoue, Kinji

    2008-01-01

    Class III phosphatidylinositol 3-kinase (PI3-kinase) regulates multiple membrane trafficking. In yeast, two distinct PI3-kinase complexes are known: complex I (Vps34, Vps15, Vps30/Atg6, and Atg14) is involved in autophagy, and complex II (Vps34, Vps15, Vps30/Atg6, and Vps38) functions in the vacuolar protein sorting pathway. Atg14 and Vps38 are important in inducing both complexes to exert distinct functions. In mammals, the counterparts of Vps34, Vps15, and Vps30/Atg6 have been identified as Vps34, p150, and Beclin 1, respectively. However, orthologues of Atg14 and Vps38 remain unknown. We identified putative mammalian homologues of Atg14 and Vps38. The Vps38 candidate is identical to UV irradiation resistance-associated gene (UVRAG), which has been reported as a Beclin 1-interacting protein. Although both human Atg14 and UVRAG interact with Beclin 1 and Vps34, Atg14, and UVRAG are not present in the same complex. Although Atg14 is present on autophagic isolation membranes, UVRAG primarily associates with Rab9-positive endosomes. Silencing of human Atg14 in HeLa cells suppresses autophagosome formation. The coiled-coil region of Atg14 required for binding with Vps34 and Beclin 1 is essential for autophagy. These results suggest that mammalian cells have at least two distinct class III PI3-kinase complexes, which may function in different membrane trafficking pathways. PMID:18843052

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

    PubMed Central

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

    1996-01-01

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

  15. Differentiation of Keratinocytes Modulates Skin HPA Analog.

    PubMed

    Wierzbicka, Justyna M; Żmijewski, Michał A; Antoniewicz, Jakub; Sobjanek, Michal; Slominski, Andrzej T

    2017-01-01

    It is well established, that epidermal keratinocytes express functional equivalent of hypothalamus-pituitary-adrenal axis (HPA) in order to respond to changing environment and maintain internal homeostasis. We are presenting data indicating that differentiation of primary neonatal human keratinocytes (HPEKp), induced by prolonged incubation or calcium is accompanied by significant changes in the expression of the elements of skin analog of HPA (sHPA). Expression of CRF, UCN1-3, POMC, ACTH, CRFR1, CRFR2, MC1R, MC2R, and GR (coded by NR3C1 gene) were observed on gene/protein levels along differentiation of keratinocytes in culture with similar pattern seen by immunohistochemistry on full thickness skin biopsies. Expression of CRF was more pronounced in less differentiated keratinocytes, which corresponded to the detection of CRF immunoreactivity preferentially in the stratum basale. POMC expression was enhanced in more differentiated keratinocytes, which corresponded to detection of ACTH immunoreactivity, predominantly in the stratum spinosum and stratum granulosum. Expression of urocortins was also affected by induction of HPEKp differentiation. Immunohistochemical studies showed high prevalence of CRFR1 in well differentiated keratinocytes, while smaller keratinocytes showed predominantly CRFR2 immunoreactivity. MC2R mRNA levels were elevated from days 4 to 8 of in vitro incubation, while MC2R immunoreactivity was the highest in the upper layers of epidermis. Similar changes in mRNA/protein levels of sHPA elements were observed in HPEKp keratinocytes treated with calcium. Summarizing, preferential expression of CRF and POMC (ACTH) by populations of keratinocytes on different stage of differentiation resembles organization of central HPA axis suggesting their distinct role in physiology and pathology of the epidermis. J. Cell. Physiol. 232: 154-166, 2017. © 2016 Wiley Periodicals, Inc.

  16. 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. PMID:20728993

  17. 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

    2015-01-01

    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.

  18. A collagen-related peptide regulates phospholipase Cgamma2 via phosphatidylinositol 3-kinase in human platelets.

    PubMed Central

    Pasquet, J M; Bobe, R; Gross, B; Gratacap, M P; Tomlinson, M G; Payrastre, B; Watson, S P

    1999-01-01

    The collagen receptor glycoprotein VI (GPVI) induces platelet activation through a similar pathway to that used by immune receptors. In the present study we have investigated the role of phosphatidylinositol 3-kinase (PI 3-kinase) in GPVI signalling. Our results show that collagen-related peptide ¿CRP: [GCP*(GPP*)(10)GCP*G](n); P*=hydroxyproline¿, which is selective to GPVI, induces formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 3,4-bisphosphate [PI(3, 4)P(2)] in platelets. The increase in the two 3-phosphorylated lipids is inhibited completely by wortmannin and by LY294002, two structurally unrelated inhibitors of PI 3-kinase. The formation of inositol phosphates and phosphatidic acid (PA), two markers of phospholipase C (PLC) activation, by CRP are inhibited by between 50 and 85% in the presence of wortmannin and LY294002. This is associated with inhibition of elevation of intracellular Ca(2+) ([Ca(2+)](i)) and aggregation. Wortmannin and LY294002 also partially inhibit elevation of Ca(2+) by CRP in murine megakaryocytes. Microinjection of the pleckstrin-homology PH domain of Bruton's tyrosine kinase, which binds selectively to PI(3,4, 5)P(3), but not the R28C (Arg(28)-->Cys) mutant which binds to PI(3, 4,5)P(3) with low affinity, also inhibits elevation of [Ca(2+)](i) in megakaryocytes, suggesting that it is this lipid species which mediates the action of the PI 3-kinase pathway. Studies in platelets show that the action of wortmannin and LY294002 is not mediated through an alteration in tyrosine phosphorylation of PLCgamma2. These results demonstrate that PI 3-kinase is required for full activation of PLCgamma2 by GPVI in platelets and megakaryocytes. PMID:10432314

  19. 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.

  20. 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

  1. 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.

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

    PubMed

    Lebel, Emily A; Rusek, Adam; Sivertz, Michael B; Yip, Kin; Thompson, Keith H; Tafrov, Stefan T

    2011-01-01

    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.

  3. 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.

  4. 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.

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

    PubMed

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

    1998-05-15

    Receptor-mediated endocytosis of albumin is an important function of the kidney proximal tubule epithelium. We have measured endocytosis of [125I]-albumin in opossum kidney cells and examined the regulation of this process by phosphatidylinositide 3-kinase (PI 3-kinase). Albumin endocytosis was inhibited by both wortmannin (IC50 6.9 nM) and LY294002 (IC50 6.5 microM) at concentrations that suggested the involvement of PI 3-kinase in its regulation. Recycling rates were unaffected. We transfected OK cells with either a wild-type p85 subunit of PI 3-kinase, or a dominant negative form of the p85 subunit (Deltap85) using the LacSwitch expression system. Transfects were screened by immunoblotting with anti-PI 3-kinase antibodies. Under basal conditions, transfects demonstrated no expression of p85 or Deltap85, but expression was briskly induced by treatment of the cells with IPTG (EC50 13.7 microM). Inhibition of PI 3-kinase activity by Deltap85 was confirmed by in vitro kinase assay of anti-phosphotyrosine immunoprecipitates from transfected cells stimulated with insulin. Expression of Deltap85 resulted in marked inhibition of albumin endocytosis, predominantly as a result of reduction of the Vmax of the transport process. Expression of p85 had no significant effect on albumin uptake. The results demonstrate that PI 3-kinase regulates an early step in the receptor-mediated endocytosis of albumin by kidney proximal tubular cells. PMID:9593770

  6. Tissue- and cell-specific expression of Ins(1,4,5)P3 3-kinase isoenzymes.

    PubMed Central

    Vanweyenberg, V; Communi, D; D'Santos, C S; Erneux, C

    1995-01-01

    The phosphorylation of Ins(1,4,5)P3 (InsP3) to Ins(1,3,4,5)P4 (InsP4) is catalysed by InsP3 3-kinase. Molecular-biological data have shown the presence of two human isoenzymes of InsP3 3-kinase, namely InsP3 3-kinases A and B. We have isolated from a rat thymus cDNA library a 2235 bp cDNA (clone B15) encoding rat InsP3 3-kinase B. Northern-blot analysis of mRNA isolated from rat tissues (thymus, testis, brain, spleen, liver, kidney, heart, lung and intestine) revealed that a rat InsP3 3-kinase B probe hybridized to a 6 kb mRNA in lung, thymus, testis, brain and heart. In contrast, Northern-blot analysis of the same tissues probed under stringent conditions with a rat InsP3 3-kinase A probe hybridized to a 2 kb mRNA only in brain and a 1.8-2.0 kb mRNA species in testis. Northern-blot analysis of three human cell lines (HL-60, SH-SY5Y and HTB-138) probed with a human InsP3 3-kinase B probe showed the presence of a 6 kb mRNA in all cell lines, except in the human neuroblastoma cell line (SH-SY5Y), where two mRNA species of 5.7 and 6 kb were detected. Using the same blot, no hybridization signal could be seen with a human InsP3 3-kinase A probe. Altogether, our data are consistent with the notion that the two InsP3 3-kinase isoenzymes, A and B, are specifically expressed in different tissues and cells. Images Figure 3 Figure 4 PMID:7887896

  7. p21ras initiates Rac-1 but not phosphatidyl inositol 3 kinase/PKB, mediated signaling pathways in T lymphocytes.

    PubMed

    Genot, E; Reif, K; Beach, S; Kramer, I; Cantrell, D

    1998-10-01

    p21ras is activated by the T cell antigen receptor (TCR) and then co-ordinates important signaling pathways for T lymphocyte activation. Effector pathways for this guanine nucleotide binding protein in T cells are mediated by the serine/threonine kinase Raf-1 and the Ras-related GTPase Rac-1. In fibroblasts, an important effector for the Ras oncogene is Phosphatidylinositol 3-kinase (PtdIns 3-kinase). Activation of this lipid kinase is able to induce critical Rac-1 signaling pathways and can couple p21ras to cell survival mechanisms via the serine/threonine kinase Akt/PKB. The role of PtdIns 3-kinase in Ras signaling in T cells has not been explored. In the present study, we examined the ability of PtdIns 3-kinase to initiate the Rac-1 signaling pathways important for T cell activation. We also examined the possibility that Akt/PKB is regulated by Ras signaling pathways in T lymphocytes. The results show that Ras can initiate a Rac-1 mediated pathway that regulates the transcriptional function of AP-1 complexes. PtdIns 3-kinase signals cannot mimic p21ras and induce the Rac mediated responses of AP-1 transcriptional activation. Moreover, neither TCR or Ras activation of AP-1 is dependent on PtdIns 3-kinase. PKB is activated in response to triggering of the T cell antigen receptor; PtdIns 3-kinase activity is both required and sufficient for this TCR response. In contrast, p21ras signals are unable to induce Akt/PKB activity in T cell nor is Ras function required for Akt/PKB activation in response to the TCR. The present data thus highlight that PtdIns 3-kinase and Akt/PKB are not universal Ras effector molecules. Ras can initiate Rac-1 regulated signaling pathways in the context of T cell antigen receptor function independently of PtdIns 3-kinase activity.

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

    PubMed

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

    2014-07-01

    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. PMID:24932672

  9. TYK2-STAT1-BCL2 Pathway Dependence in T-Cell Acute Lymphoblastic Leukemia

    PubMed Central

    Sanda, Takaomi; Tyner, Jeffrey W.; Gutierrez, Alejandro; Ngo, Vu N.; Glover, Jason; Chang, Bill H.; Yost, Arla; Ma, Wenxue; Fleischman, Angela G.; Zhou, Wenjun; Yang, Yandan; Kleppe, Maria; Ahn, Yebin; Tatarek, Jessica; Kelliher, Michelle A.; Neuberg, Donna S.; Levine, Ross L.; Moriggl, Richard; Müller, Mathias; Gray, Nathanael S.; Jamieson, Catriona H. M.; Weng, Andrew P.; Staudt, Louis M.; Druker, Brian J.; Look, A. Thomas

    2013-01-01

    Targeted molecular therapy has yielded remarkable outcomes in certain cancers, but specific therapeutic targets remain elusive for many others. As a result of two independent RNA interference (RNAi) screens, we identified pathway dependence on a member of the JAK tyrosine kinase family, TYK2, and its downstream effector STAT1 in T-cell acute lymphoblastic leukemia (T-ALL). Gene knockdown experiments consistently demonstrated TYK2 dependence in both T-ALL primary specimens and cell lines, and a small-molecule inhibitor of JAK kinase activity induced T-ALL cell death. Activation of this TYK2-STAT1 pathway i n T-ALL cell lines occurs by gain-of-function TYK2 mutations or activation of IL-10 receptor signaling, and this pathway mediates T-ALL cell survival through upregulation of the anti-apoptotic protein BCL2. These findings indicate that in many T-ALL cases, the leukemic cells are dependent upon the TYK2-STAT1-BCL2 pathway for continued survival, supporting the development of molecular therapies targeting TYK2 and other components of this pathway. PMID:23471820

  10. Igf2 pathway dependency of the Trp53 developmental and tumour phenotypes

    PubMed Central

    Haley, Victoria L; Barnes, David J; Sandovici, Ionel; Constancia, Miguel; Graham, Christopher F; Pezzella, Francesco; Bühnemann, Claudia; Carter, Emma J; Hassan, A Bassim

    2012-01-01

    Insulin-like growth factor 2 (IGF2) and the transformation related protein 53 (Trp53) are potent regulators of cell growth and metabolism in development and cancer. In vitro evidence suggests several mechanistic pathway interactions. Here, we tested whether loss of function of p53 leads to IGF2 ligand pathway dependency in vivo. Developmental lethality occurred in p53 homozygote null mice that lacked the paternal expressed allele of imprinted Igf2. Further lethality due to post-natal lung haemorrhage occurred in female progeny with Igf2 paternal null allele only if derived from double heterozygote null fathers, and was associated with a specific gene expression signature. Conditional deletion of Igf2fl/fl attenuated the rapid tumour onset promoted by homozygous deletion of p53fl/fl. Accelerated carcinoma and sarcoma tumour formation in p53+/− females with bi-allelic Igf2 expression was associated with reductions in p53 loss of heterozygosity and apoptosis. Igf2 genetic dependency of the p53 null phenotype during development and tumour formation suggests that targeting the IGF2 pathway may be useful in the prevention and treatment of human tumours with a disrupted Trp53 pathway. PMID:22674894

  11. Nuclear translocation of phosphatidylinositol 3-kinase in rat pheochromocytoma PC 12 cells after treatment with nerve growth factor.

    PubMed

    Neri, L M; Milani, D; Bertolaso, L; Stroscio, M; Bertagnolo, V; Capitani, S

    1994-07-01

    Immunocytochemical analysis of PI 3-kinase localization in PC 12 cells demonstrates that the enzyme translocates to the nucleus after cell treatment with differentiating doses of NGF. The association of PI 3-kinase to the nucleus occurs rapidly (within minutes) and increases with the time of exposure of NGF. We suggest that PI-3 kinase specific localization may determine the production of novel phosphoinositides in cell compartments targeted to effect diverse cell responses. The nuclear translocation is consistent with accumulating data on the existence of a nuclear inositol lipid cycle which could also include 3-phosphorylated inositides, participating to the modulation of the cell response to extracellular stimuli.

  12. 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.

  13. 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

  14. PEGylated Cationic Liposome – DNA Complexation in Brine is Pathway-Dependent

    PubMed Central

    Silva, Bruno F.B.; Majzoub, Ramsey N.; Chan, Chia-Ling; Li, Youli; Olsson, Ulf; Safinya, Cyrus R.

    2013-01-01

    Cationic liposome-DNA (CL-DNA) complexes, are regarded as promising materials for safe and efficient delivery of genes for therapeutical applications. In order to be used in vivo, these complexes may be coated with a hydrophilic polymer (e.g. polyethylene-glycol, PEG) that provides steric stabilization towards adhesion of proteins and removal by the immune system. In this work we study the influence of the initial salt concentration (Cs) – which modulates the electrostatic interaction between oppositely charged vesicles and DNA – on the structure and stability of PEGylated CL-DNA particles. Previous small-angle X-ray scattering has shown that if non-PEGylated or PEGylated CL-DNA lamellar complexes are prepared in water, their structure is well defined with a high number of lipid membrane-DNA layers (larger than 20). Here we show that if these complexes are transferred to saline media (150 mM NaCl or DMEM, both near physiological conditions), this structure remains nearly unchanged. Conversely, if PEGylated complexes are prepared in saline media, their lamellar structure is much looser, with fewer number of layers. This pathway dependent behavior of PEGylated complex formation in brine is modulated by the liposome membrane charge density and the mole fraction of PEG 2000 in the membranes, with the average number of layers decreasing with increasing Cs and in going from 5 mol% to 10 mol% PEG-lipid. Each of these structures (high and low number of layers) is stable with time, suggesting that despite complex formation being thermodynamically favored, the complexation process in PEGylated membranes, which determines the number of layers per particle, is kinetically controlled. In the extreme case (when polymer repulsions from 10 mol% PEG-lipid are maximized and electrostatic attraction between PEGylated CLs and DNA are minimized at low membrane charge density) complex formation is suppressed at high Cs=150 mM. PMID:24060564

  15. 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

  16. [Effect of trypsin on the rat keratinocyte separation and subculture].

    PubMed

    Ouyang, An-Li; Zhou, Yan; Hua, Ping; Tan, Wen-Song

    2002-01-01

    The effect of trypsin on the separation an subculture of the keratinocytes was investigated in this work. It was found that when 0.25% trypsin was employed for 5 minutes to separate keratinocytes, the number of active keratinocytes and the cells capable of forming colony were higher than those of other experimental conditions. The maximum attached ratio of primary keratinocytes was obtained when skin tissues were treated at 0.05% concentration of trypsin. With the increase of the trypsin concentrations, the attached ratio, attachment rate constant, and colony forming efficiency were all increased. Thus, 0.25% concentration of trypsin was recommended for separating and subculturing the keratinocytes.

  17. Insulin accelerates inter-endosomal GLUT4 traffic via phosphatidylinositol 3-kinase and protein kinase B.

    PubMed

    Foster, L J; Li, D; Randhawa, V K; Klip, A

    2001-11-23

    Insulin enhances plasmalemmal-directed traffic of glucose transporter-4 (GLUT4), but it is unknown whether insulin regulates GLUT4 traffic through endosomal compartments. In L6 myoblasts expressing Myc-tagged GLUT4, insulin markedly stimulated the rate of GLUT4myc recycling. In myoblasts stimulated with insulin to maximize surface GLUT4myc levels, we followed the rates of surface-labeled GLUT4myc endocytosis and chased its intracellular distribution in space and time using confocal immunofluorescence microscopy. Surface-labeled GLUT4myc internalized rapidly (t(12) 3 min), reaching the early endosome by 2 min and the transferrin receptor-rich, perinuclear recycling endosome by 20 min. Upon re-addition of insulin, the t(12) of GLUT4 disappearance from the plasma membrane was unchanged (3 min), but strikingly, GLUT4myc reached the recycling endosome by 10 and left by 20 min. This effect of insulin was blocked by the phosphatidylinositol 3-kinase inhibitor LY294002 or by transiently transfected dominant-negative phosphatidylinositol 3-kinase and protein kinase B mutants. In contrast, insulin did not alter the rate of arrival of rhodamine-labeled transferrin at the recycling endosome. These results reveal a heretofore unknown effect of insulin to accelerate inter-endosomal travel rates of GLUT4 and identify the recycling endosome as an obligatory stage in insulin-dependent GLUT4 recycling.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    Li, Ying-Yi; Mukaida, Naofumi

    2014-01-01

    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. PMID:25071334

  1. Cultured human keratinocytes synthesize and secrete endothelin-1.

    PubMed

    Yohn, J J; Morelli, J G; Walchak, S J; Rundell, K B; Norris, D A; Zamora, M R

    1993-01-01

    The human epidermal-melanin unit exists as a complex interplay of cell-cell interactions. Melanocytes synthesize melanin and transfer it to the surrounding keratinocytes, which, in turn, produce factors that affect melanocyte homeostasis, growth, and melanization. Endothelin-1 (ET-1), a vasoconstrictor peptide produced by endothelial cells, has recently been shown to stimulate human melanocyte proliferation and tyrosinase activity. To investigate the possibility that keratinocytes synthesize and secrete ET-1, we grew human keratinocytes in a defined serum-free medium and measured ET-1 levels in the keratinocytes and the keratinocyte-conditioned medium. Northern analysis of keratinocyte total RNA also was performed. We found that human keratinocytes express preproET-1 mRNA and translate the message to ET-1 protein, which is secreted into the keratinocyte medium. Human keratinocytes produced ET-1 in a time-dependent manner with total production of 20.1 +/- 1.1 pg ET-1/10(6) cells at 24 h (n = 7). Although total ET-1 production (secreted plus cell-associated ET-1) was similar, the proportion of secreted versus cell-associated ET-1 varied widely among the different donors. We have found that human keratinocytes synthesize and secrete ET-1 in vitro. From these data we believe that the keratinocyte could be an in vivo epidermal source of this melanocyte growth and pigmentation factor.

  2. Romidepsin (FK228) and its analogs directly inhibit phosphatidylinositol 3-kinase activity and potently induce apoptosis as histone deacetylase/phosphatidylinositol 3-kinase dual inhibitors.

    PubMed

    Saijo, Ken; Katoh, Tadashi; Shimodaira, Hideki; Oda, Akifumi; Takahashi, Ohgi; Ishioka, Chikashi

    2012-11-01

    Activation of phosphatidylinositol 3-kinase (PI3K) signaling is involved in carcinogenesis and cancer progression. The PI3K inhibitors are considered candidate drugs for cancer treatment. Here, we describe a drug screening system for novel PI3K inhibitors using Saccharomyces cerevisiae strains with deleterious mutations in the ATP-binding cassette transporter genes, because wild-type S. cerevisiae uses drug efflux pumps for reducing intracellular drug concentrations. By screening the chemical library of the Screening Committee of Anticancer Drugs, we identified the histone deacetylase (HDAC) inhibitor romidepsin (FK228) and its novel analogs. In vitro PI3K activity assays confirmed that these compounds directly inhibit PI3K activity at μM-range concentrations. FK-A5 analog was the most potent inhibitor. Western blotting revealed that these compounds inhibit phosphorylation of protein kinase B and downstream signaling components. Molecular modeling of the PI3K-FK228 complex indicated that FK228 binds to the ATP-binding pocket of PI3K. At μM-range concentrations, FK228 and FK-A5 show potent cytotoxicity, inducing apoptosis even in HDAC inhibitor-resistant cells. Furthermore, HDAC/PI3K dual inhibition by FK228 and FK-A5 at μM-range concentrations potentiates the apoptosis induction, mimicking the effect of combining specific HDAC and PI3K inhibitors. In this study, we showed that FK228 and its analogs directly inhibit PI3K activity and induce apoptosis at μM-range concentrations, similar to HDAC/PI3K dual inhibition. In future, optimizing the potency of FK228 and its analogs against PI3K may contribute to the development of novel HDAC/PI3K dual inhibitors for cancer treatment.

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

    PubMed

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

    2008-08-01

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

  4. 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

  5. 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.

  6. 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.

  7. Targeting PI3 kinase/AKT/mTOR signaling in cancer.

    PubMed

    Sheppard, Karen; Kinross, Kathryn M; Solomon, Benjamin; Pearson, Richard B; Phillips, Wayne A

    2012-01-01

    The phosphatidylinositol 3 kinase (PI3K) pathway is one of the major pathways modulating cell growth, proliferation, metabolism, survival, and angiogenesis. Hyperactivation of this pathway is one of the most frequent occurrences in human cancer and is thus an obvious target for treatment of this disease. Currently there are 26 novel compounds targeting the PI3K pathway being assessed in more than 150 cancer-related clinical trials. Although this pathway is involved in many vital biologic functions, data emanating from these clinical trials indicate that these drugs are well tolerated. This review outlines the interaction of the PI3K pathway with other signaling cascades, highlights mechanisms involved in hyperactivation, discusses current therapeutics in cancer-related clinical trials that target this pathway, and, based on preclinical data, discusses possible leads on patient selection and combinational therapy, including targeting multiple components of the associated signaling network.

  8. Depolarization and neurotrophins converge on the phosphatidylinositol 3-kinase-Akt pathway to synergistically regulate neuronal survival.

    PubMed

    Vaillant, A R; Mazzoni, I; Tudan, C; Boudreau, M; Kaplan, D R; Miller, F D

    1999-09-01

    In this report, we have examined the mechanisms whereby neurotrophins and neural activity coordinately regulate neuronal survival, focussing on sympathetic neurons, which require target-derived NGF and neural activity for survival during development. When sympathetic neurons were maintained in suboptimal concentrations of NGF, coincident depolarization with concentrations of KCl that on their own had no survival effect, synergistically enhanced survival. Biochemical analysis revealed that depolarization was sufficient to activate a Ras-phosphatidylinositol 3-kinase-Akt pathway (Ras-PI3-kinase-Akt), and function-blocking experiments using recombinant adenovirus indicated that this pathway was essential for approximately 50% of depolarization-mediated neuronal survival. At concentrations of NGF and KCl that promoted synergistic survival, these two stimuli converged to promote increased PI3-kinase-dependent Akt phosphorylation. This convergent PI3-kinase-Akt pathway was essential for synergistic survival. In contrast, inhibition of calcium/calmodulin-dependent protein kinase II revealed that, while this molecule was essential for depolarization-induced survival, it had no role in KCl- induced Akt phosphorylation, nor was it important for synergistic survival by NGF and KCl. Thus, NGF and depolarization together mediate survival of sympathetic neurons via intracellular convergence on a Ras-PI3-kinase-Akt pathway. This convergent regulation of Akt may provide a general mechanism for coordinating the effects of growth factors and neural activity on neuronal survival throughout the nervous system.

  9. 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.

  10. 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.

  11. 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

  12. Phosphatidylinositol 3-kinase signals activation of p70 S6 kinase in situ through site-specific p70 phosphorylation.

    PubMed Central

    Weng, Q P; Andrabi, K; Klippel, A; Kozlowski, M T; Williams, L T; Avruch, J

    1995-01-01

    The p70 S6 kinase is activated by insulin and mitogens through multisite phosphorylation of the enzyme. One set of activating phosphorylations occurs in a putative autoinhibitory domain in the noncatalytic carboxyl-terminal tail. Deletion of this tail yields a variant (p70 delta CT104) that nevertheless continues to be mitogen regulated. Coexpression with a recombinant constitutively active phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) gives substantial activation of both full-length p70 and p70 delta CT104 but not Rsk. Activation of p70 delta CT104 by PI 3-kinase and inhibition by wortmannin are each accompanied by parallel and selective changes in the phosphorylation of p70 Thr-252. A Thr or Ser at this site, in subdomain VIII of the catalytic domain just amino-terminal to the APE motif, is necessary for p70 40S kinase activity. The inactive ATP-binding site mutant K123M p70 delta CT104 undergoes phosphorylation of Thr-252 in situ but does not undergo direct phosphorylation by the active PI 3-kinase in vitro. PI 3-kinase provides a signal necessary for the mitogen activation of the p70 S6 kinase, which directs the site-specific phosphorylation of Thr-252 in the p70 catalytic domain, through a distinctive signal transduction pathway. Images Fig. 1 Fig. 2 Fig. 3 PMID:7777579

  13. Periostin Promotes Atrioventricular Mesenchyme Matrix Invasion and Remodeling Mediated by Integrin Signaling through Rho/PI 3-Kinase

    PubMed Central

    Butcher, Jonathan T.; Norris, Russell A.; Hoffman, Stanley; Mjaatvedt, Corey H.; Markwald, Roger R.

    2007-01-01

    Recent evidence suggests that extracellular matrix components may play a signaling role in embryonic valve development. We have previously identified the spatiotemporal expression patterns of periostin in developing valves, but its function during this process is largely unknown. To evaluate the functional role periostin plays during valvulogenesis, two separate three-dimensional culture assay systems, which model chick atrioventricular cushion development, were employed. These assays demonstrated that cushion mesenchymal cells adhered and spread on purified periostin in a dose responsive manner, similar to collagen I and fibronectin via αvβ3 and β1 integrin pairs. Periostin overexpression resulted in enhanced mesenchyme invasion through 3D collagen gels and increased matrix compaction. This invasion was dependent on αvβ3 more than β1 integrin signaling, and was mediated differentially by Rho kinase and PI 3-kinase. Both matrix invasion and compaction were associated with a colocalization of periostin and β1 integrin expression to migratory cell phenotype in both surface and deep cells. The Rho/PI 3-kinase pathway also differentially mediated matrix compaction. Both Rho and PI 3-kinase were involved in normal cushion mesenchyme matrix compaction, but only PI 3-kinase was required for the enhanced matrix compaction due to periostin. Taken together, these results highlight periostin as a mediator of matrix remodeling by cushion mesenchyme towards a mature valve structure. PMID:17070513

  14. 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.

  15. 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

  16. Inhibition of phosphatidylinositol-3-kinase causes increased sensitivity to radiation through a PKB-dependent mechanism

    SciTech Connect

    Gottschalk, Alexander R. . E-mail: gottschalk@radonc17.ucsf.edu; Doan, Albert; Nakamura, Jean L.; Stokoe, David; Haas-Kogan, Daphne A.

    2005-11-15

    Purpose: To identify whether inhibition of phosphatidylinositol-3-kinase (PI3K) causes increased radiosensitivity through inhibition of protein kinase B (PKB), implicating PKB as an important therapeutic target in prostate cancer. Methods and Materials: The prostate cancer cell line LNCaP was treated with the PI3K inhibitor LY294002, radiation, and combinations of the two therapies. Apoptosis and survival were measured by cell cycle analysis, Western blot analysis for cleaved poly (ADP-ribose) polymerase, and clonogenic survival. To test the hypothesis that inhibition of PKB is responsible for LY294002-induced radiosensitivity, LNCaP cells expressing a constitutively active form of PKB were used. Results: The combination of PI3K inhibition and radiation caused an increase in apoptosis and a decrease in clonogenic survival when compared to either modality alone. The expression of constitutively activated PKB blocked apoptosis induced by combination of PI3K inhibition and radiation and prevented radiosensitization by LY294002. Conclusion: These data indicate that PI3K inhibition increases sensitivity of prostate cancer cell lines to ionizing radiation through inactivation of PKB. Therefore, PTEN mutations, which lead to PKB activation, may play an important role in the resistance of prostate cancer to radiation therapy. Targeted therapy against PKB could be beneficial in the management of prostate cancer patients.

  17. Regulation of glucose metabolism in T cells: new insight into the role of Phosphoinositide 3-kinases

    PubMed Central

    Finlay, David K.

    2012-01-01

    Naïve T cells are relatively quiescent cells that only require energy to prevent atrophy and for survival and migration. However, in response to developmental or extrinsic cues T cells can engage in rapid growth and robust proliferation, produce of a range of effector molecules and migrate through peripheral tissues. To meet the significantly increased metabolic demands of these activities, T cells switch from primarily metabolizing glucose to carbon dioxide through oxidative phosphorylation to utilizing glycolysis to convert glucose to lactate (termed aerobic glycolysis). This metabolic switch allows glucose to be used as a source of carbon to generate biosynthetic precursors for the production of protein, DNA, and phospholipids, and is crucial for T cells to meet metabolic demands. Phosphoinositide 3-kinases (PI3K) are a family of inositol lipid kinases linked with a broad range of cellular functions in T lymphocytes that include cell growth, proliferation, metabolism, differentiation, survival, and migration. Initial research described a critical role for PI3K signaling through Akt (also called protein kinase B) for the increased glucose uptake and glycolysis that accompanies T cell activation. This review article relates this original research with more recent data and discusses the evidence for and against a role for PI3K in regulating the metabolic switch to aerobic glycolysis in T cells. PMID:22891069

  18. Atg38 is required for autophagy-specific phosphatidylinositol 3-kinase complex integrity

    PubMed Central

    Araki, Yasuhiro; Ku, Wei-Chi; Akioka, Manami; May, Alexander I.; Hayashi, Yu; Arisaka, Fumio; Ishihama, Yasushi

    2013-01-01

    Autophagy is a conserved eukaryotic process of protein and organelle self-degradation within the vacuole/lysosome. Autophagy is characterized by the formation of an autophagosome, for which Vps34-dervied phosphatidylinositol 3-phosphate (PI3P) is essential. In yeast, Vps34 forms two distinct protein complexes: complex I, which functions in autophagy, and complex II, which is involved in protein sorting to the vacuole. Here we identify and characterize Atg38 as a stably associated subunit of complex I. In atg38Δ cells, autophagic activity was significantly reduced and PI3-kinase complex I dissociated into the Vps15–Vps34 and Atg14–Vps30 subcomplexes. We find that Atg38 physically interacted with Atg14 and Vps34 via its N terminus. Further biochemical analyses revealed that Atg38 homodimerizes through its C terminus and that this homodimer formation is indispensable for the integrity of complex I. These data suggest that the homodimer of Atg38 functions as a physical linkage between the Vps15–Vps34 and Atg14–Vps30 subcomplexes to facilitate complex I formation. PMID:24165940

  19. Identification and chromosome assignment of a human gene encoding a novel phosphatidylinositol-3 kinase.

    PubMed

    Seki, N; Nimura, Y; Ohira, M; Saito, T; Ichimiya, S; Nomura, N; Nakagawara, A

    1997-10-31

    We identified a novel phosphatidylinositol (PI) 3-kinase by screening human brain cDNA libraries with probes designed from the conserved kinase-domain sequence. Analysis of cDNAs indicated that two different forms of transcripts are present: one is the full-length form composed of 1,044 amino acid residues and the other is the short form that the N-terminal 216 amino acid residues including a putative p85 binding domain has been truncated (828 amino acid residues). Database search revealed the sequence of the full-length form to be identical to that recently registered by D. Chantry et al. (Accession No. U86453 in GenBank release, August 1997). Northern blot analysis showed this mRNA to be ubiquitously expressed in various tissues, with relatively higher expression was observed in spleen, thymus and leukocytes. Based on fluorescence in situ hybridization and PCR-based analyses with both human/rodent mono-chromosomal hybrid cell panels and radiation hybrid mapping panels, this gene was localized to chromosome region 1p36.2. This region is frequently lost in a variety of human malignancies, including neuroblastoma. The novel PI3K could be a candidate target of the 1p36 alteration that occurs in neuroendocrine tumors.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2004-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-02-01

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

  4. Productive Entry of Foot-and-Mouth Disease Virus via Macropinocytosis Independent of Phosphatidylinositol 3-Kinase.

    PubMed

    Han, Shi-Chong; Guo, Hui-Chen; Sun, Shi-Qi; Jin, Ye; Wei, Yan-Quan; Feng, Xia; Yao, Xue-Ping; Cao, Sui-Zhong; Xiang Liu, Ding; Liu, Xiang-Tao

    2016-01-01

    Virus entry is an attractive target for therapeutic intervention. Here, using a combination of electron microscopy, immunofluorescence assay, siRNA interference, specific pharmacological inhibitors, and dominant negative mutation, we demonstrated that the entry of foot-and-mouth disease virus (FMDV) triggered a substantial amount of plasma membrane ruffling. We also found that the internalization of FMDV induced a robust increase in fluid-phase uptake, and virions internalized within macropinosomes colocalized with phase uptake marker dextran. During this stage, the Rac1-Pak1 signaling pathway was activated. After specific inhibition on actin, Na(+)/H(+) exchanger, receptor tyrosine kinase, Rac1, Pak1, myosin II, and protein kinase C, the entry and infection of FMDV significantly decreased. However, inhibition of phosphatidylinositol 3-kinase (PI3K) did not reduce FMDV internalization but increased the viral entry and infection to a certain extent, implying that FMDV entry did not require PI3K activity. Results showed that internalization of FMDV exhibited the main hallmarks of macropinocytosis. Moreover, intracellular trafficking of FMDV involves EEA1/Rab5-positive vesicles. The present study demonstrated macropinocytosis as another endocytic pathway apart from the clathrin-mediated pathway. The findings greatly expand our understanding of the molecular mechanisms of FMDV entry into cells, as well as provide potential insights into the entry mechanisms of other picornaviruses. PMID:26757826

  5. 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

  6. Role of phosphoinositide 3-kinase in adhesion of platelets to fibrinogen stimulated by cancer procoagulant.

    PubMed

    Olas, B; Wachowicz, B; Mielicki, W P

    2001-11-01

    Cancer procoagulant, cysteine proteinase (CP; EC 3.4.22.26) activates factor X and functions in the absence of factor VII. CP may also change the platelet function. It induces an increase of platelet adhesion to collagen and fibrinogen. Using wortmannin--the inhibitor of phosphoinositide 3-kinase (PI 3-K)--we studied the role of this enzyme in the action of cancer procoagulant on blood platelet adhesion in vitro. Wortmannin (25, 50 and 100 nM, 30 min, 37 degrees C) caused a reduction of platelet adhesion to fibrinogen (P<0.01) when blood platelets were stimulated by both 0.2 U/ml thrombin (IC(50)approximately 75 nM) and by 1 microM ADP (IC(50)approximately 60 nM). We observed that after CP treatment the adhesion of thrombin-activated and ADP-stimulated platelets to fibrinogen was augmented. The potentiated by CP adhesion of activated platelets to fibrinogen was reduced after preincubation of platelets with wortmannin (50 nM, 30 min, 37 degrees C). We conclude that in adhesion of platelets to fibrinogen stimulated by CP PI 3-K take place.

  7. Effects of Novel Isoform-Selective Phosphoinositide 3-Kinase Inhibitors on Natural Killer Cell Function

    PubMed Central

    Yea, Sung Su; So, Lomon; Mallya, Sharmila; Lee, Jongdae; Rajasekaran, Kamalakannan; Malarkannan, Subramaniam; Fruman, David A.

    2014-01-01

    Phosphoinositide 3-kinases (PI3Ks) are promising targets for therapeutic development in cancer. The class I PI3K isoform p110α has received considerable attention in oncology because the gene encoding p110α (PIK3CA) is frequently mutated in human cancer. However, little is known about the function of p110α in lymphocyte populations that modulate tumorigenesis. We used recently developed investigational inhibitors to compare the function of p110α and other isoforms in natural killer (NK) cells, a key cell type for immunosurveillance and tumor immunotherapy. Inhibitors of all class I isoforms (pan-PI3K) significantly impaired NK cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity against tumor cells, whereas p110α-selective inhibitors had no effect. In NK cells stimulated through NKG2D, p110α inhibition modestly reduced PI3K signaling output as measured by AKT phosphorylation. Production of IFN-γ and NK cell-derived chemokines was blocked by a pan-PI3K inhibitor and partially reduced by a p110δinhibitor, with lesser effects of p110α inhibitors. Oral administration of mice with MLN1117, a p110α inhibitor in oncology clinical trials, had negligible effects on NK subset maturation or terminal subset commitment. Collectively, these results support the targeting of PIK3CA mutant tumors with selective p110α inhibitors to preserve NK cell function. PMID:24915189

  8. 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

  9. 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.

  10. Drosophila Spidey/Kar Regulates Oenocyte Growth via PI3-Kinase Signaling.

    PubMed

    Cinnamon, Einat; Makki, Rami; Sawala, Annick; Wickenberg, Leah P; Blomquist, Gary J; Tittiger, Claus; Paroush, Ze'ev; Gould, Alex P

    2016-08-01

    Cell growth and proliferation depend upon many different aspects of lipid metabolism. One key signaling pathway that is utilized in many different anabolic contexts involves Phosphatidylinositide 3-kinase (PI3K) and its membrane lipid products, the Phosphatidylinositol (3,4,5)-trisphosphates. It remains unclear, however, which other branches of lipid metabolism interact with the PI3K signaling pathway. Here, we focus on specialized fat metabolizing cells in Drosophila called larval oenocytes. In the presence of dietary nutrients, oenocytes undergo PI3K-dependent cell growth and contain very few lipid droplets. In contrast, during starvation, oenocytes decrease PI3K signaling, shut down cell growth and accumulate abundant lipid droplets. We now show that PI3K in larval oenocytes, but not in fat body cells, functions to suppress lipid droplet accumulation. Several enzymes of fatty acid, triglyceride and hydrocarbon metabolism are required in oenocytes primarily for lipid droplet induction rather than for cell growth. In contrast, a very long chain fatty-acyl-CoA reductase (FarO) and a putative lipid dehydrogenase/reductase (Spidey, also known as Kar) not only promote lipid droplet induction but also inhibit oenocyte growth. In the case of Spidey/Kar, we show that the growth suppression mechanism involves inhibition of the PI3K signaling pathway upstream of Akt activity. Together, the findings in this study show how Spidey/Kar and FarO regulate the balance between the cell growth and lipid storage of larval oenocytes. PMID:27500738

  11. Cardioprotective Stimuli Mediate Phosphoinositide 3-Kinase and Phosphoinositide Dependent Kinase 1 Nuclear Accumulation in Cardiomyocytes

    PubMed Central

    Rubio, Marta; Avitabile, Daniele; Fischer, Kimberlee; Emmanuel, Gregory; Gude, Natalie; Miyamoto, Shigeki; Mishra, Shikha; Schaefer, Eric M.; Brown, Joan Heller; Sussman, Mark A.

    2009-01-01

    The phosphoinositide-3-kinase (PI3K) / phosphoinositide dependent kinase 1 (PDK1) signaling pathway exerts cardioprotective effects in the myocardium through activation of key proteins including Akt. Activated Akt accumulates in nuclei of cardiomyocytes suggesting that biologically relevant targets are located in that subcellular compartment. Nuclear Akt activity could be potentiated in both intensity and duration by the presence of a nuclear-associated PI3K / PDK1 signaling cascade as has been described in other non-myocyte cell types. PI3K / PDK1 distribution was determined in vitro and in vivo by immunostaining and nuclear extraction of cultured rat neonatal cardiomyocytes or transgenic mouse hearts. Results show that PI3K and PDK1 are present at a basal level in cardiomyocytes nuclei and that cardioprotective stimulation with atrial natriuretic peptide (ANP) increases their nuclear localization. In comparison, overexpression of nuclear-targeted Akt does not mediate increased translocation of either PI3K or PDK1 indicating that accumulation of Akt does not drive PI3K or PDK1 into the nuclear compartment. Furthermore, PI3K and phospho-Akt473 show parallel temporal accumulation in the nucleus following (MI) infarction challenge. These findings demonstrate the presence of a dynamically regulated nuclear-associated signaling cascade involving PI3K and PDK that presumably influences nuclear Akt activation. PMID:19269295

  12. 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. PMID:25775137

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

    SciTech Connect

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

    2009-09-18

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

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

    PubMed

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

    2016-09-01

    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

  15. 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. PMID:25276814

  16. Insulin-Stimulated Degradation of Apolipoprotein B100: Roles of Class II Phosphatidylinositol-3-Kinase and Autophagy

    PubMed Central

    Chirieac, Doru V.; Tuyama, Ana C.; Montenont, Emilie; Brodsky, Jeffrey L.; Fisher, Edward A.

    2013-01-01

    Both in humans and animal models, an acute increase in plasma insulin levels, typically following meals, leads to transient depression of hepatic secretion of very low density lipoproteins (VLDL). One contributing mechanism for the decrease in VLDL secretion is enhanced degradation of apolipoprotein B100 (apoB100), which is required for VLDL formation. Unlike the degradation of nascent apoB100, which occurs in the endoplasmic reticulum (ER), insulin-stimulated apoB100 degradation occurs post-ER and is inhibited by pan-phosphatidylinositol (PI)3-kinase inhibitors. It is unclear, however, which of the three classes of PI3-kinases is required for insulin-stimulated apoB100 degradation, as well as the proteolytic machinery underlying this response. Class III PI3-kinase is not activated by insulin, but the other two classes are. By using a class I-specific inhibitor and siRNA to the major class II isoform in liver, we now show that it is class II PI3-kinase that is required for insulin-stimulated apoB100 degradation in primary mouse hepatocytes. Because the insulin-stimulated process resembles other examples of apoB100 post-ER proteolysis mediated by autophagy, we hypothesized that the effects of insulin in autophagy-deficient mouse primary hepatocytes would be attenuated. Indeed, apoB100 degradation in response to insulin was significantly impaired in two types of autophagy-deficient hepatocytes. Together, our data demonstrate that insulin-stimulated apoB100 degradation in the liver requires both class II PI3-kinase activity and autophagy. PMID:23516411

  17. 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.

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

    PubMed

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

    2007-12-01

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

  19. 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.

  20. 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.

  1. A short peptide GPIGS promotes proliferation of hair bulb keratinocytes and accelerates hair regrowth in mice.

    PubMed

    Tsuruda, Akinori; Kawano, Yasuhiro; Maekawa, Takaaki; Oka, Syuichi

    2005-03-01

    The aim of this study was to discover a novel agent that promotes hair growth. We carried out a screening test in 298 types of conditioned medium (CM) from cultures of bacteria by using a hair bulb keratinocyte (HBK) growth assay. As a result, we found a HBK growth factor in the CM of Bacillus sp. M18. This HBK growth factor was purified by collecting biologically active fractions in three steps, including HP-20 batch processing, LH-20 chromatography and C18 reverse-phase high-pressure liquid chromatography, and identified as a short peptide GPIGS. GPIGS increased Akt phosphorylation in HBKs. Moreover, the GPIGS-stimulated HBK growth was inhibited by the treatment with LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI-3K). These results suggest that GPIGS promotes HBK growth via the PI-3K/Akt pathway. In addition to in vitro tests, GPIGS was found to accelerate hair regrowth in telogen mice. Our results indicate that GPIGS is a potential agent to promote hair growth.

  2. 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.

  3. Stimulatory effect of boron and manganese salts on keratinocyte migration.

    PubMed

    Chebassier, Nathalie; Ouijja, El Houssein; Viegas, Isabelle; Dreno, Brigitte

    2004-01-01

    Keratinocyte proliferation and migration are essential for the reconstruction of the cutaneous barrier after skin injury. Interestingly, thermal waters which are rich in trace elements (e.g. boron and manganese), are known to be able to improve wound healing. In order to understand the mechanism of action of this effect, our study investigated the in vitro modulation of keratinocyte migration and proliferation by boron and manganese salts, which are present in high concentrations in a thermal water (Saint Gervais). Our in vitro study demonstrated that incubating keratinocytes for 24 h with boron salts at concentrations between 0.5 and 10 microg/ml or manganese salts at concentrations between 0.1 and 1.5 microg/ml accelerated wound closure compared with control medium (+20%). As this acceleration was not related to an increase in keratinocyte proliferation we suggest that boron and manganese act on wound healing mainly by increasing the migration of keratinocytes.

  4. 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.

  5. 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

  6. Mycobacterium avium MAV_2941 mimics Phosphoinositol-3-Kinase to interfere with macrophage phagosome maturation

    PubMed Central

    Danelishvili, Lia; Bermudez, Luiz E.

    2015-01-01

    Mycobacterium avium subsp hominissuis (M. avium) is a pathogen that infects and survives in macrophages. Previously, we have identified the M. avium MAV_2941 gene encoding a 73 amino acid protein exported by the oligopeptide transporter OppA to the macrophage cytoplasm. Mutations in MAV_2941 were associated with significant impairment of M. avium growth in THP-1 macrophages. In this study, we investigated the molecular mechanism of MAV_2941 action and demonstrated that MAV_2941 interacts with the vesicle trafficking proteins syntaxin-8 (STX8), adaptor-related protein complex 3 (AP-3) complex subunit beta-1 (AP3B1) and Archain 1 (ARCN1) in mononuclear phagocytic cells. Sequencing analysis revealed that the binding site of MAV_2941 is structurally homologous to the human phosphatidylinositol 3-kinase (PI3K) chiefly in the region recognized by vesicle trafficking proteins. The β3A subunit of AP-3, encoded by AP3B1, is essential for trafficking cargo proteins, including lysosomal-associated membrane protein 1 (LAMP-1), to the phagosome and lysosome-related organelles. Here, we show that while the heat-killed M. avium when ingested by macrophages co-localizes with LAMP-1 protein, transfection of MAV_2941 in macrophages results in significant decrease of LAMP-1 co-localization with the heat-killed M. avium phagosomes. Mutated MAV_2941, where the amino acids homologous to the binding region of PI3K were changed, failed to interact with trafficking proteins. Inactivation of the AP3B1 gene led to alteration in the trafficking of LAMP-1. These results suggest that M. avium MAV_2941 interferes with the protein trafficking within macrophages altering the maturation of phagosome. PMID:26043821

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

    PubMed

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

    2014-01-01

    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.

  8. Rapamycin increases CCN2 expression of lung fibroblasts via phosphoinositide 3-kinase.

    PubMed

    Xu, Xuefeng; Dai, Huaping; Geng, Jing; Wan, Xuan; Huang, Xiaoxi; Li, Fei; Jiang, Dianhua; Wang, Chen

    2015-08-01

    Excessive production of connective tissue growth factor (CTGF, CCN2) and increased motor ability of the activated fibroblast phenotype contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, molecules and signal pathways regulating CCN2 expression and migration of lung fibroblasts are still elusive. We hypothesize that rapamycin, via binding and blocking mammalian target of rapamycin (mTOR) complex (mTORC), affects CCN2 expression and migration of lung fibroblasts in vitro. Primary normal and fibrotic human lung fibroblasts were isolated from lung tissues of three patients with primary spontaneous pneumothorax and three with IPF. Cells were incubated with regular medium, or medium containing rapamycin, human recombinant transforming growth factor (TGF)-β1, or both. CCN2 and tissue inhibitor of metalloproteinase (TIMP)-1 expression in cells or supernatant was detected. Wound healing and migration assay was used to measure the migratory potential. TGF-β type I receptor (TβRI)/Smad inhibitor, SB431542 and phosphoinositide 3-kinase (PI3K) inhibitor, LY294002 were used to determine rapamycin's mechanism of action. We demonstrated that rapamycin amplified basal or TGF-β1-induced CCN2 mRNA and protein expression in normal or fibrotic fibroblasts by Smad-independent but PI3K-dependent pathway. Additionally, rapamycin also enhanced TIMP-1 expression as indicated by ELISA. However, wound healing and migrating assay showed rapamycin did not affect the mobility of fibroblasts. Collectively, this study implies a significant fibrogenic induction activity of rapamycin by activating AKT and inducing CCN2 expression in vitro and provides the possible mechanisms for the in vivo findings which previously showed no antifibrotic effect of rapamycin on lung fibrosis. PMID:26192087

  9. Class IA phosphatidylinositol 3-kinase p110α regulates phagosome maturation.

    PubMed

    Thi, Emily P; Lambertz, Ulrike; Reiner, Neil E

    2012-01-01

    Of the various phosphatidylinositol 3- kinases (PI3Ks), only the class III enzyme Vps34 has been shown to regulate phagosome maturation. During studies of phagosome maturation in THP-1 cells deficient in class IA PI3K p110α, we discovered that this PI3K isoform is required for vacuole maturation to progress beyond acquisition of Rab7 leading to delivery of lysosomal markers. Bead phagosomes from THP-1 cells acquired p110α and contained PI3P and PI(3,4,5)P3; however, p110α and PI(3,4,5)P3 levels in phagosomes from p110α knockdown cells were decreased. Phagosomes from p110α knock down cells showed normal acquisition of both Rab5 and EEA-1, but were markedly deficient in the lysosomal markers LAMP-1 and LAMP-2, and the lysosomal hydrolase, β-galactosidase. Phagosomes from p110α deficient cells also displayed impaired fusion with Texas Red dextran-loaded lysosomes. Despite lacking lysosomal components, phagosomes from p110α deficient cells recruited normal levels of Rab7, Rab-interacting lysosomal protein (RILP) and homotypic vacuole fusion and protein sorting (HOPs) components Vps41 and Vps16. The latter observations demonstrated that phagosomal Rab7 was active and capable of recruiting effectors involved in membrane fusion. Nevertheless, active Rab7 was not sufficient to bring about the delivery of lysosomal proteins to the maturing vacuole, which is shown for the first time to be dependent on a class I PI3K. PMID:22928013

  10. Phosphatidylinositol 3-Kinase Couples Localised Calcium Influx to Activation of Akt in Central Nerve Terminals.

    PubMed

    Nicholson-Fish, Jessica C; Cousin, Michael A; Smillie, Karen J

    2016-03-01

    The efficient retrieval of synaptic vesicle membrane and cargo in central nerve terminals is dependent on the efficient recruitment of a series of endocytosis modes by different patterns of neuronal activity. During intense neuronal activity the dominant endocytosis mode is activity-dependent endocytosis (ADBE). Triggering of ADBE is linked to calcineurin-mediated dynamin I dephosphorylation since the same stimulation intensities trigger both. Dynamin I dephosphorylation is maximised by a simultaneous inhibition of its kinase glycogen synthase kinase 3 (GSK3) by the protein kinase Akt, however it is unknown how increased neuronal activity is transduced into Akt activation. To address this question we determined how the activity-dependent increases in intracellular free calcium ([Ca(2+)]i) control activation of Akt. This was achieved using either trains of high frequency action potentials to evoke localised [Ca(2+)]i increases at active zones, or a calcium ionophore to raise [Ca(2+)]i uniformly across the nerve terminal. Through the use of either non-specific calcium channel antagonists or intracellular calcium chelators we found that Akt phosphorylation (and subsequent GSK3 phosphorylation) was dependent on localised [Ca(2+)]i increases at the active zone. In an attempt to determine mechanism, we antagonised either phosphatidylinositol 3-kinase (PI3K) or calmodulin. Activity-dependent phosphorylation of both Akt and GSK3 was arrested on inhibition of PI3K, but not calmodulin. Thus localised calcium influx in central nerve terminals activates PI3K via an unknown calcium sensor to trigger the activity-dependent phosphorylation of Akt and GSK3.

  11. 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.

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

    PubMed Central

    Arcaro, Alexandre; Guerreiro, Ana S

    2007-01-01

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

  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. 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

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

    PubMed Central

    2013-01-01

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

  16. Phosphoinositide 3-Kinase γ Restrains Neurotoxic Effects of Microglia After Focal Brain Ischemia.

    PubMed

    Schmidt, Caroline; Frahm, Christiane; Schneble, Nadine; Müller, Jörg P; Brodhun, Michael; Franco, Irene; Witte, Otto W; Hirsch, Emilio; Wetzker, Reinhard; Bauer, Reinhard

    2016-10-01

    Phosphoinositide 3-kinase γ (PI3Kγ) is linked to neuroinflammation and phagocytosis. This study was conducted to elucidate conjectural differences of lipid kinase-dependent and kinase-independent functions of PI3Kγ in the evolvement of brain damage induced by focal cerebral ischemia/reperfusion. Therefore, PI3Kγ wild-type, knockout, and kinase-dead mice were subjected to middle cerebral artery occlusion followed by reperfusion. Tissue damage and cellular composition were assessed by immunohistochemical stainings. In addition, microglial cells derived from respective mouse genotypes were used for analysis of PI3Kγ effects on phagocytic activity, matrix metalloproteinase-9 release, and cAMP content under conditions of oxygen/glucose deprivation and recovery. Brain infarction was more pronounced in PI3Kγ-knockout mice compared to wild-type and kinase-dead mice 48 h after reperfusion. Immunohistochemical analyses revealed a reduced amount of galectin-3/MAC-2-positive microglial cells indicating that activated phagocytosis was reduced in ischemic brains of knockout mice. Cell culture studies disclosed enhanced metalloproteinase-9 secretion in supernatants derived from microglia of PI3Kγ-deficient mice after 2-h oxygen/glucose deprivation and 48-h recovery. Furthermore, PI3Kγ-deficient microglial cells showed a failed phagocytic activation throughout the observed recovery period. Lastly, PI3Kγ-deficient microglia exhibited strongly increased cAMP levels in comparison with wild-type microglia or cells expressing kinase-dead PI3Kγ after oxygen/glucose deprivation and recovery. Our data suggest PI3Kγ kinase activity-independent control of cAMP phosphodiesterase as a crucial mediator of microglial cAMP regulation, MMP-9 expression, and phagocytic activity following focal brain ischemia/recirculation. The suppressive effect of PI3Kγ on cAMP levels appears critical for the restriction of ischemia-induced immune cell functions and in turn tissue damage.

  17. Hydrogen peroxide stimulates the epithelial sodium channel through a phosphatidylinositide 3-kinase-dependent pathway.

    PubMed

    Ma, He-Ping

    2011-09-16

    Recent studies indicate that oxidative stress mediates salt-sensitive hypertension. To test the hypothesis that the renal epithelial sodium channel (ENaC) is a target of oxidative stress, patch clamp techniques were used to determine whether ENaC in A6 distal nephron cells is regulated by hydrogen peroxide (H(2)O(2)). In the cell-attached configuration, H(2)O(2) significantly increased ENaC open probability (P(o)) and single-channel current amplitude but not the unit conductance. High concentrations of exogenous H(2)O(2) are required to elevate intracellular H(2)O(2), probably because catalase, the enzyme that promotes the decomposition of H(2)O(2) to H(2)O and O(2), is highly expressed in A6 cells. The effect of H(2)O(2) on ENaC P(o) was enhanced by 3-aminotriazole, a catalase inhibitor, and abolished by overexpression of catalase, indicating that intracellular H(2)O(2) levels are critical to produce the effect. However, H(2)O(2) did not directly activate ENaC in inside-out patches. The effects of H(2)O(2) on ENaC P(o) and amiloride-sensitive Na(+) current were abolished by inhibition of phosphatidylinositide 3-kinase (PI3K). Confocal microscopy data showed that H(2)O(2) elevated phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) in the apical membrane by stimulating PI3K. Because ENaC is stimulated by PI(3,4,5)P(3), these data suggest that H(2)O(2) stimulates ENaC via PI3K-mediated increases in apical PI(3,4,5)P(3). PMID:21795700

  18. PI3-kinase activation is critical for host barrier permissiveness to Listeria monocytogenes

    PubMed Central

    Gessain, Grégoire; Tsai, Yu-Huan; Travier, Laetitia; Bonazzi, Matteo; Grayo, Solène; Cossart, Pascale; Charlier, Caroline; Disson, Olivier

    2015-01-01

    Invasion of nonphagocytic cells, a critical property of Listeria monocytogenes (Lm) that enables it to cross host barriers, is mediated by the interaction of two bacterial surface proteins, InlA and InlB, with their respective receptors E-cadherin and c-Met. Although InlA–E-cadherin interaction is necessary and sufficient for Lm crossing of the intestinal barrier, both InlA and InlB are required for Lm crossing of the placental barrier. The mechanisms underlying these differences are unknown. Phosphoinositide 3-kinase (PI3-K) is involved in both InlA- and InlB-dependent pathways. Indeed, InlA-dependent entry requires PI3-K activity but does not activate it, whereas InlB–c-Met interaction activates PI3-K. We show that Lm intestinal target cells exhibit a constitutive PI3-K activity, rendering InlB dispensable for InlA-dependent Lm intestinal barrier crossing. In contrast, the placental barrier does not exhibit constitutive PI3-K activity, making InlB necessary for InlA-dependent Lm placental invasion. Here, we provide the molecular explanation for the respective contributions of InlA and InlB to Lm host barrier invasion, and reveal the critical role of InlB in rendering cells permissive to InlA-mediated invasion. This study shows that PI3-K activity is critical to host barrier permissiveness to microbes, and that pathogens exploit both similarities and differences of host barriers to disseminate. PMID:25624443

  19. [Role of phosphatidylinositol 3-kinase and myosin light chain kinase during the activation of thrombin receptors].

    PubMed

    Han, Yue; Gao, Hai-Li; Zhang, Wei; Bai, Xia; Dai, Lan; Sheng, Wen-Hong; Sun, Ai-Ning; Wu, De-Pei; Wang, Zhao-Yue; Ruan, Chang-Geng

    2009-06-01

    The objective of study was to compare the influences of wortmannin on platelet aggregation and platelet membrane surface glycoproteins GPIb expression after thrombin receptor activation, and to investigate the role of phosphatidylinositol 3-kinase (PI3-K) and myosin light chain kinase (MLCK) in the course of thrombin receptor activation. Peptide SFLLRN (PAR1-AP) and AYPGKF (PAR4-AP) were used for stimulating platelet, and the changes of platelet aggregation and GPIb were analyzed with 100 nmol/L wortmannin (inhibitor of PI3-K) and 10 micromol/L wortmannin (inhibitor of MLCK). The results indicated that the platelet activation was influenced by either concentration of wortmannin in response to PAR stimulation. Platelet aggregation was apparently inhibited by 10 micromol/L wortmannin through both PAR peptides, and was slightly inhibited by 100 nmol/L wortmannin only under PAR1-AP activation. In addition, GPIbalpha internalization was partly inhibited by 100 nmol/L wortmannin in response to PAR1 (p < 0.05 at 1, 2, 5 min) and PAR4 (p < 0.05 at 2, 5, 10 min) activation. Meanwhile, 10 micromol/L wortmannin induced little change for GPIbalpha centralisation in the course of PAR activation, with a delayed restoration of surface GPIbalpha observed under PAR1-AP activation, and no change of GPIbalpha redistribution existed under PAR4-AP activation. It is concluded that the different roles of PI3-K and MLCK exist in the course of thrombin receptor activation. PI3-K accelerates the short course of GPIb centralisation for two PAR signal pathways, while MLCK inhibits the restoration of GPIbalpha in PAR1 pathway. PMID:19549383

  20. 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.

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

    PubMed

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

    2013-12-01

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

  2. The role of phosphoinositide-3 kinase and PTEN in cardiovascular physiology and disease.

    PubMed

    Oudit, Gavin Y; Sun, Hui; Kerfant, Benoit-Gilles; Crackower, Michael A; Penninger, Josef M; Backx, Peter H

    2004-08-01

    Phosphoinositide-3 kinases (PI3Ks) are a family of evolutionary conserved lipid kinases that mediate many cellular responses in both physiologic and pathophysiologic states. Class I PI3K can be activated by either receptor tyrosine kinase (RTK)/cytokine receptor activation (class I(A)) or G-protein-coupled receptors (GPCR) (class I(B)). Once activated PI3Ks generate phosphatidylinositols (PtdIns) (3,4,5)P(3) leading to the recruitment and activation of Akt/protein kinase B (PKB), PDK1 and monomeric G-proteins (e.g. Rac-GTPases), which then activate a range of downstream targets including glycogen synthase kinase-3beta (GSK-3beta), mammalian target of rapamycin (mTOR), p70S6 kinase, endothelial nitric oxide synthase (eNOS) and several anti-apoptotic effectors. Class I(A) (PI3Kalpha, beta and delta) and class I(B) (PI3Kgamma) PI3Ks mediate distinct phenotypes in the heart and under negative control by the 3'-lipid phosphatase, phosphatase and tensin homolog on chromosome ten (PTEN) which dephosphorylate PtdIns(3,4,5)P(3) into PtdIns(4,5)P(2). PI3Kalpha, gamma and PTEN are expressed in cardiomyocytes, fibroblasts, endothelial cells and vascular smooth muscle cells where they modulate cell survival/apoptosis, hypertrophy, contractility, metabolism and mechanotransduction. Several transgenic and knockout models support a fundamental role of PI3K/PTEN signaling in the regulation of myocardial contractility and hypertrophy. Consequently the PI3K/PTEN signaling pathways are involved in a wide variety of diseases including cardiac hypertrophy, heart failure, preconditioning and hypertension. In this review, we discuss the biochemistry and molecular biology of PI3K (class I isoforms) and PTEN and their critical role in cardiovascular physiology and diseases.

  3. 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

  4. 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

  5. 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. PMID:25344255

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

    PubMed Central

    Erazo, Angela

    2014-01-01

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

  7. Distinct Effects of Different Phosphatidylglycerol Species on Mouse Keratinocyte Proliferation

    PubMed Central

    Xie, Ding; Seremwe, Mutsa; Edwards, John G.; Podolsky, Robert; Bollag, Wendy B.

    2014-01-01

    We have previously shown that liposomes composed of egg-derived phosphatidylglycerol (PG), with a mixed fatty acid composition (comprising mainly palmitate and oleate), inhibit the proliferation and promote the differentiation of rapidly dividing keratinocytes, and stimulate the growth of slowly proliferating epidermal cells. To determine the species of PG most effective at modulating keratinocyte proliferation, primary mouse keratinocytes were treated with different PG species, and proliferation was measured. PG species containing polyunsaturated fatty acids were effective at inhibiting rapidly proliferating keratinocytes, whereas PG species with monounsaturated fatty acids were effective at promoting proliferation in slowly dividing cells. Thus, palmitoyl-arachidonyl-PG (16∶0/20∶4), palmitoyl-linoleoyl-PG (16∶0/18∶2), dilinoleoyl-PG (18∶2/18∶2) and soy PG (a PG mixture with a large percentage of polyunsaturated fatty acids) were particularly effective at inhibiting proliferation in rapidly dividing keratinocytes. Conversely, palmitoyl-oleoyl-PG (16∶0/18∶1) and dioleoyl-PG (18∶1/18∶1) were especially effective proproliferative PG species. This result represents the first demonstration of opposite effects of different species of a single class of phospholipid and suggests that these different PG species may signal to diverse effector enzymes to differentially affect keratinocyte proliferation and normalize keratinocyte proliferation. Thus, different PG species may be useful for treating skin diseases characterized by excessive or insufficient proliferation. PMID:25233484

  8. 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

  9. 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

  10. 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

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

    PubMed Central

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

    2003-01-01

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

  12. 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

  13. 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.

  14. 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.

  15. RXRα ablation in epidermal keratinocytes enhances UVR-induced DNA damage, apoptosis, and proliferation of keratinocytes and melanocytes.

    PubMed

    Wang, Zhixing; Coleman, Daniel J; Bajaj, Gaurav; Liang, Xiaobo; Ganguli-Indra, Gitali; Indra, Arup K

    2011-01-01

    We show here that keratinocytic nuclear receptor retinoid X receptor-α (RXRα) regulates mouse keratinocyte and melanocyte homeostasis following acute UVR. Keratinocytic RXRα has a protective role in UVR-induced keratinocyte and melanocyte proliferation/differentiation, oxidative stress-mediated DNA damage, and cellular apoptosis. We discovered that keratinocytic RXRα, in a cell-autonomous manner, regulates mitogenic growth responses in skin epidermis through secretion of heparin-binding EGF-like growth factor, GM-CSF, IL-1α, and cyclooxygenase-2 and activation of mitogen-activated protein kinase pathways. We identified altered expression of several keratinocyte-derived mitogenic paracrine growth factors such as endothelin 1, hepatocyte growth factor, α-melanocyte stimulating hormone, stem cell factor, and fibroblast growth factor-2 in skin of mice lacking RXRα in epidermal keratinocytes (RXRα(ep-/-) mice), which in a non-cell-autonomous manner modulated melanocyte proliferation and activation after UVR. RXRα(ep-/-) mice represent a unique animal model in which UVR induces melanocyte proliferation/activation in both epidermis and dermis. Considered together, the results of our study suggest that RXR antagonists, together with inhibitors of cell proliferation, can be effective in preventing solar UVR-induced photocarcinogenesis.

  16. 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 (

  17. 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.

  18. Measurement of anti-inflammatory effects of glucocorticoids on human keratinocytes in vitro. Comparison of normal human keratinocytes with the keratinocyte cell line HaCaT.

    PubMed

    Stein, M; Bernd, A; Ramirez-Bosca, A; Kippenberger, S; Holzmann, H

    1997-11-01

    There are only few objective in vitro methods available for the testing of anti-inflammatory pharmaceutical products. One possibility is in the stimulation of cytokine production in cultivated human keratinocytes by UV light and the subsequent testing of suppressing activities. From the dermatological aspect the interleukins 6 and 8 are especially interesting because they are elevated in psoriatic skin. In the present work three glucocorticoids were tested in cultures of normal human keratinocytes and in the permanent keratinocyte cell line HaCaT. Both cell species produced IL-6 and IL-8 spontaneously, albeit in very small amounts. After UV irradiation the interleukin production increased in a dose dependent manner. The IL-6 and IL-8 induction could be suppressed by each of the glucocorticoids tested. The thymidine incorporation rate of the cells was not affected by the glucocorticoids indicating that the observed suppression of cytokine induction was not the result of a generalised cell damage. The response of both HaCaT keratinocytes and primary human keratinocytes to UV irradiation and glucocorticoid application was similar indicating the possible use of the generally available HaCaT cells for the pharmacological testing of anti-inflammatory activities in vitro. PMID:9428986

  19. 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.

  20. 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.

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

    PubMed Central

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

    2013-01-01

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

  2. AtVPS34, a phosphatidylinositol 3-kinase of Arabidopsis thaliana, is an essential protein with homology to a calcium-dependent lipid binding domain.

    PubMed

    Welters, P; Takegawa, K; Emr, S D; Chrispeels, M J

    1994-11-22

    The cDNA encoding phosphatidylinositol (PI) 3-kinase was cloned from Arabidopsis thaliana, and the derived amino acid sequence (AtVPS34) has a significantly higher homology to yeast PI 3-kinase (VPS34) than to the mammalian (p110). The protein has two conserved domains: a catalytic site with the ATP-binding site near the C terminus and a calcium-dependent lipid-binding domain near the N terminus. The plant cDNA does not rescue a yeast vps34 deletion mutant, but a chimeric gene in which the coding sequence for the C-terminal third of VPS34 is replaced by the corresponding sequence from the plant gene does rescue the yeast mutant. PI 3-kinase activity is detectable in extracts from plants that overexpress the plant PI 3-kinase. Expression of antisense constructs gives rise to second-generation transformed plants severely inhibited in growth and development.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. The new InsP3Kinase inhibitor BIP-4 is competitive to InsP3 and blocks proliferation and adhesion of lung cancer cells.

    PubMed

    Schröder, Dominik; Tödter, Klaus; Gonzalez, Beatriz; Franco-Echevarría, Elsa; Rohaly, Gabor; Blecher, Christine; Lin, Hong-Ying; Mayr, Georg W; Windhorst, Sabine

    2015-07-15

    As ectopic expression of the neuronal inositol-1,4,5-trisphosphate-3-kinase A (InsP3Kinase) in tumor cells increases the metastatic potential, InsP3Kinase is an interesting target for tumor therapy. Recently, we have identified a membrane-permeable InsP3Kinase inhibitor (BAMB-4) exhibiting an IC50-value of 20 μM. Here we characterized a new InsP3Kinase inhibitor which shows a 130-fold lower IC50 value (157 ± 57 nM) as compared to BAMB-4. We demonstrate that this nitrophenolic compound, BIP-4, is non-competitive to ATP but competitive to InsP3, thus exhibits a high selectivity for inhibition of InsP3Kinase activity. Docking analysis suggested a putative binding mode of this molecule into the InsP3Kinase active site. Determination of cellular uptake in lung cancer cells (H1299) revealed that 6% of extracellular BIP-4 is internalized by non-endosomal uptake, showing that BIP-4 is not trapped inside endo/lysosomes but is available to inhibit cellular InsP3Kinase activity. Interestingly, we found that BIP-4 mediated inhibition of InsP3Kinase activity in the two lung cancer cell lines H1299 and LN4323 inhibited proliferation and adhesion at IC50 values of 3 μM or 2 μM, respectively. InsP3Kinase inhibition did not alter ATP-induced calcium signals but significantly reduced the level of Ins(1,3,4,5,6)P5. From these data we conclude that the inhibitory effect of BIP-4 on proliferation and adhesion of lung cancer cells does not result from alterations of calcium but from alterations of inositol phosphate signals. In summary, we reveal that inhibition of cellular InsP3Kinase by BIP-4 impairs proliferation and adhesion and therefore BIP-4 might be a promising compound to reduce the metastatic potential of lung carcinoma cells.

  8. Phosphorylation in vitro of the 85 kDa subunit of phosphatidylinositol 3-kinase and its possible activation by insulin receptor tyrosine kinase.

    PubMed Central

    Hayashi, H; Miyake, N; Kanai, F; Shibasaki, F; Takenawa, T; Ebina, Y

    1991-01-01

    Insulin causes a dramatic and rapid increase in phosphatidylinositol 3-kinase activity in the anti-phosphotyrosine immunoprecipitates of cells overexpressing the human insulin receptor. This enzyme may therefore be a mediator of insulin signal transduction [Endemann, Yonezawa & Roth (1990) J. Biol. Chem. 265, 396-400; Ruderman, Kapeller, White & Cantley (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415]. At least two questions remain to be elucidated. Firstly, does the insulin receptor tyrosine kinase phosphorylate phosphatidylinositol 3-kinase directly, or does it phosphorylate a protein associated with the 3-kinase? Second, if the enzyme is a direct substrate for the insulin receptor tyrosine kinase, does tyrosine phosphorylation of phosphatidylinositol 3-kinase by the kinase alter the specific enzyme activity, or does the amount of the tyrosine-phosphorylated form of the phosphatidylinositol 3-kinase increase, with no change in the specific activity? We report here evidence that the 85 kDa subunit of highly purified phosphatidylinositol 3-kinase is phosphorylated on the tyrosine residue by the activated normal insulin receptor in vitro, but not by a mutant insulin receptor which lacks tyrosine kinase activity. We found that an increase in enzyme activity was detected in response to insulin not only in the anti-phosphotyrosine immunoprecipitates of the cytosol, but also in the cytosolic fraction before immunoprecipitation. In addition, we partially separated the tyrosine-phosphorylated form from the unphosphorylated form of the enzyme, by using a f.p.l.c. Mono Q column. The insulin-stimulated phosphatidylinositol 3-kinase activity was mainly detected in the fraction containing almost all of the tyrosine-phosphorylated form. This result suggests that tyrosine phosphorylation of phosphatidylinositol 3-kinase by the insulin receptor kinase may increase the specific activity of the former enzyme in vivo. Images Fig. 1. Fig. 2. Fig. 4. PMID:1722393

  9. Epidermal Growth Factor Receptor Transactivation Is Required for Mitogen-Activated Protein Kinase Activation by Muscarinic Acetylcholine Receptors in HaCaT Keratinocytes

    PubMed Central

    Ockenga, Wymke; Kühne, Sina; Bocksberger, Simone; Banning, Antje; Tikkanen, Ritva

    2014-01-01

    Non-neuronal acetylcholine plays a substantial role in the human skin by influencing adhesion, migration, proliferation and differentiation of keratinocytes. These processes are regulated by the Mitogen-Activated Protein (MAP) kinase cascade. Here we show that in HaCaT keratinocytes all five muscarinic receptor subtypes are expressed, but M1 and M3 are the subtypes involved in mitogenic signaling. Stimulation with the cholinergic agonist carbachol leads to activation of the MAP kinase extracellular signal regulated kinase, together with the protein kinase Akt. The activation is fully dependent on the transactivation of the epidermal growth factor receptor (EGFR), which even appears to be the sole pathway for the muscarinic receptors to facilitate MAP kinase activation in HaCaT cells. The transactivation pathway involves a triple-membrane-passing process, based on activation of matrix metalloproteases, and extracellular ligand release; whereas phosphatidylinositol 3-kinase, Src family kinases or protein kinase C do not appear to be involved in MAP kinase activation. Furthermore, phosphorylation, ubiquitination and endocytosis of the EGF receptor after cholinergic transactivation are different from that induced by a direct stimulation with EGF, suggesting that ligands other than EGF itself mediate the cholinergic transactivation. PMID:25421240

  10. 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

  11. 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.

  12. Keratinocyte cytoskeletal roles in cell sheet engineering

    PubMed Central

    2013-01-01

    Background There is an increasing need to understand cell-cell interactions for cell and tissue engineering purposes, such as optimizing cell sheet constructs, as well as for examining adhesion defect diseases. For cell-sheet engineering, one major obstacle to sheet function is that cell sheets in suspension are fragile and, over time, will contract. While the role of the cytoskeleton in maintaining the structure and adhesion of cells cultured on a rigid substrate is well-characterized, a systematic examination of the role played by different components of the cytoskeleton in regulating cell sheet contraction and cohesion in the absence of a substrate has been lacking. Results In this study, keratinocytes were cultured until confluent and cell sheets were generated using dispase to remove the influence of the substrate. The effects of disrupting actin, microtubules or intermediate filaments on cell-cell interactions were assessed by measuring cell sheet cohesion and contraction. Keratin intermediate filament disruption caused comparable effects on cell sheet cohesion and contraction, when compared to actin or microtubule disruption. Interfering with actomyosin contraction demonstrated that interfering with cell contraction can also diminish cell cohesion. Conclusions All components of the cytoskeleton are involved in maintaining cell sheet cohesion and contraction, although not to the same extent. These findings demonstrate that substrate-free cell sheet biomechanical properties are dependent on the integrity of the cytoskeleton network. PMID:23442760

  13. HPV-18 immortalization of human keratinocytes.

    PubMed

    Kaur, P; McDougall, J K

    1989-11-01

    The oncogenic potential of human papillomavirus type 18 which is found in a significant number of cervical and penile cancer biopsies was tested in primary human keratinocytes derived from neonatal foreskin. Viral DNA and a gene for resistance to neomycin were introduced into these cells by calcium phosphate transfection. Selection of cells in G418 led to the isolation of resistant colonies which were propagated in culture. Four cell lines termed FE-A, FEH 18L, FEP18-5, and FEP18-11 have been maintained in culture for 1 1/2-2 years and were selected for further analysis. In all cases the viral DNA was integrated into the cellular genome and the early genes were transcribed, including RNA complementary to the E2, E6, and E7 open reading frames. Radioimmunoprecipitation showed that all cell lines synthesized the E6 and E7 proteins. However, none of the cell lines tested were tumorigenic. The differentiation capacity of these cells was analyzed by assessing their ability to proliferate clonally after exposure to 1.2 mM calcium chloride. All four cell lines were resistant to this stimulus and formed colonies upon return to regular growth medium whereas normal cells differentiated terminally. K6a and K14 keratin RNA expression was down-regulated in the HPV immortalized cell lines compared to primary human epithelial cells.

  14. 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.

  15. Insulin stimulates glucose uptake via a phosphatidylinositide 3-kinase-linked signaling pathway in bovine mammary epithelial cells.

    PubMed

    Zhao, K; Liu, H-Y; Zhou, M-M; Zhao, F-Q; Liu, J-X

    2014-01-01

    The aim of this study was to investigate the effects of insulin on glucose uptake in lactating bovine mammary epithelial cells (BMEC). Primary BMEC were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F-12 and treated with different levels of insulin (0, 5, 50, and 500 ng/mL) for 48 h after a 24-h starvation without fetal calf serum. Compared with the control cells (0 ng of insulin/mL), cell proliferation was enhanced by insulin treatment at all tested levels. Insulin significantly increased glucose uptake at a concentration of 500 ng/mL. In addition, the protein synthesis inhibitor cycloheximide (0.5mg/mL) counteracted the insulin-elevated glucose uptake, thereby suggesting that newly synthesized transporter protein might take part in the insulin-induced glucose uptake. Furthermore, pretreatment of the cells with SB203580, an inhibitor of p38 mitogen-activated protein kinase, did not influence the insulin-induced glucose uptake, but LY294002, a specific inhibitor of phosphatidylinositide 3-kinase, significantly reduced the insulin-stimulated glucose uptake. These results indicated that insulin-induced glucose uptake in BMEC may involve the phosphatidylinositide 3-kinase- but not mitogen-activated protein kinase-mediated signaling pathways.

  16. 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.

  17. Two distinct intracytoplasmic regions of the T-cell adhesion molecule CD28 participate in phosphatidylinositol 3-kinase association.

    PubMed

    Pagès, F; Ragueneau, M; Klasen, S; Battifora, M; Couez, D; Sweet, R; Truneh, A; Ward, S G; Olive, D

    1996-04-19

    Through the interaction with its ligands, CD80/B7-1 and CD86/B7-2 or B70, the human CD28 molecule plays a major functional role as a costimulator of T cells along with the CD3-TcR complex. We and others have previously reported that phosphatidylinositol 3-kinase inducibly associates with CD28. This association is mediated by the SH2 domains of the p85 adaptor subunit interacting with a cytoplasmic YMNM consensus motif present in CD28 at position 173-176. Disruption of this binding site by site-directed mutagenesis abolishes CD28-induced activation events in a murine T-cell hybridoma transfected with human CD28 gene. Here we show that the last 10 residues of the intracytoplasmic domain of CD28 (residues 193-202) are required for its costimulatory function. These residues are involved in interleukin-2 secretion, p85 binding, and CD28-associated phosphatidylinositol 3-kinase activity. In contrast, the CD28/CD8O interaction is unaffected by this deletion, as is the induction of other second messengers such as the rise in intracellular calcium and tyrosine phosphorylation of CD28-specific substrates. Furthermore, we also demonstrate that, within these residues, the tyrosine at position 200 is involved in p85 binding, probably together with the short proline-rich motif present between residues 190 and 194 (PYAPP).

  18. Therapeutic implications of targeting the PI3Kinase/AKT/mTOR signaling module in melanoma therapy

    PubMed Central

    Jazirehi, Ali R; Wenn, Peter B; Damavand, Mohsen

    2012-01-01

    The PI3Kinase/AKT/mTOR signaling module is implicated in various cellular functions including cell survival, growth and proliferation, glucose metabolism, apoptosis, migration, and angiogenesis. Increased expression of AKT and its up- and downstream regulators is linked to several types of cancer. Aberrant expression of AKT is observed in nearly 60% of melanomas culminating in apoptosis resistance via deactivation of apoptotic molecules Bad and Cas-pase-9. Through cross-talk with NF-κB, ERK1/2, JNK and p38MAPK signaling pathways, AKT induces a plethora of cellular effects often leading to tumor development and progression. Due to frequently observed resistance to other common cancer treatments such as chemotherapy, immunotherapy, and radiation, and the detrimental consequences of constitutive activation of the PI3Kinase/AKT/mTOR signaling module, targeted inhibition of the effectors and substrates involved in this module has become a viable and attractive option for molecular targeted therapy in melanoma. Pharmacological inhibitors of various components of this module, either alone or in combination with other agents, have shown significant decrease in proliferation, tumorigenesis, cell growth and survival of various tumors in phases I and II clinical trials. Some inhibitors have even received their Food and Drug Administration (FDA) approval. This review summarizes the current knowledge on this module, its cross-talk with other major cell survival pathways and its targeted inhibition for therapeutic purposes in melanoma. PMID:22485197

  19. 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. PMID:26529487

  20. 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

  1. SHARPIN regulates mitochondria-dependent apoptosis in keratinocytes

    PubMed Central

    Liang, Yanhua; Sundberg, John P

    2011-01-01

    Background The chronic proliferative dermatitis mutation (CPDM) in mice, due to Sharpin deficiency (Sharpincpdm), is a multisystem disorder characterized by peripheral blood eosinophilia and eosinophil infiltration of affected tissues including the skin, bone marrow, spleen, lung, heart, and other organs. The epidermis has numerous apoptotic keratinocytes which increase with age, coalesce, form vesicles, and rupture causing ulceration. Objective To clarify the molecular pathways involved in the keratinocyte apoptosis caused by loss of function of SHARPIN in mice. Method 10-week-old Sharpincpdm and wildtype mice were used for experiments. Ultrastructural changes of skin were evaluated by transmission electron microscopy. Cross points of mitochondrial pathway were analyzed by in vitro and in vivo cellular and molecular assays. Results 77.5% skin cells in Sharpincpdm mice were functionally apoptotic and dead cells, compared to only 18.1% unhealthy skin cells in wildtype mice, indicated by annexin-V/propidium iodide FACS analysis. Mitochondria in keratinocytes were disrupted containing prominent electron dense inclusions and membrane potential depolarization, accompanied by a shift in protein expression between the anti-apoptotic BCL2 and pro-apoptotic BAX proteins. Enzymatic activities of caspases 9 and 3, but not 8, were markedly increased in Sharpincpdm keratinocytes. Caspase-3 was cleaved in most cells in skin of 10-week-old mutant mice. Conclusion The present results indicated that keratinocyte apoptosis in Sharpincpdm mice was regulated by an intrinsic caspase-dependent mitochondria pathway. PMID:21620685

  2. Quantitative analysis of laminin 5 gene expression in human keratinocytes.

    PubMed

    Akutsu, Nobuko; Amano, Satoshi; Nishiyama, Toshio

    2005-05-01

    To examine the expression of laminin 5 genes (LAMA3, LAMB3, and LAMC2) encoding the three polypeptide chains alpha3, beta3, and gamma2, respectively, in human keratinocytes, we developed novel quantitative polymerase chain reaction (PCR) methods utilizing Thermus aquaticus DNA polymerase, specific primers, and fluorescein-labeled probes with the ABI PRISM 7700 sequence detector system. Gene expression levels of LAMA3, LAMB3, and LAMC2 and glyceraldehyde-3-phosphate dehydrogenase were quantitated reproducibly and sensitively in the range from 1 x 10(2) to 1 x 10(8) gene copies. Basal gene expression level of LAMB3 was about one-tenth of that of LAMA3 or LAMC2 in human keratinocytes, although there was no clear difference among immunoprecipitated protein levels of alpha3, beta3, and gamma2 synthesized in radio-labeled keratinocytes. Human serum augmented gene expressions of LAMA3, LAMB3, and LAMC2 in human keratinocytes to almost the same extent, and this was associated with an increase of the laminin 5 protein content measured by a specific sandwich enzyme-linked immunosorbent assay. These results demonstrate that the absolute mRNA levels generated from the laminin 5 genes do not determine the translated protein levels of the laminin 5 chains in keratinocytes, and indicate that the expression of the laminin 5 genes may be controlled by common regulation mechanisms. PMID:15854126

  3. Chemosensory information processing between keratinocytes and trigeminal neurons.

    PubMed

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

    2014-06-20

    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

  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. Amarogentin Displays Immunomodulatory Effects in Human Mast Cells and Keratinocytes

    PubMed Central

    Wölfle, Ute; Haarhaus, Birgit; Schempp, Christoph M.

    2015-01-01

    Keratinocytes express the bitter taste receptors TAS2R1 and TAS2R38. Amarogentin as an agonist for TAS2R1 and other TAS2Rs promotes keratinocyte differentiation. Similarly, mast cells are known to express bitter taste receptors. The aim of this study was to assess whether bitter compounds display immunomodulatory effects on these immunocompetent cells in the skin, so that they might be a target in chronic inflammatory diseases such as atopic dermatitis and psoriasis. Here, we investigated the impact of amarogentin on substance P-induced release of histamine and TNF-α from the human mast cell line LAD-2. Furthermore, the effect of amarogentin on HaCaT keratinocytes costimulated with TNF-α and histamine was investigated. Amarogentin inhibited in LAD-2 cells substance P-induced production of newly synthesized TNF-α, but the degranulation and release of stored histamine were not affected. In HaCaT keratinocytes histamine and TNF-α induced IL-8 and MMP-1 expression was reduced by amarogentin to a similar extent as with azelastine. In conclusion amarogentin displays immunomodulatory effects in the skin by interacting with mast cells and keratinocytes. PMID:26600671

  6. Proliferation of normal human keratinocytes on silicone substrates.

    PubMed

    Rosdy, M; Grisoni, B; Clauss, L C

    1991-07-01

    Several polydimethylsiloxane elastomers and gels were tested as culture substrates for proliferating normal human epidermal keratinocytes. Growth kinetics of normal human keratinocytes (NHK) and dermal fibroblasts were compared on 'very soft', 'soft' and 'hard' silicone gels, as well as on standard cell culture polystyrene dishes. Water contact angles and chemical compositions (IRFT-HATR) of the different silicone surfaces were found to be equivalent, although very different from standard cell culture polystyrene. The topography of the surfaces as well as the shape of the keratinocytes and fibroblasts grown on the different substrates were visualized by scanning electron microscopy, and compared. Although the surface softness and topography of the substrates differed markedly, dermal fibroblasts proliferated in serum-containing medium in equivalent manner on all substrates. Again no correlation could be found between the characteristics and the attachment of the substrates and rapid proliferation of the epidermal keratinocytes in defined medium. The epidermal keratinocytes spread, secreted a structured extracellular matrix network and grew up to confluence on all silicone substrates (elastomers and gels), except the relatively 'hard' silicone gel; this could be due to a direct interference by the waves observed on the silicone gel surfaces. PMID:1654138

  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. PMID:27371895

  8. Induction of differentiation in psoriatic keratinocytes by propylthiouracil and fructose.

    PubMed

    Arul, Santhosh; Dayalan, Haripriya; Jegadeesan, Muhilan; Damodharan, Prabhavathy

    2016-12-01

    Psoriasis is characterized by uncontrolled proliferation and poor differentiation. Sirtuin1 (SIRT1) a class III deacetylase, crucial for differentiation in normal keratinocytes, is reduced in psoriasis. Down regulated SIRT1 levels may contribute to poor differentiation in psoriasis. In addition, the levels of early differentiation factors Keratin1 (K1) and Keratin10 (K10) are depleted in psoriasis. We attempted to study a possible effect of fructose, a SIRT1 upregulator and Propylthiouracil (PTU) to augment differentiation in psoriatic keratinocytes. Keratinocytes were cultured from lesional biopsies obtained from psoriatic patients and control cells were obtained from patients undergoing abdominoplasty. Cells were treated with fructose and PTU individually. K1 and K10 transcript levels were measured to evaluate early differentiation; SIRT1 protein expression was also studied to decipher its role in the mechanism of differentiation. The K1, K10 transcript levels, SIRT1 protein and transcript levels in fructose treated psoriatic keratinocytes were improved. This suggests keratinocyte differentiation was induced by fructose through SIRT1 upregulation. Whereas PTU induced differentiation, as confirmed by improved K1, K10 transcript levels followed a non-SIRT1 mechanism. We conclude that the use of fructose and PTU may be an adjunct to the existing therapies for psoriasis. PMID:27453822

  9. Methylparaben potentiates UV-induced damage of skin keratinocytes.

    PubMed

    Handa, Osamu; Kokura, Satoshi; Adachi, Satoko; Takagi, Tomohisa; Naito, Yuji; Tanigawa, Toru; Yoshida, Norimasa; Yoshikawa, Toshikazu

    2006-10-01

    For many years, methylparaben (MP) has been used as a preservative in cosmetics. In this study, we investigated the effects of ultraviolet-B (UVB) exposure on MP-treated human skin keratinocytes. HaCaT keratinocyte was cultured in MP-containing medium for 24h, exposed to UVB (15 or 30 mJ/cm(2)) and further cultured for another 24h. Subsequent cellular viability was quantified by MTT-based assay and cell death was qualified by fluorescent microscopy and flow cytometry. Oxidative stress, nitric oxide (NO) production and cellular lipid peroxidation were measured using fluorescent probes. In addition, activation of nuclear factor kappa B and activator protein-1 was assessed by electro-mobility gel-shift assay. Practical concentrations of MP (0.003%) had a little or no effect on cellular viability, oxidative stress, NO production, lipid peroxidation and activation of nuclear transcription factors in HaCaT keratinocytes. Low-dose UVB also had little or no effect on these parameters in HaCaT keratinocytes. However, UVB exposure significantly increased cell death, oxidative stress, NO production, lipid peroxidation and activation of transcription factors in MP-treated HaCaT keratinocytes. These results indicate that MP, which has been considered a safe preservative in cosmetics, may have harmful effects on human skin when exposed to sunlight.

  10. Amarogentin Displays Immunomodulatory Effects in Human Mast Cells and Keratinocytes.

    PubMed

    Wölfle, Ute; Haarhaus, Birgit; Schempp, Christoph M

    2015-01-01

    Keratinocytes express the bitter taste receptors TAS2R1 and TAS2R38. Amarogentin as an agonist for TAS2R1 and other TAS2Rs promotes keratinocyte differentiation. Similarly, mast cells are known to express bitter taste receptors. The aim of this study was to assess whether bitter compounds display immunomodulatory effects on these immunocompetent cells in the skin, so that they might be a target in chronic inflammatory diseases such as atopic dermatitis and psoriasis. Here, we investigated the impact of amarogentin on substance P-induced release of histamine and TNF-α from the human mast cell line LAD-2. Furthermore, the effect of amarogentin on HaCaT keratinocytes costimulated with TNF-α and histamine was investigated. Amarogentin inhibited in LAD-2 cells substance P-induced production of newly synthesized TNF-α, but the degranulation and release of stored histamine were not affected. In HaCaT keratinocytes histamine and TNF-α induced IL-8 and MMP-1 expression was reduced by amarogentin to a similar extent as with azelastine. In conclusion amarogentin displays immunomodulatory effects in the skin by interacting with mast cells and keratinocytes. PMID:26600671

  11. Skin explant cultures as a source of keratinocytes for cultivation.

    PubMed

    Dragúňová, J; Kabát, P; Koller, J

    2013-06-01

    Cultivated human keratinocytes can be used successfully in the treatment of burn patients, but efforts to heal burns and other wounds can be hampered by the very small skin biopsies available for cultivation of transplantable keratinocyte sheets. A small biopsy (and correspondingly small number of enzymatically isolated keratinocytes for use in classical cultivation techniques) can lead to a low yield of multilayer sheets for clinical application or unacceptably long cultivation times. One way of addressing this is to make use of skin remnants remaining after enzymatic digestion and culture cells migrating out of these skin explants. Sufficient numbers of explant-derived keratinocytes can be obtained to facilitate additional routine cultivation of these cells. Biopsy remnants can be used to initiate explant cultures repeatedly (we were able to re-use pieces of skin 10 times and still obtain useful numbers of keratinocytes) and this "passaging" yields substantially more cells for classical cultivation than would be available from conventional methodology alone, and in a comparable timeframe. Another advantage of this method is that it does not require additional biopsies to be procured from already-compromised patients and overcomes problems associated with contamination of skin samples with resistant hospital-acquired bacterial infections common during prolonged hospitalization. PMID:22850975

  12. 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

  13. 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

  14. 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

  15. Sodium butyrate induces differentiation of gastric cancer cells to intestinal cells via the PTEN/phosphoinositide 3-kinase pathway.

    PubMed

    Bai, Zhigang; Zhang, Zhongtao; Ye, Yingjiang; Wang, Shan

    2010-12-01

    NaB (sodium butyrate) inhibits cell proliferation and induces differentiation in a variety of tumour cells. In this study, we aimed to determine whether NaB induced differentiation and regulated the expression of the mucosal factor MUC2 through the PTEN/PI3K (phosphoinositide 3-kinase) pathway. BGC823 cells treated with NaB for 24-72 h showed marked inhibition of cell proliferation and alteration in cellular morphology. NaB treatment markedly increased the expression of PTEN and MUC2, but it decreased the expression of PI3K. These effects were enhanced by intervention with PI3K inhibitors and were reduced by intervention with PTEN siRNA. Hence, we conclude that NaB increased PTEN expression, promoted the expression of MUC2 and induced the differentiation of gastric cancer cells through the PTEN/PI3K signalling pathway.

  16. Turkey erythrocytes possess a membrane-associated inositol 1,4,5-trisphosphate 3-kinase that is activated by Ca2+ in the presence of calmodulin.

    PubMed Central

    Morris, A J; Downes, C P; Harden, T K; Michell, R H

    1987-01-01

    Turkey erythrocytes contain soluble and particulate kinase activities which catalyse the ATP-dependent phosphorylation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. The particle-bound activity accounts for approximately one-quarter of the total cellular Ins(1,4,5)P3 kinase, when assayed at a [Ca2+] of 10 nM. The particle-bound Ins(1,4,5)P3 kinase is not washed from the membrane by 0.6 M-KCl, yet may be solubilized by a variety of detergents. This suggests that it is an intrinsic membrane protein. The product of the membrane-bound Ins(1,4,5)P3 kinase is inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], identifying the enzyme as an Ins(1,4,5)P3 3-kinase. In the presence of calmodulin, the membrane-associated Ins(1,4,5)P3 3-kinase is activated as [Ca2+] is increased over the range 0.2-1.0 microM. Under these conditions, the rates of dephosphorylation of Ins(1,3,4,5)P4 and Ins(1,4,5)P3 by phosphatases in the membrane fraction are unchanged. PMID:2829830

  17. Inhibition of phosphatidylinositide 3-kinase in OK-cells reduces Na/Pi-cotransport but does not interfere with its regulation by parathyroid hormone.

    PubMed

    Pfister, M F; Brunskill, N J; Forgo, J; Stange, G; Biber, J; Murer, H

    1999-08-01

    The importance of phosphatidylinositide 3- kinase(s) [PI 3-kinase(s)] in membrane trafficking processes led us to examine its/their possible role in parathyroid-hormone- (PTH-) induced endocytosis and lysosomal degradation of the type IIa Na/Pi-cotransporter in opossum kidney cells (OK-cells). We used wortmannin, a potent inhibitor of several mammalian PI 3-kinase isoforms, and measured Na/Pi-cotransporter activity and type IIa Na/Pi-cotransporter protein expression; also the induction of a negative dominant subunit (Deltap85) was used to reduce PI 3-kinase activity. Wortmannin and Deltap85 led to a reduction of Na/Pi-cotransport activity but were unable to prevent its inhibition by PTH. Wortmannin led in a dose- and time-dependent manner to a reduction of Na/Pi-cotransport activity and transporter protein expression, and retarded their recovery from PTH-induced inhibition/degradation. The data suggest that a PI 3-kinase "controlled" mechanism is involved in the synthesis (and/or routing) of the apical type IIa Na/Pi-cotransporter in OK-cells. PMID:10398872

  18. Schizosaccharomyces pombe Sst4p, a Conserved Vps27/Hrs Homolog, Functions Downstream of Phosphatidylinositol 3-Kinase Pik3p To Mediate Proper Spore Formation▿ †

    PubMed Central

    Onishi, Masayuki; Iida, Michihiro; Koga, Takako; Yamada, Sadayuki; Hirata, Aiko; Iwaki, Tomoko; Takegawa, Kaoru; Fukui, Yasuhisa; Tachikawa, Hiroyuki

    2007-01-01

    Sporulation of the fission yeast Schizosaccharomyces pombe is a developmental process that generates gametes and that includes the formation of spore envelope precursors called the forespore membranes. Assembly and development of forespore membranes require vesicular trafficking from other intracellular membrane compartments. We have shown that phosphatidylinositol 3-kinase (PtdIns 3-kinase) is required for efficient and proper development of forespore membranes. The role of a FYVE domain protein, Sst4p, a homolog of Vps27p/Hrs, as a downstream factor for PtdIns 3-kinase in sporulation was investigated. sst4Δ asci formed spores with oval-shaped morphology and with reduced viability compared to that of the wild-type spores. The extension of forespore membranes was inefficient, and bubble-like structures emerged from the leading edges of the forespore membranes. Sst4p localization was examined using fluorescent protein fusions and was found to be adjacent to the forespore membranes during sporulation. The localization and function of Sst4p were dependent on its FYVE domain and on PtdIns 3-kinase. Sst4p colocalized and interacted with Hse1p, a homolog of Saccharomyces cerevisiae Hse1p and of mammalian STAM. Mutations in all three UIM domains of the Sst4p/Hse1p complex resulted in formation of spores with abnormal morphology. These results suggest that Sst4p is a downstream factor of PtdIns 3-kinase and functions in forespore membrane formation. PMID:17951524

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

    PubMed Central

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

    2016-01-01

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

  20. Phophatidylinositol-3 kinase/mammalian target of rapamycin/p70S6K regulates contractile protein accumulation in airway myocyte differentiation.

    PubMed

    Halayko, Andrew J; Kartha, Sreedharan; Stelmack, Gerald L; McConville, John; Tam, John; Camoretti-Mercado, Blanca; Forsythe, Sean M; Hershenson, Marc B; Solway, Julian

    2004-09-01

    Increased airway smooth muscle in airway remodeling results from myocyte proliferation and hypertrophy. Skeletal and vascular smooth muscle hypertrophy is induced by phosphatidylinositide-3 kinase (PI(3) kinase) via mammalian target of rapamycin (mTOR) and p70S6 kinase (p70S6K). We tested the hypothesis that this pathway regulates contractile protein accumulation in cultured canine airway myocytes acquiring an elongated contractile phenotype in serum-free culture. In vitro assays revealed a sustained activation of PI(3) kinase and p70S6K during serum deprivation up to 12 d, with concomitant accumulation of SM22 and smooth muscle myosin heavy chain (smMHC) proteins. Immunocytochemistry revealed that activation of PI3K/mTOR/p70S6K occurred almost exclusively in myocytes that acquire the contractile phenotype. Inhibition of PI(3) kinase or mTOR with LY294002 or rapamycin blocked p70S6K activation, prevented formation of large elongated contractile phenotype myocytes, and blocked accumulation of SM22 and smMHC. Inhibition of MEK had no effect. Steady-state mRNA abundance for SM22 and smMHC was unaffected by blocking p70S6K activation. These studies provide primary evidence that PI(3) kinase and mTOR activate p70S6K in airway myocytes leading to the accumulation of contractile apparatus proteins, differentiation, and growth of large, elongated contractile phenotype airway smooth muscle cells. PMID:15105162

  1. Monosodium urate activates Src/Pyk2/PI3 kinase and cathepsin dependent unconventional protein secretion from human primary macrophages.

    PubMed

    Välimäki, Elina; Miettinen, Juho J; Lietzén, Niina; Matikainen, Sampsa; Nyman, Tuula A

    2013-03-01

    Monosodium urate (MSU) is an endogenous danger signal that is crystallized from uric acid released from injured cells. MSU is known to activate inflammatory response in macrophages but the molecular mechanisms involved have remained uncharacterized. Activated macrophages start to secrete proteins to activate immune response and to recruit other immune cells to the site of infection and/or tissue damage. Secretome characterization after activation of innate immune system is essential to unravel the details of early phases of defense responses. Here, we have analyzed the secretome of human primary macrophages stimulated with MSU using quantitative two-dimensional gel electrophoresis based proteomics as well as high-throughput qualitative GeLC-MS/MS approach combining protein separation by SDS-PAGE and protein identification by liquid chromatography-MS/MS. Both methods showed that MSU stimulation induced robust protein secretion from lipopolysaccharide-primed human macrophages. Bioinformatic analysis of the secretome data showed that MSU stimulation strongly activates unconventional, vesicle mediated protein secretion. The unconventionally secreted proteins included pro-inflammatory cytokines like IL-1β and IL-18, interferon-induced proteins, and danger signal proteins. Also active forms of lysosomal proteases cathepsins were secreted on MSU stimulation, and cathepsin activity was essential for MSU-induced unconventional protein secretion. Additionally, proteins associated to phosphorylation events including Src family tyrosine kinases were increased in the secretome of MSU-stimulated cells. Our functional studies demonstrated that Src, Pyk2, and PI3 kinases act upstream of cathepsins to activate the overall protein secretion from macrophages. In conclusion, we provide the first comprehensive characterization of protein secretion pathways activated by MSU in human macrophages, and reveal a novel role for cathepsins and Src, Pyk2, PI3 kinases in the activation of

  2. 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.

  3. Endothelin-1 production by normal human cultured keratinocytes and its regulation.

    PubMed

    Inoue, H; Wakisaka, N; Tane, N; Ando, K; Isono, E; Yamanaka, M; Aihara, M; Ishida, H

    1994-01-01

    The possibility that cultured keratinocytes produce endothelins were investigated. The results showed that cultured keratinocytes derived from normal human skin produce endothelin-1. Moreover, keratinocyte endothelin-1 production was completely inhibited by the presence of actinomycin D in the medium. As in the case of endothelial cells, recombinant interleukin-1beta was capable of promoting endothelin-1 production in keratinocytes, whereas herapin inhibited it. Thrombin also inhibited endothelin-1 production. These results indicate that the mechanism of endothelin-1 production in keratinocytes is slightly different from the mechanism in vascular endothelial cells.

  4. High commitment of embryonic keratinocytes to terminal differentiation through a Notch1-caspase 3 regulatory mechanism.

    PubMed

    Okuyama, Ryuhei; Nguyen, Bach-Cuc; Talora, Claudio; Ogawa, Eisaku; Tommasi di Vignano, Alice; Lioumi, Maria; Chiorino, Giovanna; Tagami, Hachiro; Woo, Minna; Dotto, G Paolo

    2004-04-01

    Embryonic cells are expected to possess high growth/differentiation potential, required for organ morphogenesis and expansion during development. However, little is known about the intrinsic properties of embryonic epithelial cells due to difficulties in their isolation and cultivation. We report here that pure keratinocyte populations from E15.5 mouse embryos commit irreversibly to differentiation much earlier than newborn cells. Notch signaling, which promotes keratinocyte differentiation, is upregulated in embryonic keratinocyte and epidermis, and elevated caspase 3 expression, which we identify as a transcriptional Notch1 target, accounts in part for the high commitment of embryonic keratinocytes to terminal differentiation. In vivo, lack of caspase 3 results in increased proliferation and decreased differentiation of interfollicular embryonic keratinocytes, together with decreased activation of PKC-delta, a caspase 3 substrate which functions as a positive regulator of keratinocyte differentiation. Thus, a Notch1-caspase 3 regulatory mechanism underlies the intrinsically high commitment of embryonic keratinocytes to terminal differentiation.

  5. 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.

  6. Hydrogen peroxide activates focal adhesion kinase and c-Src by a phosphatidylinositol 3 kinase-dependent mechanism and promotes cell migration in Caco-2 cell monolayers.

    PubMed

    Basuroy, Shyamali; Dunagan, Mitzi; Sheth, Parimal; Seth, Ankur; Rao, R K

    2010-07-01

    Recent studies showed that c-Src and phosphatidylinositol 3 (PI3) kinase mediate the oxidative stress-induced disruption of tight junctions in Caco-2 cell monolayers. The present study evaluated the roles of PI3 kinase and Src kinase in the oxidative stress-induced activation of focal adhesion kinase (FAK) and acceleration of cell migration. Oxidative stress, induced by xanthine and xanthine oxidase system, rapidly increased phosphorylation of FAK on Y397, Y925, and Y577 in the detergent-insoluble and soluble fractions and increased its tyrosine kinase activity. The PI3 kinase inhibitors, wortmannin and LY294002, and the Src kinase inhibitor, 4-amino-5[chlorophyll]-7-[t-butyl]pyrazolo[3-4-d]pyrimidine, attenuated tyrosine phosphorylation of FAK. Oxidative stress induced phosphorylation of c-Src on Y418 by a PI3 kinase-dependent mechanism, whereas oxidative stress-induced activation of PI3 kinase was independent of Src kinase activity. Hydrogen peroxide accelerated Caco-2 cell migration in a concentration-dependent manner. Promotion of cell migration by hydrogen peroxide was attenuated by LY294002 and PP2. Reduced expression of FAK by siRNA attenuated hydrogen peroxide-induced acceleration of cell migration. The expression of constitutively active c-Src(Y527F) enhanced cell migration, whereas the expression of dominant negative c-Src(K296R/Y528F) attenuated hydrogen peroxide-induced stimulation of cell migration. Oxidative stress-induced activation of c-Src and FAK was associated with a rapid increase in the tyrosine phosphorylation and the levels of paxillin and p130(CAS) in actin-rich, detergent-insoluble fractions. This study shows that oxidative stress activates FAK and accelerates cell migration in an intestinal epithelium by a PI3 kinase- and Src kinase-dependent mechanism. PMID:20378826

  7. Effect of silver nanoparticles on human primary keratinocytes.

    PubMed

    Szmyd, Radoslaw; Goralczyk, Anna Grazyna; Skalniak, Lukasz; Cierniak, Agnieszka; Lipert, Barbara; Filon, Francesca Larese; Crosera, Matteo; Borowczyk, Julia; Laczna, Eliza; Drukala, Justyna; Klein, Andrzej; Jura, Jolanta

    2013-01-01

    Silver nanoparticles (AgNPs) have many biological applications in biomedicine, biotechnology and other life sciences. Depending on the size, shape and the type of carrier, AgNPs demonstrate different physical and chemical properties. AgNPs have strong antimicrobial, antiviral and antifungal activity, thus they are used extensively in a range of medical settings, particularly in wound dressings but also in cosmetics. This study was undertaken to examine the potential toxic effects of 15 nm polyvinylpyrrolidone-coated AgNPs on primary normal human epidermal keratinocytes (NHEK). Cells were treated with different concentrations of AgNPs and then cell viability, metabolic activity and other biological and biochemical aspects of keratinocytes functioning were studied. We observed that AgNPs decrease keratinocyte viability, metabolism and also proliferatory and migratory potential of these cells. Moreover, longer exposure resulted in activation of caspase 3/7 and DNA damage. Our studies show for the first time, that AgNPs may present possible danger for primary keratinocytes, concerning activation of genotoxic and cytotoxic processes depending on the concentration.

  8. 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

  9. 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. PMID:26763450

  10. Absence of premature senescence in Werner's syndrome keratinocytes.

    PubMed

    Ibrahim, Badr; Sheerin, Angela N; Jennert-Burston, Katrin; Bird, Joe L E; Massala, M V; Illsley, Matthew; James, S Elizabeth; Faragher, Richard G A

    2016-10-01

    Werner's syndrome (WS) is an autosomal recessive genetic disorder caused by loss of function mutation in wrn and is a useful model of premature in vivo ageing. Cellular senescence is a plausible causal mechanism of mammalian ageing and, at the cellular level, WS fibroblasts show premature senescence resulting from a combination of telomeric attrition and replication fork stalling. Over 90% of WS fibroblast cultures achieve <20 population doublings (PD) in vitro compared to wild type human fibroblast cultures. It has been proposed that some cell types, capable of proliferation, will fail to show a premature senescence phenotype in response to wrn mutations. To test this hypothesis, human dermal keratinocytes (derived from both WS and wild type patients) were cultured long term. WS Keratinocytes showed a replicative lifespan in excess of 100 population doublings but maintained functional growth arrest mechanisms based on p16 and p53. The karyotype of the cells was superficially normal and the cultures retained markers characteristic of keratinocyte holoclones (stem cells) including p63 expression and telomerase activity. Accordingly we conclude that, in contrast to WS fibroblasts, WS keratinocytes do not demonstrate slow growth rates or features of premature senescence. These findings suggest that the epidermis is among the tissue types that do not display symptoms of premature ageing caused by loss of function of wrn. This is in support that Werner's syndrome is a segmental progeroid syndrome. PMID:27492502

  11. Dental metal-induced innate reactivity in keratinocytes.

    PubMed

    Rachmawati, Dessy; Buskermolen, Jeroen K; Scheper, Rik J; Gibbs, Susan; von Blomberg, B Mary E; van Hoogstraten, Ingrid M W

    2015-12-25

    Gold, nickel, copper and mercury, i.e. four metals frequently used in dental applications, were explored for their capacity to induce innate immune activation in keratinocytes (KC). Due to their anatomical location the latter epithelial cells are key in primary local irritative responses of skin and mucosa. Fresh foreskin-derived keratinocytes and skin and gingiva KC cell lines were studied for IL-8 release as a most sensitive parameter for NF-kB activation. First, we verified that viral-defense mediating TLR3 is a key innate immune receptor in both skin- and mucosa derived keratinocytes. Second, we found that, in line with our earlier finding that ionized gold can mimic viral dsRNA in triggering TLR3, gold is very effective in KC activation. It would appear that epithelial TLR3 can play a key role in both skin- and mucosa localized irritation reactivities to gold. Subsequently we found that not only gold, but also nickel, copper and mercury salts can activate innate immune reactivity in keratinocytes, although the pathways involved remain unclear. Although current alloys have been optimized for minimal leakage of metal ions, secondary factors such as mechanical friction and acidity may still facilitate such leakage. Subsequently, these metal ions may create local irritation, itching and swelling by triggering innate immune reactions, potentially also facilitating the development of metal specific adaptive immunity.

  12. 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.

  13. Calcium-induced assembly of adherens junctions in keratinocytes

    PubMed Central

    1987-01-01

    Extracellular calcium concentration has been shown to control the stratification of cultured keratinocytes, presumably by regulation of formation of desmosomes. Previous studies have shown that keratinocytes cultured in medium containing 0.1 mM Ca++ form loose colonies without desmosomes. If the Ca++ is raised to 1 mM, desmosomes are assembled and the distribution of keratin filaments is altered. We have examined the disposition of vinculin and actin in keratinocytes under similar conditions. Using immunofluorescence microscopy we show that raising [Ca++] in the medium dramatically alters the distribution of vinculin and actin and results in the formation of adherens-type junctions within 15 min after switching to high calcium medium. Borders of cells at the edge of colonies, which are not proximal to other cells, are not affected, while cells in the interior of the colony form junctions around their periphery. Attachment plaques in keratinocytes grown in low calcium medium are located at the ventral plane of the cell, but junctions formed after switching to high calcium are not, as demonstrated by interference reflection microscopy. In cells colabeled with antibodies against vinculin and desmoplakin, vinculin-containing adherens junctions were visible before desmosomal junctions when cells were switched to high calcium. Although newly formed vinculin- containing structures in high calcium cells, like desmosomes, colocalize with phase-dense structures, superimposition of video fluorescence images using digitized fluorescence microscopy indicates that adherens junctions and desmosomes are discrete structures. Adherens junctions, like desmosomes, may play an essential role in controlling stratification of keratinocytes. PMID:2442175

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

    SciTech Connect

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

    2014-09-10

    -3 kinase inhibitors reverse the lymphoproliferative phenotype in vivo. - Highlights: • First genetic evidence that PTEN controls LPS/TLR4 signaling in B lymphocytes. • Evidence that PTEN regulates LPS induced lymphoproliferation in vivo. • PI-3 kinase inhibitors block LPS induced lymphoproliferation in vivo.

  15. Calcium-induced changes in cytoskeleton and motility of cultured human keratinocytes.

    PubMed

    Magee, A I; Lytton, N A; Watt, F M

    1987-09-01

    In normal epidermis keratinocytes migrate upward from the basal layer as they undergo terminal differentiation, yet they also have the capacity for lateral movement during wound healing. The purpose of our experiments was to investigate these two types of movement by manipulating the calcium ion concentration of the medium so that keratinocytes formed monolayers (0.1 mM calcium) or stratified sheets (2.0 mM calcium). Time-lapse video recording indicated that keratinocytes in low-calcium medium were laterally more motile than keratinocytes in normal medium. This was consistent with the ultrastructural appearance of the cells and the lack of desmosomal junctions, determined by scanning and transmission electron microscopy. During calcium-induced stratification keratinocytes moved upward from the basal layer by gliding over their neighbors and forming contacts with other suprabasal cells. Keratinocytes in low-calcium medium migrated into wounds made in the cultures, a process which was inhibited by monensin; however, stratified keratinocytes in normal medium did not enter wounds. Cytochalasin D caused rapid cell rounding and disruption of actin filaments in keratinocytes grown in low-calcium but not in normal medium, indicating more rapid treadmilling of actin and consistent with the greater motility of keratinocytes in low-calcium medium. Our results suggest that desmosome formation may place constraints on the movement of individual keratinocytes and that the actomyosin cytoskeleton is involved in lateral migration.

  16. The tumor suppressor p53 fine-tunes reactive oxygen species levels and neurogenesis via PI3 kinase signaling.

    PubMed

    Forsberg, Kirsi; Wuttke, Anja; Quadrato, Giorgia; Chumakov, Peter M; Wizenmann, Andrea; Di Giovanni, Simone

    2013-09-01

    Mounting evidence points to a role for endogenous reactive oxygen species (ROS) in cell signaling, including in the control of cell proliferation, differentiation, and fate. However, the function of ROS and their molecular regulation in embryonic mouse neural progenitor cells (eNPCs) has not yet been clarified. Here, we describe that physiological ROS are required for appropriate timing of neurogenesis in the developing telencephalon in vivo and in cultured NPCs, and that the tumor suppressor p53 plays a key role in the regulation of ROS-dependent neurogenesis. p53 loss of function leads to elevated ROS and early neurogenesis, while restoration of p53 and antioxidant treatment partially reverse the phenotype associated with premature neurogenesis. Furthermore, we describe that the expression of a number of neurogenic and oxidative stress genes relies on p53 and that both p53 and ROS-dependent induction of neurogenesis depend on PI3 kinase/phospho-Akt signaling. Our results suggest that p53 fine-tunes endogenous ROS levels to ensure the appropriate timing of neurogenesis in eNPCs. This may also have implications for the generation of tumors of neurodevelopmental origin.

  17. Sequential Activities of Phosphoinositide 3-Kinase, PKB/Akt, and Rab7 during Macropinosome Formation in Dictyostelium

    PubMed Central

    Rupper, Adam; Lee, Kyung; Knecht, David; Cardelli, James

    2001-01-01

    Macropinocytosis plays an important role in the internalization of antigens by dendritic cells and is the route of entry for many bacterial pathogens; however, little is known about the molecular mechanisms that regulate the formation or maturation of macropinosomes. Like dendritic cells, Dictyostelium amoebae are active in macropinocytosis, and various proteins have been identified that contribute to this process. As described here, microscopic analysis of null mutants have revealed that the class I phosphoinositide 3-kinases, PIK1 and PIK2, and the downstream effector protein kinase B (PKB/Akt) are important in regulating completion of macropinocytosis. Although actin-rich membrane protrusions form in these cell lines, they recede without forming macropinosomes. Imaging of cells expressing green fluorescent protein (GFP) fused to the pleckstrin homology domain (PH) of PKB (GFP-PHPKB) indicates that D3 phosphoinositides are enriched in the forming macropinocytic cup and remain associated with newly formed macropinosomes for <1 minute. A fusion protein, consisting of GFP fused to an F-actin binding domain, overlaps with GFP-PHPKB in the timing of association with forming macropinosomes. Although macropinocytosis is reduced in cells expressing dominant negative Rab7, microscopic imaging studies reveal that GFP-Rab7 associates only with formed macropinosomes at approximately the time that F-actin and D3 phosphoinositide levels decrease. These results support a model in which F-actin modulating proteins and vesicle trafficking proteins coordinately regulate the formation and maturation of macropinosomes. PMID:11553719

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

    PubMed

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

    2016-01-01

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

  19. Regulation of the Tumor-Suppressor Function of the Class III Phosphatidylinositol 3-Kinase Complex by Ubiquitin and SUMO

    PubMed Central

    Reidick, Christina; El Magraoui, Fouzi; Meyer, Helmut E.; Stenmark, Harald; Platta, Harald W.

    2014-01-01

    The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept. PMID:25545884

  20. Genetic enhancement of behavioral itch responses in mice lacking phosphoinositide 3-kinase-γ (PI3Kγ)

    PubMed Central

    2011-01-01

    Phosphoinositide 3-kinases (PI3Ks) are important for synaptic plasticity and various brain functions. The only class IB isoform of PI3K, PI3Kγ, has received the most attention due to its unique roles in synaptic plasticity and cognition. However, the potential role of PI3Kγ in sensory transmission, such as pain and itch has not been examined. In this study, we present the evidence for the first time, that genetic deletion of PI3Kγ enhanced scratching behaviours in histamine-dependent and protease-activated receptor 2 (PAR-2)-dependent itch. In contrast, PI3Kγ-deficient mice did not exhibit enhanced scratching in chloroquine-induced itch, suggesting that PI3Kγ selectively contributes to certain types of behavioal itch response. Furthermore, PI3Kγ-deficient mice exhibited normal acute nociceptive responses to thermal and mechanical noxious stimuli. Behavioral licking responses to intraplantar injections of formalin and mechanical allodynia in a chronic inflammatory pain model (CFA) were also not affected by PI3Kγ gene deletion. Our findings indicate that PI3Kγ selectively contributes to behavioral itching induced by histamine and PAR-2 agonist, but not chloroquine agonist. PMID:22168443

  1. Dual PI-3 kinase/mTOR inhibition impairs autophagy flux and induces cell death independent of apoptosis and necroptosis.

    PubMed

    Button, Robert W; Vincent, Joseph H; Strang, Conor J; Luo, Shouqing

    2016-02-01

    The PI-3 kinase (PI-3K)/mTOR pathway is critical for cell growth and proliferation. Strategies of antagonising this signaling have proven to be detrimental to cell survival. This observation, coupled with the fact many tumours show enhanced growth signaling, has caused dual inhibitors of PI-3K and mTOR to be implicated in cancer treatment, and have thus been studied across various tumour models. Since PI-3K (class-I)/mTOR pathway negatively regulates autophagy, dual inhibitors of PI-3K/mTOR are currently believed to be autophagy activators. However, our present data show that the dual PI-3K/mTOR inhibition (DKI) potently suppresses autophagic flux. We further confirm that inhibition of Vps34/PI3KC3, the class-III PI-3K, causes the blockade to autophagosome-lysosome fusion. Our data suggest that DKI induces cell death independently of apoptosis and necroptosis, whereas autophagy perturbation by DKI may contribute to cell death. Given that autophagy is critical in cellular homeostasis, our study not only clarifies the role of a dual PI-3K/mTOR inhibitor in autophagy, but also suggests that its autophagy inhibition needs to be considered if such an agent is used in cancer chemotherapy.

  2. NRBF2 Regulates Autophagy and Prevents Liver Injury by Modulating Atg14L-Linked Phosphatidylinositol-3 Kinase III Activity

    PubMed Central

    Lu, Jiahong; He, Liqiang; Behrends, Christian; Araki, Masatake; Araki, Kimi; Wang, Qing Jun; Catanzaro, Joseph M.; Friedman, Scott L.; Zong, Wei-Xing; Fiel, M. Isabel; Li, Min; Yue, Zhenyu

    2015-01-01

    The Beclin 1-Vps34 complex, the core component of the class III phosphatidylinositol-3 kinase (PI3K-III), binds Atg14L or UVRAG to control different steps of autophagy. However, the mechanism underlying the control of PI3K-III activity remains elusive. Here we report the identification of NRBF2 as a component in the specific PI3K-III complex and a modulator of PI3K-III activity. Through its MIT domain, NRBF2 binds Atg14L directly and enhances Atg14L-linked Vps34 kinase activity and autophagy induction. NRBF2 deficient cells exhibit enhanced vulnerability to ER stress that is reversed by re-introducing exogenous NRBF2. NRBF2 deficient mice develop focal liver necrosis and ductular reaction, accompanied by impaired Atg14L-linked Vps34 activity and autophagy, though the mice show no increased mortality. Our data reveals a key role for NRBF2 in the assembly of the specific Atg14L-Beclin 1-Vps34-Vps15 complex for autophagy induction. Thus, NRBF2 modulates autophagy via regulation of PI3K-III and prevents ER stress-mediated cytotoxicity and liver injury. PMID:24849286

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

    PubMed Central

    Yu, Ming

    2012-01-01

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

  4. Discovery of a small molecule agonist of phosphatidylinositol 3-kinase p110α that reactivates latent HIV-1.

    PubMed

    Doyon, Geneviève; Sobolewski, Michele D; Huber, Kelly; McMahon, Deborah; Mellors, John W; Sluis-Cremer, Nicolas

    2014-01-01

    Combination antiretroviral therapy (cART) can effectively suppress HIV-1 replication, but the latent viral reservoir in resting memory CD4(+) T cells is impervious to cART and represents a major barrier to curing HIV-1 infection. Reactivation of latent HIV-1 represents a possible strategy for elimination of this reservoir. In this study we describe the discovery of 1,2,9,10-tetramethoxy-7H-dibenzo[de,g]quinolin-7-one (57704) which reactivates latent HIV-1 in several cell-line models of latency (J89GFP, U1 and ACH-2). 57704 also increased HIV-1 expression in 3 of 4 CD8(+)-depleted blood mononuclear cell preparations isolated from HIV-1-infected individuals on suppressive cART. In contrast, vorinostat increased HIV-1 expression in only 1 of the 4 donors tested. Importantly, 57704 does not induce global T cell activation. Mechanistic studies revealed that 57704 reactivates latent HIV-1 via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. 57704 was found to be an agonist of PI3K with specificity to the p110α isoform, but not the p110β, δ or γ isoforms. Taken together, our work suggests that 57704 could serve as a scaffold for the development of more potent activators of latent HIV-1. Furthermore, it highlights the involvement of the PI3K/Akt pathway in the maintenance of HIV-1 latency.

  5. 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

  6. Novel anti-microbial peptide SR-0379 accelerates wound healing via the PI3 kinase/Akt/mTOR pathway.

    PubMed

    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

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

    PubMed

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

    2012-01-01

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

  8. 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

  9. Requirement for Class II Phosphoinositide 3-Kinase C2α in Maintenance of Glomerular Structure and Function▿

    PubMed Central

    Harris, David P.; Vogel, Peter; Wims, Marie; Moberg, Karen; Humphries, Juliane; Jhaver, Kanchan G.; DaCosta, Christopher M.; Shadoan, Melanie K.; Xu, Nianhua; Hansen, Gwenn M.; Balakrishnan, Sanjeevi; Domin, Jan; Powell, David R.; Oravecz, Tamas

    2011-01-01

    An early lesion in many kidney diseases is damage to podocytes, which are critical components of the glomerular filtration barrier. A number of proteins are essential for podocyte filtration function, but the signaling events contributing to development of nephrotic syndrome are not well defined. Here we show that class II phosphoinositide 3-kinase C2α (PI3KC2α) is expressed in podocytes and plays a critical role in maintaining normal renal homeostasis. PI3KC2α-deficient mice developed chronic renal failure and exhibited a range of kidney lesions, including glomerular crescent formation and renal tubule defects in early disease, which progressed to diffuse mesangial sclerosis, with reduced podocytes, widespread effacement of foot processes, and modest proteinuria. These findings were associated with altered expression of nephrin, synaptopodin, WT-1, and desmin, indicating that PI3KC2α deficiency specifically impacts podocyte morphology and function. Deposition of glomerular IgA was observed in knockout mice; importantly, however, the development of severe glomerulonephropathy preceded IgA production, indicating that nephropathy was not directly IgA mediated. PI3KC2α deficiency did not affect immune responses, and bone marrow transplantation studies also indicated that the glomerulonephropathy was not the direct consequence of an immune-mediated disease. Thus, PI3KC2α is critical for maintenance of normal glomerular structure and function by supporting normal podocyte function. PMID:20974805

  10. Requirement for class II phosphoinositide 3-kinase C2alpha in maintenance of glomerular structure and function.

    PubMed

    Harris, David P; Vogel, Peter; Wims, Marie; Moberg, Karen; Humphries, Juliane; Jhaver, Kanchan G; DaCosta, Christopher M; Shadoan, Melanie K; Xu, Nianhua; Hansen, Gwenn M; Balakrishnan, Sanjeevi; Domin, Jan; Powell, David R; Oravecz, Tamas

    2011-01-01

    An early lesion in many kidney diseases is damage to podocytes, which are critical components of the glomerular filtration barrier. A number of proteins are essential for podocyte filtration function, but the signaling events contributing to development of nephrotic syndrome are not well defined. Here we show that class II phosphoinositide 3-kinase C2α (PI3KC2α) is expressed in podocytes and plays a critical role in maintaining normal renal homeostasis. PI3KC2α-deficient mice developed chronic renal failure and exhibited a range of kidney lesions, including glomerular crescent formation and renal tubule defects in early disease, which progressed to diffuse mesangial sclerosis, with reduced podocytes, widespread effacement of foot processes, and modest proteinuria. These findings were associated with altered expression of nephrin, synaptopodin, WT-1, and desmin, indicating that PI3KC2α deficiency specifically impacts podocyte morphology and function. Deposition of glomerular IgA was observed in knockout mice; importantly, however, the development of severe glomerulonephropathy preceded IgA production, indicating that nephropathy was not directly IgA mediated. PI3KC2α deficiency did not affect immune responses, and bone marrow transplantation studies also indicated that the glomerulonephropathy was not the direct consequence of an immune-mediated disease. Thus, PI3KC2α is critical for maintenance of normal glomerular structure and function by supporting normal podocyte function.

  11. Insulin-like growth factor-1 delays Fas-mediated apoptosis in human neutrophils through the phosphatidylinositol-3 kinase pathway.

    PubMed

    Himpe, Eddy; Degaillier, Céline; Coppens, Astrid; Kooijman, Ron

    2008-10-01

    Apoptosis of human neutrophils is a crucial mechanism for the resolution of inflammation. We previously showed that insulin-like growth factor-1 (IGF1) delays spontaneous neutrophil apoptosis without influencing the secretion of cytokines by these cells. In the present study, we further addressed the role of IGF1 in regulating neutrophil survival in the presence of other factors present during inflammation, and the mechanism involved in delaying apoptosis. We show that IGF1 delays neutrophil apoptosis triggered by the agonistic anti-Fas antibody CH11 and that the effect of IGF1 is comparable in magnitude to that of the acknowledged anti-apoptotic cytokines interferon-gamma (IFNG) and granulocyte-macrophage colony-stimulating factor (GM-CSF; now known as CSF2). Furthermore, IGF1 exerted additional effects on cell survival in the presence of these cytokines. IGF1 did not affect Fas expression or activation by anti-Fas of caspase-8, but inhibited the depolarization of the mitochondrial membrane. Inhibitor studies indicate that the phosphatidylinositol-3 kinase (PI3K) pathway, but not the MEK-ERK pathway, mediates the effects of IGF1. However, in contrast to CSF2, IGF1 did not induce phosphorylation and translocation to the membrane of AKT, the canonical downstream target of PI3K. We therefore speculate that other downstream targets of PI3K are involved in the delay of neutrophil apoptosis by IGF1, possibly through stabilization of the mitochondrial membrane.

  12. TLR signalling affects sperm mitochondrial function and motility via phosphatidylinositol 3-kinase and glycogen synthase kinase-3α.

    PubMed

    Zhu, Xingxing; Shi, Dongyan; Li, Xiaoqian; Gong, Weijuan; Wu, Fengjiao; Guo, Xuejiang; Xiao, Hui; Liu, Lixin; Zhou, Hong

    2016-03-01

    Infection in male and female genital tracts can lead to infertility. The underlying mechanisms of this process remain unclear. Toll-like receptors (TLRs) recognize conserved structures and respond to pathogens by initiating signals that activate inflammatory gene transcription. Here, we demonstrate that TLR activation in sperm reduces sperm motility via signalling through myeloid differentiation factor 88 (MyD88), phosphatidylinositol 3-kinase (PI3K), and glycogen synthase kinase (GSK)-3α. Upon TLR activation, phosphorylated forms of PI3K and GSK3α were detected in the mitochondria, and the mitochondrial membrane potential was impaired in sperm. In addition, mitochondrial ATP levels were decreased after TLR agonist stimulation. Furthermore, blocking PI3K or GSK3α activation abrogated these effects and reversed the TLR-induced reduction in sperm motility. These results identify a previously unrecognized TLR signalling pathway that leads to dysfunctional sperm mitochondria, which reduce sperm motility. Our study reveals a novel mechanism by which pathogenic infection affects sperm motility and possibly leads to infertility.

  13. RIPK1 and RIPK3 Kinases Promote Cell-Death-Independent Inflammation by Toll-like Receptor 4.

    PubMed

    Najjar, Malek; Saleh, Danish; Zelic, Matija; Nogusa, Shoko; Shah, Saumil; Tai, Albert; Finger, Joshua N; Polykratis, Apostolos; Gough, Peter J; Bertin, John; Whalen, Michael J; Pasparakis, Manolis; Balachandran, Siddharth; Kelliher, Michelle; Poltorak, Alexander; Degterev, Alexei

    2016-07-19

    Macrophages are a crucial component of the innate immune system in sensing pathogens and promoting local and systemic inflammation. RIPK1 and RIPK3 are homologous kinases, previously linked to activation of necroptotic death. In this study, we have described roles for these kinases as master regulators of pro-inflammatory gene expression induced by lipopolysaccharide, independent of their well-documented cell death functions. In primary macrophages, this regulation was elicited in the absence of caspase-8 activity, required the adaptor molecule TRIF, and proceeded in a cell autonomous manner. RIPK1 and RIPK3 kinases promoted sustained activation of Erk, cFos, and NF-κB, which were required for inflammatory changes. Utilizing genetic and pharmacologic tools, we showed that RIPK1 and RIPK3 account for acute inflammatory responses induced by lipopolysaccharide in vivo; notably, this regulation did not require exogenous manipulation of caspases. These findings identified a new pharmacologically accessible pathway that may be relevant to inflammatory pathologies. PMID:27396959

  14. BKM-120 (Buparlisib): A Phosphatidyl-Inositol-3 Kinase Inhibitor with Anti-Invasive Properties in Glioblastoma.

    PubMed

    Speranza, Maria-Carmela; Nowicki, Michal O; Behera, Prajna; Cho, Choi-Fong; Chiocca, E Antonio; Lawler, Sean E

    2016-01-01

    Glioblastoma is an aggressive, invasive tumor of the central nervous system (CNS). There is a widely acknowledged need for anti-invasive therapeutics to limit glioblastoma invasion. BKM-120 is a CNS-penetrant pan-class I phosphatidyl-inositol-3 kinase (PI3K) inhibitor in clinical trials for solid tumors, including glioblastoma. We observed that BKM-120 has potent anti-invasive effects in glioblastoma cell lines and patient-derived glioma cells in vitro. These anti-migratory effects were clearly distinguishable from cytostatic and cytotoxic effects at higher drug concentrations and longer durations of drug exposure. The effects were reversible and accompanied by changes in cell morphology and pronounced reduction in both cell/cell and cell/substrate adhesion. In vivo studies showed that a short period of treatment with BKM-120 slowed tumor spread in an intracranial xenografts. GDC-0941, a similar potent and selective PI3K inhibitor, only caused a moderate reduction in glioblastoma cell migration. The effects of BKM-120 and GDC-0941 were indistinguishable by in vitro kinase selectivity screening and phospho-protein arrays. BKM-120 reduced the numbers of focal adhesions and the velocity of microtubule treadmilling compared with GDC-0941, suggesting that mechanisms in addition to PI3K inhibition contribute to the anti-invasive effects of BKM-120. Our data suggest the CNS-penetrant PI3K inhibitor BKM-120 may have anti-invasive properties in glioblastoma. PMID:26846842

  15. Ephexin4 and EphA2 mediate resistance to anoikis through RhoG and phosphatidylinositol 3-kinase.

    PubMed

    Harada, Kohei; Hiramoto-Yamaki, Nao; Negishi, Manabu; Katoh, Hironori

    2011-07-15

    Disruption of cell-extracellular matrix interaction causes epithelial cells to undergo apoptosis called anoikis, and resistance to anoikis has been suggested to be a critical step for cancer cells to metastasize. EphA2 is frequently overexpressed in a variety of human cancers, and recent studies have found that overexpression of EphA2 contributes to malignant cellular behavior, including resistance to anoikis, in several different types of cancer cells. Here we show that Ephexin4, a guanine nucleotide exchange factor for the small GTPase RhoG that interacts with EphA2, plays an important role in the regulation of anoikis. Knockdown of Ephexin4 promoted anoikis in HeLa cells, and experiments using a knockdown-rescue approach showed that activation of RhoG, phosphatidylinositol 3-kinase (PI3K), and Akt was required for the Ephexin4-mediated suppression of anoikis. Indeed, Ephexin4 knockdown caused a decrease in RhoG activity and Akt phosphorylation in HeLa cells cultured in suspension. In addition, Ephexin4 was involved in the EphA2-mediated suppression of anoikis. Taken together, these results suggest that Ephexin4 mediates resistance to anoikis through activation of RhoG and PI3K downstream of EphA2. PMID:21621533

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

    PubMed Central

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

    2015-01-01

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

  17. Dual PI-3 kinase/mTOR inhibition impairs autophagy flux and induces cell death independent of apoptosis and necroptosis

    PubMed Central

    Button, Robert W.; Vincent, Joseph H.; Strang, Conor J.; Luo, Shouqing

    2016-01-01

    The PI-3 kinase (PI-3K)/mTOR pathway is critical for cell growth and proliferation. Strategies of antagonising this signaling have proven to be detrimental to cell survival. This observation, coupled with the fact many tumours show enhanced growth signaling, has caused dual inhibitors of PI-3K and mTOR to be implicated in cancer treatment, and have thus been studied across various tumour models. Since PI-3K (class-I)/mTOR pathway negatively regulates autophagy, dual inhibitors of PI-3K/mTOR are currently believed to be autophagy activators. However, our present data show that the dual PI-3K/mTOR inhibition (DKI) potently suppresses autophagic flux. We further confirm that inhibition of Vps34/PI3KC3, the class-III PI-3K, causes the blockade to autophagosome-lysosome fusion. Our data suggest that DKI induces cell death independently of apoptosis and necroptosis, whereas autophagy perturbation by DKI may contribute to cell death. Given that autophagy is critical in cellular homeostasis, our study not only clarifies the role of a dual PI-3K/mTOR inhibitor in autophagy, but also suggests that its autophagy inhibition needs to be considered if such an agent is used in cancer chemotherapy. PMID:26814436

  18. Phosphatidylinositol 3-kinase regulation of gastrin-releasing peptide-induced cell cycle progression in neuroblastoma cells.

    PubMed

    Ishola, Titilope A; Kang, JungHee; Qiao, Jingbo; Evers, B Mark; Chung, Dai H

    2007-06-01

    Gastrin-releasing peptide (GRP), the mammalian equivalent of bombesin (BBS), is an autocrine growth factor for neuroblastoma; its receptor is up-regulated in undifferentiated neuroblastomas. Phosphatidylinositol 3-kinase (PI3K) is a critical cell survival pathway; it is negatively regulated by the PTEN tumor suppressor gene. We have recently found that poorly differentiated neuroblastomas express decreased PTEN protein levels. Moreover, overexpression of the GRP receptor, a member of the G-protein coupled receptor family, down-regulates PTEN expression, resulting in increased neuroblastoma cell growth. Therefore, we sought to determine whether GRP or BBS activates PI3K in neuroblastoma cells (BE(2)-C, LAN-1, SK-N-SH). GRP or BBS treatment rapidly increased phosphorylation of Akt and GSK-3beta in neuroblastoma cells. Inhibition of GRP receptor, with antagonist GRP-H2756 or siRNA, attenuated BBS-induced phosphorylation of Akt. LY294002, a PI3K inhibitor, also abrogated BBS-stimulated phospho-Akt as well as its cell cycle targets. GRP increased G1/S phase progression in SK-N-SH cells. BBS-mediated BrdU incorporation was blocked by LY294002. Our findings identify PI3K as an important signaling pathway for GRP-mediated neuroblastoma cell growth. A novel therapy targeted at GRP/GRP receptor may prove to be an effective treatment option to inhibit PI3K in neuroblastomas.

  19. Choline phosphate potentiates sphingosine-1-phosphate-induced Raf-1 kinase activation dependent of Ras--phosphatidylinositol-3-kinase pathway.

    PubMed

    Lee, Michael; Han, Sang Seop

    2002-04-01

    In NIH3T3 cells, sphingosine-1-phosphate (S1P) caused a significant increase of Raf-1 kinase activity as early as 2 min. Interestingly, choline phosphate (ChoP) produced synergistic increase of S1P-stimulated Raf-1 kinase activation in the presence of ATP while showing additive effect in the absence of ATP. However, Raf-1 kinase activation induced by S1P decreased in the presence of ATP when applied alone. The overexpression of N-terminal fragment of Raf-1 (RfI) to inhibit Raf--Ras interaction caused the inhibition of S1P-induced Raf-1 kinase activation. Also, wortmannin, phosphatidylinositol-3-kinase (PI3K) inhibitor, exhibited inhibitory effects on S1P-induced activation of Raf-1 kinase. In addition, we demonstrated that the chemical antioxidant, N-acetylcysteine attenuated Raf-1 activation induced by S1P, suggesting that H(2)O(2) may be required for the signalling pathway leading to Raf-1 activation. This H(2)O(2)-induced Raf-1 kinase activation was also blocked by inhibition of Ras--PI3K signalling pathway using alpha-hydroxyfarnesylphosphonic acid and wortmannin. Taken together, these results indicate that S1P-induced Raf-1 kinase activation is mediated by H(2)O(2) stimulation of Ras--PI3K pathway, and is enhanced by ChoP in the presence of ATP.

  20. Dehydroglyasperin D Inhibits the Proliferation of HT-29 Human Colorectal Cancer Cells Through Direct Interaction With Phosphatidylinositol 3-kinase

    PubMed Central

    Jung, Sung Keun; Jeong, Chul-Ho

    2016-01-01

    Background: Despite recent advances in therapy, colorectal cancer still has a grim prognosis. Although licorice has been used in East Asian traditional medicine, the molecular properties of its constituents including dehydroglyasperin D (DHGA-D) remain unknown. We sought to evaluate the inhibitory effect of DHGA-D on colorectal cancer cell proliferation and identify the primary signaling molecule targeted by DHGA-D. Methods: We evaluated anchorage-dependent and -independent cell growth in HT-29 human colorectal adenocarcinoma cells. The target protein of DHGA-D was identified by Western blot analysis with a specific antibody, and direct interaction between DHGA-D and the target protein was confirmed by kinase and pull-down assays. Cell cycle analysis by flow cytometry and further Western blot analysis was performed to identify the signaling pathway involved. Results: DHGA-D significantly suppressed anchorage-dependent and -independent HT-29 colorectal cancer cell proliferation. DHGA-D directly suppressed phosphatidylinositol 3-kinase (PI3K) activity and subsequent Akt phosphorylation and bound to the p110 subunit of PI3K. DHGA-D also significantly induced G1 cell cycle arrest, together with the suppression of glycogen synthase kinase 3β and retinoblastoma phosphorylation and cyclin D1 expression. Conclusions: DHGA-D has potent anticancer activity and targets PI3K in human colorectal adenocarcinoma HT-29 cells. To our knowledge, this is the first report to detail the molecular basis of DHGA-D in suppressing colorectal cancer cell growth. PMID:27051646

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

    PubMed

    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.

  2. p87 and p101 subunits are distinct regulators determining class IB phosphoinositide 3-kinase (PI3K) specificity.

    PubMed

    Shymanets, Aliaksei; Prajwal; Bucher, Kirsten; Beer-Hammer, Sandra; Harteneck, Christian; Nürnberg, Bernd

    2013-10-25

    Class IB phosphoinositide 3-kinase γ (PI3Kγ) comprises a single catalytic p110γ subunit, which binds to two non-catalytic subunits, p87 or p101, and controls a plethora of fundamental cellular responses. The non-catalytic subunits are assumed to be redundant adaptors for Gβγ enabling G-protein-coupled receptor-mediated regulation of PI3Kγ. Growing experimental data provide contradictory evidence. To elucidate the roles of the non-catalytic subunits in determining the specificity of PI3Kγ, we tested the impact of p87 and p101 in heterodimeric p87-p110γ and p101-p110γ complexes on the modulation of PI3Kγ activity in vitro and in living cells. RT-PCR, biochemical, and imaging data provide four lines of evidence: (i) specific expression patterns of p87 and p101, (ii) up-regulation of p101, providing the basis to consider p87 as a protein forming a constitutively and p101 as a protein forming an inducibly expressed PI3Kγ, (iii) differences in basal and stimulated enzymatic activities, and (iv) differences in complex stability, all indicating apparent diversity within class IB PI3Kγ. In conclusion, expression and activities of PI3Kγ are modified differently by p87 and p101 in vitro and in living cells, arguing for specific regulatory roles of the non-catalytic subunits in the differentiation of PI3Kγ signaling pathways. PMID:24014027

  3. The Phosphatidylinositol 3-Kinase/mTor Pathway as a Therapeutic Target for Brain Aging and Neurodegeneration

    PubMed Central

    Heras-Sandoval, David; Avila-Muñoz, Evangelina; Arias, Clorinda

    2011-01-01

    Many pathological conditions are associated with phosphatidylinositol 3-kinase (PI3K) dysfunction, providing an incentive for the study of the effects of PI3K modulation in different aspects of diabetes, cancer, and aging. The PI3K/AKT/mTOR pathway is a key transducer of brain metabolic and mitogenic signals involved in neuronal proliferation, differentiation, and survival. In several models of neurodegenerative diseases associated with aging, the PI3K/AKT pathway has been found to be dysregulated, suggesting that two or more initiating events may trigger disease formation in an age-related manner. The search for chemical compounds able to modulate the activity of the PI3K/AKT/mTOR pathway is emerging as a potential therapeutic strategy for the treatment and/or prevention of some metabolic defects associated with brain aging. In the current review, we summarize some of the critical actions of PI3K in brain function as well as the evidence of its involvement in aging and Alzheimer's disease.

  4. 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

  5. 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

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

    PubMed

    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

  7. BKM-120 (Buparlisib): A Phosphatidyl-Inositol-3 Kinase Inhibitor with Anti-Invasive Properties in Glioblastoma

    PubMed Central

    Speranza, Maria-Carmela; Nowicki, Michal O.; Behera, Prajna; Cho, Choi-Fong; Chiocca, E. Antonio; Lawler, Sean E.

    2016-01-01

    Glioblastoma is an aggressive, invasive tumor of the central nervous system (CNS). There is a widely acknowledged need for anti-invasive therapeutics to limit glioblastoma invasion. BKM-120 is a CNS-penetrant pan-class I phosphatidyl-inositol-3 kinase (PI3K) inhibitor in clinical trials for solid tumors, including glioblastoma. We observed that BKM-120 has potent anti-invasive effects in glioblastoma cell lines and patient-derived glioma cells in vitro. These anti-migratory effects were clearly distinguishable from cytostatic and cytotoxic effects at higher drug concentrations and longer durations of drug exposure. The effects were reversible and accompanied by changes in cell morphology and pronounced reduction in both cell/cell and cell/substrate adhesion. In vivo studies showed that a short period of treatment with BKM-120 slowed tumor spread in an intracranial xenografts. GDC-0941, a similar potent and selective PI3K inhibitor, only caused a moderate reduction in glioblastoma cell migration. The effects of BKM-120 and GDC-0941 were indistinguishable by in vitro kinase selectivity screening and phospho-protein arrays. BKM-120 reduced the numbers of focal adhesions and the velocity of microtubule treadmilling compared with GDC-0941, suggesting that mechanisms in addition to PI3K inhibition contribute to the anti-invasive effects of BKM-120. Our data suggest the CNS-penetrant PI3K inhibitor BKM-120 may have anti-invasive properties in glioblastoma. PMID:26846842

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

    PubMed Central

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

    2016-01-01

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

  9. Cbl participates in shikonin-induced apoptosis by negatively regulating phosphoinositide 3-kinase/protein kinase B signaling.

    PubMed

    Qu, Dan; Xu, Xiao-Man; Zhang, Meng; Jiang, Ting-Shu; Zhang, Yi; Li, Sheng-Qi

    2015-07-01

    Shikonin, a naturally occurring naphthoquinone, exhibits anti-tumorigenic activity. However, its precise mechanisms of action have remained elusive. In the present study, the involvement in the action of shikonin of the ubiquitin ligases Cbl-b and c-Cbl, which are negative regulators of phosphoinositide 3-kinase (PI3K) activation, was investigated. Shikonin was observed to reduce cell viability and induce apoptosis and G2/M phase arrest in lung cancer cells. In addition, shikonin increased the protein levels of B-cell lymphoma 2 (Bcl-2)-associated X and p53 and reduced those of Bcl-2. Additionally, shikonin inhibited PI3k/Akt activity and upregulated Cbl protein expression. In addition, a specific inhibitor of PI3K, LY294002, was observed to have a synergistic effect on the proliferation inhibition and apoptotic induction of A549 cells with shikonin. In conclusion, the results of the present study suggested that Cbl proteins promote shikonin-induced apoptosis by negatively regulating PI3K/Akt signaling in lung cancer cells.

  10. Inositol 1,4,5-trisphosphate 3-kinase B controls survival and prevents anergy in B cells.

    PubMed

    Maréchal, Yoann; Quéant, Séverine; Polizzi, Selena; Pouillon, Valérie; Schurmans, Stéphane

    2011-01-01

    Inositol 1,4,5-trisphosphate 3-kinase B (or Itpkb) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), its reaction product, play an important role in the control of B lymphocyte fate and function in vivo. In order to investigate the fine mechanisms of Itpkb and Ins(1,3,4,5)P4 action in B cells, we crossed Itpkb(-/-) mice with transgenic mice expressing a 3-83μδ B cell receptor (BCR) specific for membrane-bound MHC-I H2-K(b) and H2-K(k) molecules. On a non-deleting H2-K(d) genetic background, we show that Itpkb is important for the control of Bim protein expression and B cell survival rather than for the control of B cell development from one stage to another. Analyses of cell surface markers expression, proapoptotic Bim protein expression, in vitro survival and in vivo turnover demonstrated that BCR transgenic Itpkb(-/-) B cells exhibit an anergic phenotype with the notable exception of their enhanced antigen-induced calcium signalling. On a deleting H2-K(b) genetic background, we show that Itpkb is not essential for BCR editing or negative selection. These data establish Itpkb as an important regulator of B cell survival and anergy in vivo.

  11. Suppression of Nkx3.2 by phosphatidylinositol-3-kinase signaling regulates cartilage development by modulating chondrocyte hypertrophy

    PubMed Central

    Kim, Jeong-Ah; Im, Suhjean; Cantley, Lewis C.; Kim, Dae-Won

    2016-01-01

    Phosphatidylinositol-3-kinase (PI3K) is a key regulator of diverse biological processes including cell proliferation, migration, survival, and differentiation. While a role of PI3K in chondrocyte differentiation has been suggested, its precise mechanisms of action are poorly understood. Here we show that PI3K signaling can down-regulate Nkx3.2 at both mRNA and protein levels in various chondrocyte cultures in vitro. In addition, we have intriguingly found that p85β, not p85α, is specifically employed as a regulatory subunit for PI3K-mediated Nkx3.2 suppression. Furthermore, we found that regulation of Nkx3.2 by PI3K requires Rac1–PAK1, but not Akt, signaling downstream of PI3K. Finally, using embryonic limb bud cultures, ex vivo long bone cultures, and p85β knockout mice, we demonstrated that PI3K-mediated suppression of Nkx3.2 in chondrocytes plays a role in the control of cartilage hypertrophy during skeletal development in vertebrates. PMID:26363466

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

    PubMed

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

    2013-01-01

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

  13. PI3 Kinase Disease

    MedlinePlus

    ... Patients Procedure for Accessing Lab Services Data Package Requirements AIDS Therapies Resource Guide In Vitro Efficacy Evaluations ... Assurances to Users Application and Approval Process User Requirements Malaria Vaccine Production Services Data Sharing and Release ...

  14. ERK 1/2 and PI-3 kinase pathways as a potential mechanism of ghrelin action on cell proliferation and apoptosis in the porcine ovarian follicular cells.

    PubMed

    Rak-Mardyla, A; Gregoraszczuk, E L

    2010-08-01

    Recently, we reported the stimulatory effect of ghrelin on ovarian cell proliferation in parallel with the inhibitory action of ghrelin on cell apoptosis. The aim of the presented data propose local activation of extracellular signal-regulated protein kinase 1 and 2 (ERK 1/2) and phosphoinositide-3 (PI-3) kinase pathways as a mechanism of ghrelin effect in the porcine ovary. To test this hypothesis, action of ghrelin on levels of ERK 1/2 with PI-3 kinase activity and protein expression using ELISA and western blot analysis, respectively, was examined. Additionally, to determine which pathways (ERK 1/2 or PI-3 kinase) are the potential signals of ghrelin-mediated cell proliferation and apoptosis in ovarian cells, we used PD098059 (50 microM) and wortmannin (200 microM), well-known inhibitors of these kinases. Treatment of ovarian coculture cells with ghrelin (100, 250, 500 and 1000 pg/ml) showed stimulation of phospho-ERK 1/2 levels and PI-3 kinase activity, with the maximum effect observed after 15 min of cell incubation. Additionally, western blot analysis indicated that ghrelin increased expression of both kinases. Moreover, ghrelin used in combination with PD098059 or wortmannin significantly decreased cell proliferation, which was measured by the Alamar Blue assay and increased apoptosis, which was measured by caspase - 3 activity and DNA fragmentation. In conclusion, these results suggest that the ERK 1/2 and PI-3 kinase pathways may be potential signals of ghrelin mediate the cell proliferation and apoptosis of ovary cells.

  15. 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.

  16. Norepinephrine and endothelin activate diacylglycerol kinases in caveolae/rafts of rat mesenteric arteries: agonist-specific role of PI3-kinase.

    PubMed

    Clarke, Christopher J; Ohanian, Vasken; Ohanian, Jacqueline

    2007-05-01

    The phosphatidylinositol (PI) signaling pathway mediates norepinephrine (NE)- and endothelin-1 (ET-1)-stimulated vascular smooth muscle contraction through an inositol-trisphosphate-induced rise in intracellular calcium and diacylglycerol (DG) activation of protein kinase C (PKC). Subsequent activation of DG kinases (DGKs) metabolizes DG to phosphatidic acid (PA), potentially regulating PKC activity. Because precise regulation and spatial restriction of the PI pathway is necessary for specificity, we have investigated whether this occurs within caveolae/rafts, specialized plasma membrane microdomains implicated in vascular smooth muscle contraction. We show that components of the PI signaling cascade-phosphatidylinositol 4,5-bisphosphate (PIP(2)), PA, and DGK-theta are present in caveolae/rafts prepared from rat mesenteric small arteries. Stimulation with NE or ET-1 induced [(33)P]PIP(2) hydrolysis solely within caveolae/rafts. NE stimulated an increase in DGK activity in caveolae/rafts alone, whereas ET-1 activated DGK in caveolae/rafts and noncaveolae/rafts; however, [(33)P]PA increased in all fractions with both agonists. Previously, we reported that NE activated DGK-theta in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner; here, we describe PI3-kinase-dependent DGK activation and [(33)P]PA production in caveolae/rafts in response to NE but not ET-1. Additionally, PKB, a potential activator of DGK-theta, translocated to caveolae/rafts in response to NE but not ET-1, and PI3-kinase inhibition prevented this. Furthermore, PI3-kinase inhibition reduced the sensitivity of contraction to NE but not ET-1. Our study shows that caveolae/rafts are major sites of vasoconstrictor hormone activation of the PI pathway in intact small arteries and suggest a link between lipid signaling events within caveolae/rafts and contraction. PMID:17208990

  17. The EphA8 Receptor Regulates Integrin Activity through p110γ Phosphatidylinositol-3 Kinase in a Tyrosine Kinase Activity-Independent Manner

    PubMed Central

    Gu, Changkyu; Park, Soochul

    2001-01-01

    Recent genetic studies suggest that ephrins may function in a kinase-independent Eph receptor pathway. Here we report that expression of EphA8 in either NIH 3T3 or HEK293 cells enhanced cell adhesion to fibronectin via α5β1- or β3 integrins. Interestingly, a kinase-inactive EphA8 mutant also markedly promoted cell attachment to fibronectin in these cell lines. Using a panel of EphA8 point mutants, we have demonstrated that EphA8 kinase activity does not correlate with its ability to promote cell attachment to fibronectin. Analysis using EphA8 extracellular and intracellular domain mutants has revealed that enhanced cell adhesion is dependent on ephrin A binding to the extracellular domain and the juxtamembrane segment of the cytoplasmic domain of the receptor. EphA8-promoted adhesion was efficiently inhibited by wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. Additionally, we found that EphA8 had associated PI 3-kinase activity and that the p110γ isoform of PI 3-kinase is associated with EphA8. In vitro binding experiments revealed that the EphA8 juxtamembrane segment was sufficient for the formation of a stable complex with p110γ. Similar results were obtained in assay using cells stripped of endogenous ephrin A ligands by treatment with preclustered ephrin A5-Fc proteins. In addition, a membrane-targeted lipid kinase-inactive p110γ mutant was demonstrated to stably associate with EphA8 and suppress EphA8-promoted cell adhesion to fibronectin. Taken together, these results suggest the presence of a novel mechanism by which the EphA8 receptor localizes p110γ PI 3-kinase to the plasma membrane in a tyrosine kinase-independent fashion, thereby allowing access to lipid substrates to enable the signals required for integrin-mediated cell adhesion. PMID:11416136

  18. Rho-kinase-dependent F-actin rearrangement is involved in the inhibition of PI3-kinase/Akt during ischemia–reperfusion-induced endothelial cell apoptosis

    PubMed Central

    Versteilen, Amanda M. G.; Sipkema, Pieter; van Nieuw Amerongen, Geerten P.; Musters, Rene J. P.; Groeneveld, A. B. Johan

    2007-01-01

    Activation of cytoskeleton regulator Rho-kinase during ischemia–reperfusion (I/R) plays a major role in I/R injury and apoptosis. Since Rho-kinase is a negative regulator of the pro-survival phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, we hypothesized that inhibition of Rho-kinase can prevent I/R-induced endothelial cell apoptosis by maintaining PI3-kinase/Akt activity and that protective effects of Rho-kinase inhibition are facilitated by prevention of F-actin rearrangement. Human umbilical vein endothelial cells were subjected to 1 h of simulated ischemia and 1 or 24 h of simulated reperfusion after treatment with Rho-kinase inhibitor Y-27632, PI3-kinase inhibitor wortmannin, F-actin depolymerizers cytochalasinD and latrunculinA and F-actin stabilizer jasplakinolide. Intracellular ATP levels decreased following I/R. Y-27632 treatment reduced I/R-induced apoptosis by 31% (P < 0.01) and maintained Akt activity. Both effects were blocked by co-treatment with wortmannin. Y-27632 treatment prevented the formation of F-actin bundles during I/R. Similar results were observed with cytochalasinD treatment. In contrast, latrunculinA and jasplakinolide treatment did not prevent the formation of F-actin bundles during I/R and had no effect on I/R-induced apoptosis. Apoptosis and Akt activity were inversely correlated (R2 = 0.68, P < 0.05). In conclusion, prevention of F-actin rearrangement by Rho-kinase inhibition or by cytochalasinD treatment attenuated I/R-induced endothelial cell apoptosis by maintaining PI3-kinase and Akt activity. PMID:18165899

  19. Zinc oxide nanoparticle induced genotoxicity in primary human epidermal keratinocytes.

    PubMed

    Sharma, Vyom; Singh, Suman K; Anderson, Diana; Tobin, Desmond J; Dhawan, Alok

    2011-05-01

    Zinc oxide (ZnO) nanoparticles are widely used in cosmetics and sunscreens. Human epidermal keratinocytes may serve as the first portal of entry for these nanoparticles either directly through topically applied cosmetics or indirectly through any breaches in the skin integrity. Therefore, the objective of the present study was to assess the biological interactions of ZnO nanoparticles in primary human epidermal keratinocytes (HEK) as they are the most abundant cell type in the human epidermis. Cellular uptake of nanoparticles was investigated by scanning electron microscopy using back scattered electrons imaging as well as transmission electron microscopy. The electron microscopy revealed the internalization of ZnO nanoparticles in primary HEK after 6 h exposure at 14 microg/ml concentration. ZnO nanoparticles exhibited a time (6-24 h) as well as concentration (8-20 microg/ml) dependent inhibition of mitochondrial activity as evident by the MTT assay. A significant (p < 0.05) induction in DNA damage was observed in cells exposed to ZnO nanoparticles for 6 h at 8 and 14 microg/ml concentrations compared to control as evident in the Comet assay. This is the first study providing information on biological interactions of ZnO nanoparticles with primary human epidermal keratinocytes. Our findings demonstrate that ZnO nanoparticles are internalized by the human epidermal keratinocytes and elicit a cytotoxic and genotoxic response. Therefore, caution should be taken while using consumer products containing nanoparticles as any perturbation in the skin barrier could expose the underlying cells to nanoparticles.

  20. 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.

  1. Salivary trefoil factor 3 enhances migration of oral keratinocytes.

    PubMed

    Storesund, Trond; Hayashi, Katsuhiko; Kolltveit, Kristin M; Bryne, Magne; Schenck, Karl

    2008-04-01

    Trefoil factor 3 (TFF3) is a member of the mammalian TFF family. Trefoil factors are secreted onto mucosal surfaces of the entire body and exert different effects according to tissue location. Trefoil factors may enhance mucosal healing by modulating motogenic activity, inhibiting apoptosis, and promoting angiogenesis. Trefoil factor 3 is secreted from the submandibular gland and is present in whole saliva. The aim of this study was to assess the migratory and proliferative effects of TFF3 on primary oral human keratinocytes and oral cancer cell lines. The addition of TFF3 increased the migration of both normal oral keratinocytes and the cancer cell line D12, as evaluated by a two-dimensional scratch assay. By contrast, no increase in proliferation or energy metabolism was observed after stimulation with TFF3. Trefoil factor 3-enhanced migration was found to be driven partly by the extracellular signal-related kinase (Erk1/2) pathway, as shown by addition of the mitogen-activated protein kinase (MAPK) inhibitor PD 98059. Previous functional studies on trefoil peptides have all been based on cells from monolayered epithelium like the intestinal mucosa; this is the first report to show that normal and cancerous keratinocytes from stratified epithelium respond to TFF stimuli. Taken together, salivary TFF3 is likely to contribute to oral wound healing. PMID:18353006

  2. A high yield method for growing primary canine keratinocytes.

    PubMed

    Watson, Adrian; Baker, Claire; Bailey, Julie; Fray, Tim; Markwell, Peter

    2004-07-01

    From a small amount of starting material, a large quantity of canine keratinocytes can be generated for experimental purposes using a refined method of explant culture to initiate the growth of basal cells with a high proliferative potential. The dividing capacity of cultures was promoted by a system selecting clonogenic cells onto an i3T3 feeder layer in combination with carefully monitoring cell morphology and passaging to select out excessive numbers of differentiated keratinocytes. Levels of contaminating dermal fibroblasts, which if left unchecked will overgrow keratinocytes, were kept to a minimum by a combination of careful explant micro-dissection to remove dermis, eliminating explants with signs of fibroblast growth as well as using cholera toxin, EGF and i3T3 feeder layers. The advantage of the method described is that it does not rely on the provision of large quantities of starting material thereby reducing the need for repeated tissue sampling, and passage numbers of five or six can be routinely achieved. This technique can therefore be useful to experimenters who require a regular and reliable source of cells for their studies. PMID:15158212

  3. Salivary trefoil factor 3 enhances migration of oral keratinocytes.

    PubMed

    Storesund, Trond; Hayashi, Katsuhiko; Kolltveit, Kristin M; Bryne, Magne; Schenck, Karl

    2008-04-01

    Trefoil factor 3 (TFF3) is a member of the mammalian TFF family. Trefoil factors are secreted onto mucosal surfaces of the entire body and exert different effects according to tissue location. Trefoil factors may enhance mucosal healing by modulating motogenic activity, inhibiting apoptosis, and promoting angiogenesis. Trefoil factor 3 is secreted from the submandibular gland and is present in whole saliva. The aim of this study was to assess the migratory and proliferative effects of TFF3 on primary oral human keratinocytes and oral cancer cell lines. The addition of TFF3 increased the migration of both normal oral keratinocytes and the cancer cell line D12, as evaluated by a two-dimensional scratch assay. By contrast, no increase in proliferation or energy metabolism was observed after stimulation with TFF3. Trefoil factor 3-enhanced migration was found to be driven partly by the extracellular signal-related kinase (Erk1/2) pathway, as shown by addition of the mitogen-activated protein kinase (MAPK) inhibitor PD 98059. Previous functional studies on trefoil peptides have all been based on cells from monolayered epithelium like the intestinal mucosa; this is the first report to show that normal and cancerous keratinocytes from stratified epithelium respond to TFF stimuli. Taken together, salivary TFF3 is likely to contribute to oral wound healing.

  4. Micronucleus formation in human keratinocytes is dependent on radiation quality and tissue architecture.

    PubMed

    Snijders, Antoine M; Mannion, Brandon J; Leung, Stanley G; Moon, Sol C; Kronenberg, Amy; Wiese, Claudia

    2015-01-01

    The cytokinesis-block micronucleus (MN) assay was used to assess the genotoxicity of low doses of different types of space radiation. Normal human primary keratinocytes and immortalized keratinocytes grown in 2D monolayers each were exposed to graded doses of 0.3 or 1.0 GeV/n silicon ions or similar energies of iron ions. The frequencies of induced MN were determined and compared to γ-ray data. RBE(max) values ranged from 1.6 to 3.9 for primary keratinocytes and from 2.4 to 6.3 for immortalized keratinocytes. At low radiation doses ≤ 0.4 Gy, 0.3 GeV/n iron ions were the most effective at inducing MN in normal keratinocytes. An "over-kill effect" was observed for 0.3 GeV/n iron ions at higher doses, wherein 1.0 GeV/n iron ions were most efficient in inducing MN. In immortalized keratinocytes, 0.3 GeV/n iron ions produced MN with greater frequency than 1.0 GeV/n iron ions, except at the highest dose tested. MN formation was higher in immortalized keratinocytes than in normal keratinocytes for all doses and radiation qualities investigated. MN induction was also assessed in human keratinocytes cultured in 3D to simulate the complex architecture of human skin. RBE values for MN formation in 3D were reduced for normal keratinocytes exposed to iron ions, but were elevated for immortalized keratinocytes. Overall, MN induction was significantly lower in keratinocytes cultured in 3D than in 2D. Together, the results suggest that tissue architecture and immortalization status modulate the genotoxic response to space radiation, perhaps via alterations in DNA repair fidelity. PMID:25041929

  5. Enhanced Keratinocyte Proliferation and Migration in Co-culture with Fibroblasts

    PubMed Central

    Wang, Zhenxiang; Wang, Ying; Farhangfar, Farhang; Zimmer, Monica; Zhang, Yongxin

    2012-01-01

    Wound healing is primarily controlled by the proliferation and migration of keratinocytes and fibroblasts as well as the complex interactions between these two cell types. To investigate the interactions between keratinocytes and fibroblasts and the effects of direct cell-to-cell contact on the proliferation and migration of keratinocytes, keratinocytes and fibroblasts were stained with different fluorescence dyes and co-cultured with or without transwells. During the early stage (first 5 days) of the culture, the keratinocytes in contact with fibroblasts proliferated significantly faster than those not in contact with fibroblasts, but in the late stage (11th to 15th day), keratinocyte growth slowed down in all cultures unless EGF was added. In addition, keratinocyte migration was enhanced in co-cultures with fibroblasts in direct contact, but not in the transwells. Furthermore, the effects of the fibroblasts on keratinocyte migration and growth at early culture stage correlated with heparin-binding EGF-like growth factor (HB-EGF), IL-1α and TGF-β1 levels in the cultures where the cells were grown in direct contact. These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition. Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation. These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage. PMID:22911722

  6. Extracellular signal-regulated kinase and phosphoinositol-3 kinase mediate IGF-1 induced proliferation of fetal sheep cardiomyocytes.

    PubMed

    Sundgren, Nathan C; Giraud, George D; Schultz, Jess M; Lasarev, Michael R; Stork, Philip J S; Thornburg, Kent L

    2003-12-01

    Growth of the fetal heart involves cardiomyocyte enlargement, division, and maturation. Insulin-like growth factor-1 (IGF-1) is implicated in many aspects of growth and is likely to be important in developmental heart growth. IGF-1 stimulates the IGF-1 receptor (IGF1R) and downstream signaling pathways, including extracellular signal-regulated kinase (ERK) and phosphoinositol-3 kinase (PI3K). We hypothesized that IGF-1 stimulates cardiomyocyte proliferation and enlargement through stimulation of the ERK cascade and stimulates cardiomyocyte differentiation through the PI3K cascade. In vivo administration of Long R3 IGF-1 (LR3 IGF-1) did not stimulate cardiomyocyte hypertrophy but led to a decreased percentage of cells that were binucleated in vivo. In culture, LR3 IGF-1 increased myocyte bromodeoxyuridine (BrdU) uptake by three- to five-fold. The blockade of either ERK or PI3K signaling (by UO-126 or LY-294002, respectively) completely abolished BrdU uptake stimulated by LR3 IGF-1. LR3 IGF-1 did not increase footprint area, but as expected, phenylephrine stimulated an increase in binucleated cardiomyocyte size. We conclude that 1) IGF-1 through IGF1R stimulates cardiomyocyte division in vivo; hyperplastic growth is the most likely explanation of IGF-1 stimulated heart growth in vivo; 2) IGF-1 through IGF1R does not stimulate binucleation in vitro or in vivo; 3) IGF-1 through IGF1R does not stimulate hypertrophy either in vivo or in vitro; and 4) IGF-1 through IGF1R requires both ERK and PI3K signaling for proliferation of near-term fetal sheep cardiomyocytes in vitro. PMID:12947030

  7. The fibrotic role of phosphatidylinositol-3-kinase/Akt pathway in injured skeletal muscle after acute contusion.

    PubMed

    Li, H-Y; Zhang, Q-G; Chen, J-W; Chen, S-Q; Chen, S-Y

    2013-09-01

    Transforming growth factor β (TGF-β) is a multifunctional cytokine with fibrogenic properties. Previous studies demonstrated that Phosphatidylinositol 3-Kinase (PI3K)/Akt/ mammalian target of Ramycin (mTOR), a non-Smad TGF-β pathway, plays an important role in the fibrotic pathogenesis of different organs such as the lung, kidney, skin and liver. However, the role of PI3k-Akt pathway in fibrosis in injured skeletal muscle is still unclear. In this study, we determined the fibrotic role of PI3K-Akt pathway in injured skeletal muscle. We established a mouse model for acute muscle contusion. Western blotting analysis showed that TGF-β, phosphorylated Akt and phosphorylated mTOR were increased in muscles after acute contusion, which indicated that the PI3K-Akt- mTOR pathway was activated in skeletal muscle after acute contusion. The pathway was inhibited by a PI3K inhibitor, LY294002. Moreover, the expression of fibrosis markers vimentin, α SMA and collagen I and the area of scar decreased in injured skeletal muscle after PI3K pathway was blocked. The muscle function improved in terms of both fast-twitch and tetanic strength after PI3K/Akt pathway was inhibited in injured skeletal muscle. In conclusion, activation of PI3K-Akt-mTOR pathway might promote collagen production and scar formation in the acute contused skeletal muscle. Blocking of PI3K-Akt-mTOR pathway could improve the function of injured skeletal muscle. PMID:23444088

  8. Expression Regulation of the Metastasis-Promoting Protein InsP3-Kinase-A in Tumor Cells.

    PubMed

    Chang, Lydia; Schwarzenbach, Heidi; Meyer-Staeckling, Sönke; Brandt, Burkard; Mayr, Georg W; Weitzel, Joachim M; Windhorst, Sabine

    2011-04-01

    Under physiologic conditions, the inositol-1,4,5-trisphosphate (InsP(3))-metabolizing, F-actin-bundling InsP(3)-kinase-A (ITPKA) is expressed only in neurons. Tumor cells that have gained the ability to express ITPKA show an increased metastatic potential due to the migration-promoting properties of ITPKA. Here we investigated the mechanism how tumor cells have gained the ability to reexpress ITPKA by using a breast cancer cell line (T47D) with no expression and a lung carcinoma cell line (H1299) with ectopic ITPKA expression. Cloning of a 1,250-bp ITPKA promoter fragment revealed that methylation of CpG islands was reduced in H1299 as compared with T47D cells, but DNA demethylation did not alter the expression of ITPKA. Instead, we showed that the repressor-element-1-silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF), which suppresses expression of neuronal genes in nonneuronal tissues, regulates expression of ITPKA. Knockdown of REST/NRSF induced expression of ITPKA in T47D cells, whereas its overexpression in H1299 cells strongly reduced the level of ITPKA. In T47D cells, REST/NRSF was bound to the RE-1 site of the ITPKA promoter and strongly reduced its activity. In H1299 cells, in contrast, expressing comparable REST/NRSF levels as T47D cells, REST/NRSF only slightly reduced ITPKA promoter activity. This reduced suppressor activity most likely results from expression of a dominant-negative isoform of REST/NRSF, REST4, which impairs binding of REST/NRSF to the RE-1 site. Thus, ITPKA may belong to the neuronal metastasis-promoting proteins whose ectopic reexpression in tumor cells is associated with impaired REST/NRSF activity. Mol Cancer Res; 9(4); 1-10. ©2011 AACR. PMID:21460179

  9. LY294002 inhibits glucocorticoid-induced COX-2 gene expression in cardiomyocytes through a phosphatidylinositol 3 kinase-independent mechanism

    SciTech Connect

    Sun Haipeng; Xu Beibei; Sheveleva, Elena; Chen, Qin M.

    2008-10-01

    Glucocorticoids induce COX-2 expression in rat cardiomyocytes. While investigating whether phosphatidylinositol 3 kinase (PI3K) plays a role in corticosterone (CT)-induced COX-2, we found that LY294002 (LY29) but not wortmannin (WM) attenuates CT from inducing COX-2 gene expression. Expression of a dominant-negative mutant of p85 subunit of PI3K failed to inhibit CT from inducing COX-2 expression. CT did not activate PI3K/AKT signaling pathway whereas LY29 and WM decreased the activity of PI3K. LY303511 (LY30), a structural analogue and a negative control for PI3K inhibitory activity of LY29, also suppressed COX-2 induction. These data suggest PI3K-independent mechanisms in regulating CT-induced COX-2 expression. LY29 and LY30 do not inhibit glucocorticoid receptor transactivity. Both compounds have been reported to inhibit Casein Kinase 2 activity and modulate potassium and calcium levels independent of PI3K, while LY29 has been reported to inhibit mammalian Target of Rapamycin (mTOR), and DNA-dependent Protein Kinase (DNA-PK). Inhibitor of Casein Kinase 2 (CK2), mTOR or DNA-PK failed to prevent CT from inducing COX-2 expression. Tetraethylammonium (TEA), a potassium channel blocker, and nimodipine, a calcium channel blocker, both attenuated CT from inducing COX-2 gene expression. CT was found to increase intracellular Ca{sup 2+} concentration, which can be inhibited by LY29, TEA or nimodipine. These data suggest a possible role of calcium instead of PI3K in CT-induced COX-2 expression in cardiomyocytes.

  10. Exploration of a potent PI3 kinase/mTOR inhibitor as a novel anti-fibrotic agent in IPF

    PubMed Central

    Mercer, Paul F; Woodcock, Hannah V; Eley, Jessica D; Platé, Manuela; Sulikowski, Michal G; Durrenberger, Pascal F; Franklin, Linda; Nanthakumar, Carmel B; Man, Yim; Genovese, Federica; McAnulty, Robin J; Yang, Shuying; Maher, Toby M; Nicholson, Andrew G; Blanchard, Andy D; Marshall, Richard P; Lukey, Pauline T; Chambers, Rachel C

    2016-01-01

    Rationale Idiopathic pulmonary fibrosis (IPF) is the most rapidly progressive and fatal of all fibrotic conditions with no curative therapies. Common pathomechanisms between IPF and cancer are increasingly recognised, including dysfunctional pan-PI3 kinase (PI3K) signalling as a driver of aberrant proliferative responses. GSK2126458 is a novel, potent, PI3K/mammalian target of rapamycin (mTOR) inhibitor which has recently completed phase I trials in the oncology setting. Our aim was to establish a scientific and dosing framework for PI3K inhibition with this agent in IPF at a clinically developable dose. Methods We explored evidence for pathway signalling in IPF lung tissue and examined the potency of GSK2126458 in fibroblast functional assays and precision-cut IPF lung tissue. We further explored the potential of IPF patient-derived bronchoalveolar lavage (BAL) cells to serve as pharmacodynamic biosensors to monitor GSK2126458 target engagement within the lung. Results We provide evidence for PI3K pathway activation in fibrotic foci, the cardinal lesions in IPF. GSK2126458 inhibited PI3K signalling and functional responses in IPF-derived lung fibroblasts, inhibiting Akt phosphorylation in IPF lung tissue and BAL derived cells with comparable potency. Integration of these data with GSK2126458 pharmacokinetic data from clinical trials in cancer enabled modelling of an optimal dosing regimen for patients with IPF. Conclusions Our data define PI3K as a promising therapeutic target in IPF and provide a scientific and dosing framework for progressing GSK2126458 to clinical testing in this disease setting. A proof-of-mechanism trial of this agent is currently underway. Trial registration number NCT01725139, pre-clinical. PMID:27103349

  11. 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

  12. Involvement of phosphatidylinositol 3-kinase, but not RalGDS, in TC21/R-Ras2-mediated transformation.

    PubMed

    Murphy, Gretchen A; Graham, Suzanne M; Morita, Staeci; Reks, Sarah E; Rogers-Graham, Kelley; Vojtek, Anne; Kelley, Grant G; Der, Channing J

    2002-03-22

    Oncogenic Ras and activated forms of the Ras-related protein TC21/R-Ras2 share similar abilities to alter cell proliferation. However, in contrast to Ras, we found previously that TC21 fails to activate the Raf-1 serine/threonine kinase. Thus, TC21 must utilize non-Raf effectors to regulate cell function. In this study, we determined that TC21 interacts strongly with some (RalGDS, RGL, RGL2/Rlf, AF6, and the phosphatidylinositol 3-kinase (PI3K) catalytic subunit p110delta), and weakly with other Ras small middle dotGTP-binding proteins. In addition, library screening identified novel TC21-interacting proteins. We also determined that TC21, similar to Ras, mediates activation of phospholipase Cepsilon. We then examined if RalGDS, a RalA guanine nucleotide exchange factor, or PI3K are effectors for TC21-mediated signaling and cell proliferation in murine fibroblasts. We found that overexpression of full-length RalGDS reduced the focus forming activity of activated TC21. Furthermore, expression of activated Ras, but not TC21, enhanced GTP loading on RalA. In fact, TC21 attenuated insulin-stimulated RalA small middle dotGTP formation. In contrast, like Ras, expression of activated TC21 resulted in membrane translocation and an increase in the PI3K-dependent phosphorylation of Akt, and inhibition of PI3K activity interfered with TC21 focus formation. Finally, unlike Ras, TC21 did not activate the Rac small GTPase, indicating that Ras may not activate Rac by PI3K. Taken together, these results suggest that PI3K, but not RalGDS, is an important mediator of cell proliferation by TC21.

  13. Augmentation of Sodium Butyrate-induced Apoptosis by Phosphatidylinositol 3-kinase Inhibition in the Human Cervical Cancer Cell-line

    PubMed Central

    Park, Jung Kyu; Cho, Chi Heum; Ramachandran, Sabarish; Shin, So Jin; Kwon, Sang Hoon; Kwon, Sun Young

    2006-01-01

    Purpose Sodium butyrate (NaBT) is principally a histone deacetylase (HDAC) inhibitor, and it has the potential to arrest HPV-positive carcinoma cells at the G1 to S phase transition of the cell cycle. The aim of study was to determine whether phosphatidylinositol 3-kinase (PI3K) inhibition can enhance the inhibitory effect of NaBT on a human cervical cancer cell line (HeLa). Materials and Methods Cervical cancer cells (HeLa) were treated with NaBT alone or in combination with the PI3K inhibitors wortmannin or LY294002. Cell viability analysis and FACS analysis were carried out. The expressions of the cell cycle related proteins were evaluated by Western-blot analysis. Results Inhibition of PI3K enhanced NaBT-mediated apoptosis and this decreased the HeLa cell viability. Either wortmannin or LY294002, combined with NaBT, enhanced the activation of caspase 3 and caspase 9, and this enhanced the subsequent cleavage of poly (ADP-ribose) polymerase (PARP). Cervical cancer cells were arrested in the subG1 and G2/M phase, as was detected by FACS analysis. NaBT treatment in combination with PI3K inhibitors showed the increased expression of the CDK inhibitors p21Cip1/Waf1 and p27Kip1, in a p53 dependent manner, and also the increased dephosphorylation of Rb whereas there was a reduction in the expression levels of cyclin A, cyclin D1 and cyclin B1. Conclusion The results demonstrate that inhibition of PI3K enhances NaBT-mediated cervical cancer cell apoptosis through the activation of the caspase pathway. Moreover, these findings will support future investigation using the PI3K inhibitors in combination with adjuvant treatment for treating carcinoma of the cervix. PMID:19771269

  14. Cell autonomous phosphoinositide 3-kinase activation in oocytes disrupts normal ovarian function through promoting survival and overgrowth of ovarian follicles.

    PubMed

    Kim, So-Youn; Ebbert, Katherine; Cordeiro, Marilia H; Romero, Megan; Zhu, Jie; Serna, Vanida Ann; Whelan, Kelly A; Woodruff, Teresa K; Kurita, Takeshi

    2015-04-01

    In this study, we explored the effects of oocytic phosphoinositide 3-kinase (PI3K) activation on folliculogensis by generating transgenic mice, in which the oocyte-specific Cre-recombinase induces the expression of constitutively active mutant PI3K during the formation of primordial follicles. The ovaries of neonatal transgenic (Cre+) mice showed significantly reduced apoptosis in follicles, which resulted in an excess number of follicles per ovary. Thus, the elevation of phosphatidylinositol (3,4,5)-trisphosphate levels within oocytes promotes the survival of follicles during neonatal development. Despite the increase in AKT phosphorylation, primordial follicles in neonatal Cre+ mice remained dormant demonstrating a nuclear accumulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). These primordial follicles containing a high level of nuclear PTEN persisted in postpubertal females, suggesting that PTEN is the dominant factor in the maintenance of female reproductive lifespan through the regulation of primordial follicle recruitment. Although the oocytic PI3K activity and PTEN levels were elevated, the activation of primordial follicles and the subsequent accumulation of antral follicles with developmentally competent oocytes progressed normally in prepubertal Cre+ mice. However, mature Cre+ female mice were anovulatory. Because postnatal day 50 Cre+ mice released cumulus-oocyte complexes with developmentally competent oocytes in response to super-ovulation treatment, the anovulatory phenotype was not due to follicular defects but rather endocrine abnormalities, which were likely caused by the excess number of overgrown follicles. Our current study has elucidated the critical role of oocytic PI3K activity in follicular function, as well as the presence of a PTEN-mediated mechanism in the prevention of immature follicle activation.

  15. MicroRNA-21 promotes phosphatase gene and protein kinase B/phosphatidylinositol 3-kinase expression in colorectal cancer

    PubMed Central

    Sheng, Wei-Zhong; Chen, Yu-Sheng; Tu, Chuan-Tao; He, Juan; Zhang, Bo; Gao, Wei-Dong

    2016-01-01

    AIM: To explore the regulatory mechanism of the target gene of microRNA-21 (miR-21), phosphatase gene (PTEN), and its downstream proteins, protein kinase B (AKT) and phosphatidylinositol 3-kinase (PI3K), in colorectal cancer (CRC) cells. METHODS: Quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the expression levels of miR-21 and PTEN in HCT116, HT29, Colo32 and SW480 CRC cell lines. Also, the expression levels of PTEN mRNA and its downstream proteins AKT and PI3K in HCT116 cells after downregulating miR-21 were investigated. RESULTS: Comparing the miR-21 expression in CRC cells, the expression levels of miR-21 were highest in HCT116 cells, and the expression levels of miR-21 were lowest in SW480 cells. In comparing miR-21 and PTEN expression in CRC cells, we found that the protein expression levels of miR-21 and PTEN were inversely correlated (P < 0.05); when miR-21 expression was reduced, mRNA expression levels of PTEN did not significantly change (P > 0.05), but the expression levels of its protein significantly increased (P < 0.05). In comparing the levels of PTEN protein and downstream AKT and PI3K in HCT116 cells after downregulation of miR-21 expression, the levels of AKT and PI3K protein expression significantly decreased (P < 0.05). CONCLUSION: PTEN is one of the direct target genes of miR-21. Thus, phosphatase gene and its downstream AKT and PI3K expression levels can be regulated by regulating the expression levels of miR-21, which in turn regulates the development of CRC. PMID:27350731

  16. Leptin induces macrophage lipid body formation by a phosphatidylinositol 3-kinase- and mammalian target of rapamycin-dependent mechanism.

    PubMed

    Maya-Monteiro, Clarissa M; Almeida, Patricia E; D'Avila, Heloisa; Martins, Aline S; Rezende, Ana Paula; Castro-Faria-Neto, Hugo; Bozza, Patricia T

    2008-01-25

    Leptin is an adipocyte-derived hormone/cytokine that links nutritional status with neuroendocrine and immune functions. Lipid bodies (lipid droplets) are emerging as dynamic organelles with roles in lipid metabolism and inflammation. Here we investigated the roles of leptin in signaling pathways involved in cytoplasmic lipid body biogenesis and leukotriene B(4) synthesis in macrophages. Our results demonstrated that leptin directly activated macrophages and induced the formation of adipose differentiation-related protein-enriched lipid bodies. Newly formed lipid bodies were sites of 5-lipoxygenase localization and correlated with an enhanced capacity of leukotriene B(4) production. We demonstrated that leptin-induced macrophage activation was dependent on phosphatidylinositol 3-kinase (PI3K) activity, since the lipid body formation was inhibited by LY294002 and was absent in the PI3K knock-out mice. Leptin induces phosphorylation of p70(S6K) and 4EBP1 key downstream signaling intermediates of the mammalian target of rapamycin (mTOR) pathway in a rapamycin-sensitive mechanism. The mTOR inhibitor, rapamycin, inhibited leptin-induced lipid body formation, both in vivo and in vitro. In addition, rapamycin inhibited leptin-induced adipose differentiation-related protein accumulation in macrophages and lipid body-dependent leukotriene synthesis, demonstrating a key role for mTOR in lipid body biogenesis and function. Our results establish PI3K/mTOR as an important signaling pathway for leptin-induced cytoplasmic lipid body biogenesis and adipose differentiation-related protein accumulation. Furthermore, we demonstrate a previously unrecognized link between intracellular (mTOR) and systemic (leptin) nutrient sensors in macrophage lipid metabolism. Leptin-induced increased formation of cytoplasmic lipid bodies and enhanced inflammatory mediator production in macrophages may have implications for obesity-related cardiovascular diseases. PMID:18039669

  17. Propofol mediates signal transducer and activator of transcription 3 activation and crosstalk with phosphoinositide 3-kinase/AKT

    PubMed Central

    Shravah, Jayant; Wang, Baohua; Pavlovic, Marijana; Kumar, Ujendra; Chen, David DY; Luo, Honglin; Ansley, David M

    2014-01-01

    We previously demonstrated that propofol, an intravenous anesthetic with anti-oxidative properties, activated the phosphoinositide 3-kinase (PI3K)/AKT pathway to increase the expression of B cell lymphoma (Bcl)-2 and, therefore the anti-apoptotic potential on cardiomyocytes. Here, we wanted to determine if propofol can also activate the Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3 pathway, another branch of cardioprotective signaling. The cellular response of nuclear factor kappa B (NFκB) and STAT3 was also evaluated. Cardiac H9c2 cells were treated by propofol alone or in combination with pretreatment by inhibitors for JAK2/STAT3 or PI3K/AKT pathway. STAT3 and AKT phosphorylation, and STAT3 translocation were measured by western blotting and immunofluorescence staining, respectively. Propofol treatment significantly increased STAT3 phosphorylation at both tyrosine 705 and serine 727 residues. Sustained early phosphorylation of STAT3 was observed with 25~75 μM propofol at 10 and 30 min. Nuclear translocation of STAT3 was seen at 4 h after treatment with 50 μM propofol. In cultured H9c2 cells, we further demonstrated that propofol-induced STAT3 phosphorylation was reduced by pretreatment with PI3K/AKT pathway inhibitors wortmannin or API-2. Conversely, pretreatment with JAK2/STAT3 pathway inhibitor AG490 or stattic inhibited propofol-induced AKT phosphorylation. In addition, propofol induced NFκB p65 subunit perinuclear translocation. Inhibition or knockdown of STAT3 was associated with increased levels of the NFκB p65 subunit. Our results suggest that propofol induces an adaptive response by dual activation and crosstalk of cytoprotective PI3K/AKT and JAK2/STAT3 pathways. Rationale to apply propofol clinically as a preemptive cardioprotectant during cardiac surgery is supported by our findings. PMID:25105067

  18. 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.

  19. 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.

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

    PubMed

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

    2014-04-01

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

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

    PubMed

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

    2009-07-15

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

  2. 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.

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

    PubMed

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

    2016-06-01

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

  4. Delphinidin suppresses ultraviolet B-induced cyclooxygenases-2 expression through inhibition of MAPKK4 and PI-3 kinase.

    PubMed

    Kwon, Jung Yeon; Lee, Ki Won; Kim, Jong-Eun; Jung, Sung Keun; Kang, Nam Joo; Hwang, Mun Kyung; Heo, Yong-Seok; Bode, Ann M; Dong, Zigang; Lee, Hyong Joo

    2009-11-01

    Cyclooxygenase-2 (COX-2), a key mediator of inflammation, and its product, prostaglandin E(2) (PGE(2)), enhance carcinogenesis, particularly in skin. Ultraviolet (UV) B is the most carcinogenic component of solar irradiation, and a crucial role of COX-2 in UVB-mediated skin carcinogenesis has been reported. Here, we investigated the effects of delphinidin, an abundant dietary anthocyanin, on UVB-induced COX-2 upregulation and the underlying molecular mechanism. We found that delphinidin suppressed UVB-induced COX-2 expression in JB6 P+ mouse epidermal cells. COX-2 promoter activity and PGE(2) production were also suppressed by delphinidin treatment within non-cytotoxic concentrations. Activator protein-1 and nuclear factor-kappaB, crucial transcription factors involved in COX-2 expression, were activated by UVB and delphinidin abolished this activation. UVB-induced phosphorylation of c-Jun N-terminal kinase, p38 kinase and Akt was inhibited by delphinidin. The activities of mitogen-activated protein kinase kinase (MAPKK) 4 and phosphatidylinositol-3 kinase (PI-3K) were inhibited markedly by delphinidin. A pull-down assay using delphinidin-Sepharose beads revealed that delphinidin binds directly with MAPKK4 or PI-3K in a manner that was competitive with adenosine triphosphate. Moreover, in vivo investigations using mouse skin revealed that the upregulation of COX-2 expression, MAPKK4 activity and PI-3K activity induced by UVB was abolished with delphinidin treatment. Collectively, our results demonstrated that delphinidin targets MAPKK4 and PI-3K directly to suppress COX-2 overexpression, suggesting a potential protective role for delphinidin against UVB-mediated skin carcinogenesis.

  5. Activation of PI3-kinase stimulates endocytosis of ROMK via Akt1/SGK1-dependent phosphorylation of WNK1.

    PubMed

    Cheng, Chih-Jen; Huang, Chou-Long

    2011-03-01

    WNK kinases stimulate endocytosis of ROMK channels to regulate renal K+ handling. Phosphatidylinositol 3-kinase (PI3K)-activating hormones, such as insulin and IGF 1, phosphorylate WNK1, but how this affects the regulation of ROMK abundance is unknown. Here, serum starvation of ROMK-transfected HEK cells led to an increase of ROMK current density; subsequent addition of insulin or IGF1 inhibited ROMK currents in a PI3K-dependent manner. Serum and insulin also increased phosphorylation of the downstream kinases Akt1 and SGK1 as well as WNK1. A biotinylation assay suggested that insulin and IGF1 inhibit ROMK by enhancing its endocytosis, a process that WNK1 may mediate. Knockdown of WNK1 with siRNA or expression of a phospho-deficient WNK1 mutant (T58A) both prevented insulin-induced inhibition of ROMK currents, suggesting that phosphorylation at Threonine-58 of WNK1 is important to mediate the inhibition of ROMK by PI3K-activating hormones or growth factors. In vitro and in vivo kinase assays supported the notion that Akt1 and SGK1 can phosphorylate WNK1 at this site, and we established that Akt1 and SGK1 synergistically inhibit ROMK through WNK1. We used dominant-negative intersectin and dynamin constructs to show that SGK1-mediated phosphorylation of WNK1 inhibits ROMK by promoting its endocytosis. Taken together, these results suggest that PI3K-activating hormones inhibit ROMK by enhancing its endocytosis via a mechanism that involves phosphorylation of WNK1 by Akt1 and SGK1. PMID:21355052

  6. Cell Autonomous Phosphoinositide 3-Kinase Activation in Oocytes Disrupts Normal Ovarian Function Through Promoting Survival and Overgrowth of Ovarian Follicles

    PubMed Central

    Ebbert, Katherine; Cordeiro, Marilia H.; Romero, Megan; Zhu, Jie; Serna, Vanida Ann; Whelan, Kelly A.; Woodruff, Teresa K.

    2015-01-01

    In this study, we explored the effects of oocytic phosphoinositide 3-kinase (PI3K) activation on folliculogensis by generating transgenic mice, in which the oocyte-specific Cre-recombinase induces the expression of constitutively active mutant PI3K during the formation of primordial follicles. The ovaries of neonatal transgenic (Cre+) mice showed significantly reduced apoptosis in follicles, which resulted in an excess number of follicles per ovary. Thus, the elevation of phosphatidylinositol (3,4,5)-trisphosphate levels within oocytes promotes the survival of follicles during neonatal development. Despite the increase in AKT phosphorylation, primordial follicles in neonatal Cre+ mice remained dormant demonstrating a nuclear accumulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). These primordial follicles containing a high level of nuclear PTEN persisted in postpubertal females, suggesting that PTEN is the dominant factor in the maintenance of female reproductive lifespan through the regulation of primordial follicle recruitment. Although the oocytic PI3K activity and PTEN levels were elevated, the activation of primordial follicles and the subsequent accumulation of antral follicles with developmentally competent oocytes progressed normally in prepubertal Cre+ mice. However, mature Cre+ female mice were anovulatory. Because postnatal day 50 Cre+ mice released cumulus-oocyte complexes with developmentally competent oocytes in response to super-ovulation treatment, the anovulatory phenotype was not due to follicular defects but rather endocrine abnormalities, which were likely caused by the excess number of overgrown follicles. Our current study has elucidated the critical role of oocytic PI3K activity in follicular function, as well as the presence of a PTEN-mediated mechanism in the prevention of immature follicle activation. PMID:25594701

  7. 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

  8. Membrane translocation of TRPC6 channels and endothelial migration are regulated by calmodulin and PI3 kinase activation

    PubMed Central

    Chaudhuri, Pinaki; Rosenbaum, Michael A.; Sinharoy, Pritam; Damron, Derek S.; Birnbaumer, Lutz; Graham, Linda M.

    2016-01-01

    Lipid oxidation products, including lysophosphatidylcholine (lysoPC), activate canonical transient receptor potential 6 (TRPC6) channels leading to inhibition of endothelial cell (EC) migration in vitro and delayed EC healing of arterial injuries in vivo. The precise mechanism through which lysoPC activates TRPC6 channels is not known, but calmodulin (CaM) contributes to the regulation of TRPC channels. Using site-directed mutagenesis, cDNAs were generated in which Tyr99 or Tyr138 of CaM was replaced with Phe, generating mutant CaM, Phe99-CaM, or Phe138-CaM, respectively. In ECs transiently transfected with pcDNA3.1-myc-His-Phe99-CaM, but not in ECs transfected with pcDNA3.1-myc-His-Phe138-CaM, the lysoPC-induced TRPC6-CaM dissociation and TRPC6 externalization was disrupted. Also, the lysoPC-induced increase in intracellular calcium concentration was inhibited in ECs transiently transfected with pcDNA3.1-myc-His-Phe99-CaM. Blocking phosphorylation of CaM at Tyr99 also reduced CaM association with the p85 subunit and subsequent activation of phosphatidylinositol 3-kinase (PI3K). This prevented the increase in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and the translocation of TRPC6 to the cell membrane and reduced the inhibition of EC migration by lysoPC. These findings suggest that lysoPC induces CaM phosphorylation at Tyr99 by a Src family kinase and that phosphorylated CaM activates PI3K to produce PIP3, which promotes TRPC6 translocation to the cell membrane. PMID:26858457

  9. Isoginkgetin inhibits tumor cell invasion by regulating phosphatidylinositol 3-kinase/Akt-dependent matrix metalloproteinase-9 expression.

    PubMed

    Yoon, Sang-Oh; Shin, Sejeong; Lee, Ho-Jae; Chun, Hyo-Kon; Chung, An-Sik

    2006-11-01

    Matrix metalloproteinase (MMP)-9 plays a key role in tumor invasion. Inhibitors of MMP-9 were screened from Metasequoia glyptostroboides (Dawn redwood) and one potent inhibitor, isoginkgetin, a biflavonoid, was identified. Noncytotoxic levels of isoginkgetin decreased MMP-9 production profoundly, but up-regulated the level of tissue inhibitor of metalloproteinase (TIMP)-1, an inhibitor of MMP-9, in HT1080 human fibrosarcoma cells. The major mechanism of Ras-dependent MMP-9 production in HT1080 cells was phosphatidylinositol 3-kinase (PI3K)/Akt/nuclear factor-kappaB (NF-kappaB) activation. Expression of dominant-active H-Ras and p85 (a subunit of PI3K) increased MMP-9 activity, whereas dominant-negative forms of these molecules decreased the level of MMP-9. H-Ras did not increase MMP-9 in the presence of a PI3K inhibitor, LY294002, and a NF-kappaB inhibitor, SN50. Further studies showed that isoginkgetin regulated MMP-9 production via PI3K/Akt/NF-kappaB pathway, as evidenced by the findings that isoginkgetin inhibited activities of both Akt and NF-kappaB. PI3K/Akt is a well-known key pathway for cell invasion, and isoginkgetin inhibited HT1080 tumor cell invasion substantially. Isoginkgetin was also quite effective in inhibiting the activities of Akt and MMP-9 in MDA-MB-231 breast carcinomas and B16F10 melanoma. Moreover, isoginkgetin treatment resulted in marked decrease in invasion of these cells. In summary, PI3K/Akt is a major pathway for MMP-9 expression and isoginkgetin markedly decreased MMP-9 expression and invasion through inhibition of this pathway. This suggests that isoginkgetin could be a potential candidate as a therapeutic agent against tumor invasion.

  10. Ras is an indispensable coregulator of the class IB phosphoinositide 3-kinase p87/p110γ

    PubMed Central

    Kurig, Barbara; Shymanets, Aliaksei; Bohnacker, Thomas; Prajwal; Brock, Carsten; Ahmadian, Mohammad Reza; Schaefer, Michael; Gohla, Antje; Harteneck, Christian; Wymann, Matthias P.; Jeanclos, Elisabeth; Nürnberg, Bernd

    2009-01-01

    Class IB phosphoinositide 3-kinase γ (PI3Kγ) elicits various immunologic and cardiovascular responses; however, the molecular basis for this signal heterogeneity is unclear. PI3Kγ consists of a catalytic p110γ and a regulatory p87PIKAP (p87, also p84) or p101 subunit. Hitherto p87 and p101 are generally assumed to exhibit redundant functions in receptor-induced and G protein βγ (Gβγ)-mediated PI3Kγ regulation. Here we investigated the molecular mechanism for receptor-dependent p87/p110γ activation. By analyzing GFP-tagged proteins expressed in HEK293 cells, PI3Kγ-complemented bone marrow–derived mast cells (BMMCs) from p110γ-/- mice, and purified recombinant proteins reconstituted to lipid vesicles, we elucidated a novel pathway of p87-dependent, G protein–coupled receptor (GPCR)-induced PI3Kγ activation. Although p101 strongly interacted with Gβγ, thereby mediating PI3Kγ membrane recruitment and stimulation, p87 exhibited only a weak interaction, resulting in modest kinase activation and lack of membrane recruitment. Surprisingly, Ras-GTP substituted the missing Gβγ-dependent membrane recruitment of p87/p110γ by direct interaction with p110γ, suggesting the indispensability of Ras for activation of p87/p110γ. Consequently, interference with Ras signaling indeed selectively blocked p87/p110γ, but not p101/p110γ, kinase activity in HEK293 and BMMC cells, revealing an important crosstalk between monomeric and trimeric G proteins for p87/p110γ activation. Our data display distinct signaling requirements of p87 and p101, conferring signaling specificity to PI3Kγ that could open up new possibilities for therapeutic intervention. PMID:19906996

  11. Receptor Interacting Protein 3 Suppresses Vascular Smooth Muscle Cell Growth by Inhibition of the Phosphoinositide 3-Kinase-Akt Axis*

    PubMed Central

    Li, Qian; Li, Geng; Lan, Xiaomei; Zheng, Ming; Chen, Kuang-Hueih; Cao, Chun-Mei; Xiao, Rui-Ping

    2010-01-01

    Proliferation of vascular smooth muscle cells (VSMCs) is a primary mechanism underlying cardiovascular proliferative disorders. Phosphoinositide 3-kinase (PI3K)-Akt (or protein kinase B) axis has been assigned at the center of pathways that regulate cell proliferation. Here we demonstrate that enhanced PI3K-Akt signaling by mitogenic stimulation or arterial injury profoundly elevates expression of receptor interacting protein 3 (RIP3) in primary cultured rat VSMCs and in vivo and that the up-regulation of RIP3 leads to VSMC growth arrest and apoptosis via inhibiting the PI3K-Akt signaling pathway, thereby alleviating balloon injury-induced neointimal formation. Specifically, mitogenic stimulation with platelet-derived growth factor-BB or angiotensin II leads to a profound increase in RIP3 expression, which is abolished by inhibition of PI3K or Akt, and increased PI3K-Akt signaling by expression of a constitutively active PI3K mutant also elevates RIP3 expression. Importantly, adenoviral overexpression of RIP3 not only triggers apoptosis but also causes cell cycle arrest at G1/G0 phases that is associated with suppressed Akt activation. In sharp contrast, RIP3 gene silencing enhances serum- and platelet-derived growth factor-induced cell proliferation and Akt activation. In vivo adenoviral gene delivery of rat RIP3 (rRIP3) increased apoptosis and reduced VSMC proliferation, thus, effectively alleviating balloon injury-induced neointimal formation. The growth-suppressive and pro-apoptotic effects are independent of rRIP3 Ser/Thr kinase activity, because overexpression of a kinase-inactive mutant of rRIP3, similar to its wild type, is sufficient to induce growth arrest and apoptosis. These findings reveal a novel growth-suppressive action of RIP3, marking RIP3 as an important factor to prevent excessive mitogenic stimulation- or injury-induced vascular smooth muscle cells hyperplasia. PMID:20042608

  12. Inhibition of the PI3 kinase cascade in corticolimbic circuit: temporal and differential effects on contextual fear and extinction.

    PubMed

    Kritman, Milly; Maroun, Mouna

    2013-05-01

    We studied the role of PI3K cascade in the basolateral amygdala (BLA) and the infralimbic region of the medial prefrontal cortex (IL-mPFC), in contextual fear learning and extinction in the rat. To that end, we micro-infused the phosphoinositide-3-kinase (PIK3) inhibitor LY294002 into either the mPFC or the BLA. Infusion of LY294002 into the BLA following fear conditioning was associated with enhanced freezing levels and impaired extinction in the subsequent sessions. Similarly, inhibition of PI3K in the BLA before the retrieval of fear memory was associated with impaired retrieval of the fear memory, which was expressed as reduced freezing levels that persisted over 2 d. In the IL-mPFC, only consolidation of fear extinction was impaired: micro-infusion of PI3K inhibitor following the retrieval of fear was associated with impaired extinction on the following days. These results indicate differences in the temporal parameters of the effects of PI3K inhibition in the IL-mPFC and in the BLA, which suggest differential involvement of these structures in long-term fear and in extinction of fear memory. Our findings provide additional evidence for the critical roles played by PI3K in intact formation of fear memory and in its extinction and add new evidence for a role of PI3K in consolidation of memory of extinction. Better understanding of the differential involvement of the PI3K cascade during acquisition and extinction of fear conditioning in the mPFC-amygdala circuit could potentially contribute to the understanding and treatment of anxiety disorders.

  13. 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.

  14. Regulation of Mammalian Autophagy by Class II and III PI 3-Kinases through PI3P Synthesis

    PubMed Central

    Devereaux, Kelly; Ogasawara, Yuta; Zhou, Xiang; Wang, Fan; Yamamoto, Akitsugu; De Camilli, Pietro; Di Paolo, Gilbert

    2013-01-01

    Synthesis of phosphatidylinositol-3-phosphate (PI3P) by Vps34, a class III phosphatidylinositol 3-kinase (PI3K), is critical for the initial steps of autophagosome (AP) biogenesis. Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. Here we generated Vps34 knockout mouse embryonic fibroblasts (MEFs) and using a higher affinity 4x-FYVE finger PI3P-binding probe found a Vps34-independent pool of PI3P accounting for ~35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in Vps34 knockout MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and β isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis. PMID:24098492

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

    PubMed

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

    2016-06-01

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

  16. 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

  17. Stimulation of Toll-like receptor 2 in human platelets induces a thromboinflammatory response through activation of phosphoinositide 3-kinase.

    PubMed

    Blair, Price; Rex, Sybille; Vitseva, Olga; Beaulieu, Lea; Tanriverdi, Kahraman; Chakrabarti, Subrata; Hayashi, Chie; Genco, Caroline A; Iafrati, Mark; Freedman, Jane E

    2009-02-13

    Cells of the innate immune system use Toll-like receptors (TLRs) to initiate the proinflammatory response to microbial infection. Recent studies have shown acute infections are associated with a transient increase in the risk of vascular thrombotic events. Although platelets play a central role in acute thrombosis and accumulating evidence demonstrates their role in inflammation and innate immunity, investigations into the expression and functionality of platelet TLRs have been limited. In the present study, we demonstrate that human platelets express TLR2, TLR1, and TLR6. Incubation of isolated platelets with Pam(3)CSK4, a synthetic TLR2/TLR1 agonist, directly induced platelet aggregation and adhesion to collagen. These functional responses were inhibited in TLR2-deficient mice and, in human platelets, by pretreatment with TLR2-blocking antibody. Stimulation of platelet TLR2 also increased P-selectin surface expression, activation of integrin alpha(IIb)beta(3), generation of reactive oxygen species, and, in human whole blood, formation of platelet-neutrophil heterotypic aggregates. TLR2 stimulation also activated the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway in platelets, and inhibition of PI3-K significantly reduced Pam(3)CSK4-induced platelet responses. In vivo challenge with live Porphyromonas gingivalis, a Gram-negative pathogenic bacterium that uses TLR2 for innate immune signaling, also induced significant formation of platelet-neutrophil aggregates in wild-type but not TLR2-deficient mice. Together, these data provide the first demonstration that human platelets express functional TLR2 capable of recognizing bacterial components and activating the platelet thrombotic and/or inflammatory pathways. This work substantiates the role of platelets in the immune and inflammatory response and suggests a mechanism by which bacteria could directly activate platelets.

  18. A Yeast GSK-3 Kinase Mck1 Promotes Cdc6 Degradation to Inhibit DNA Re-Replication

    PubMed Central

    Ikui, Amy E.; Rossio, Valentina; Schroeder, Lea; Yoshida, Satoshi

    2012-01-01

    Cdc6p is an essential component of the pre-replicative complex (pre-RC), which binds to DNA replication origins to promote initiation of DNA replication. Only once per cell cycle does DNA replication take place. After initiation, the pre-RC components are disassembled in order to prevent re-replication. It has been shown that the N-terminal region of Cdc6p is targeted for degradation after phosphorylation by Cyclin Dependent Kinase (CDK). Here we show that Mck1p, a yeast homologue of GSK-3 kinase, is also required for Cdc6 degradation through a distinct mechanism. Cdc6 is an unstable protein and is accumulated in the nucleus only during G1 and early S-phase in wild-type cells. In mck1 deletion cells, CDC6p is stabilized and accumulates in the nucleus even in late S phase and mitosis. Overexpression of Mck1p induces rapid Cdc6p degradation in a manner dependent on Threonine-368, a GSK-3 phosphorylation consensus site, and SCFCDC4. We show evidence that Mck1p-dependent degradation of Cdc6 is required for prevention of DNA re-replication. Loss of Mck1 activity results in synthetic lethality with other pre-RC mutants previously implicated in re-replication control, and these double mutant strains over-replicate DNA within a single cell cycle. These results suggest that a GSK3 family protein plays an unexpected role in preventing DNA over-replication through Cdc6 degradation in Saccharomyces cerevisiae. We propose that both CDK and Mck1 kinases are required for Cdc6 degradation to ensure a tight control of DNA replication. PMID:23236290

  19. Paracrine regulation of fibroblast aminopeptidase N/CD13 expression by keratinocyte-releasable stratifin.

    PubMed

    Lai, Amy; Ghaffari, Abdi; Li, Yunyuan; Ghahary, Aziz

    2011-12-01

    As wound healing proceeds into the tissue remodeling phase, cellular interactions become dominated by the interplay of keratinocytes with fibroblasts in the skin, which is largely mediated through paracrine signaling and greatly affects the molecular constitution of the extracellular matrix. We have recently identified aminopeptidase N (APN)/CD13 as a potential fibroblast receptor for 14-3-3 sigma (also known as stratifin), a keratinocyte-releasable protein with potent matrix metalloproteinase 1 (MMP1) stimulatory activity. The present study demonstrates that the expression of APN on dermal fibroblasts is regulated through paracrine signaling by keratinocyte-derived soluble factors. By using an in vitro keratinocyte-fibroblast co-culture system, we showed that APN expression in dermal fibroblasts is induced in the presence of keratinocytes or in response to keratinocyte-conditioned medium. Conditioned medium collected from differentiated keratinocytes further increases APN protein production, suggesting an amplified stimulatory effect by keratinocyte differentiation. Recombinant stratifin potently induces APN synthesis in a dose-dependent manner. A consistent correlation between the protein expression levels of APN and MMP1 was also observed. These results confirm paracrine regulation of APN expression in dermal fibroblasts by keratinocyte-derived stimuli, in particular stratifin, and provide evidence that APN may serve as a target in the regulation of MMP1 expression in epidermal-mesenchymal communication. PMID:21302309

  20. AMF/PGI transactivates the MMP-3 gene through the activation of Src-RhoA-phosphatidylinositol 3-kinase signaling to induce hepatoma cell migration.

    PubMed

    Shih, Wen-Ling; Liao, Ming-Huei; Yu, Feng-Ling; Lin, Ping-Yuan; Hsu, Hsue-Yin; Chiu, Shu-Jun

    2008-11-01

    We have previously shown that AMF/PGI induces hepatoma cell migration through the induction of MMP-3. This work investigates how AMF/PGI activates the MMP-3 gene. We demonstrated that AMF/PGI transactivates the MMP-3 gene promoter through AP-1. The transactivation and induction of cell migration effect of AMF/PGI directly correlates with its enzymatic activity. Various analyses showed that AMF/PGI stimulated the Src-RhoA-PI3-kinase signaling pathway, and these three signaling molecules could form a complex. Our results demonstrate a new mechanism of AMF/PGI-induced cell migration and a link between Src-RhoA-PI3-kinase, AP-1, MMP-3 and hepatoma cell migration.

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

    PubMed

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

    1999-04-01

    Hexamethylenebisacetamide (HMBA) induces in murine erythroleukemia cells (MELC) the commitment to terminal differentiation leading to globin gene expression. In the thyroid, HMBA acts as a growth factor and also as a differentiating agent. In the present paper, we studied the effect of HMBA on the very specific thyroid marker thyroglobulin (Tg) in two different thyroid cell systems, i.e., porcine cells in primary culture and ovine cells in long term culture. Using wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, we investigated whether this enzyme is involved in HMBA mode of action. We found that HMBA is a positive modulator of Tg production in porcine cells, but a negative effector in the OVNIS cell line. As all HMBA effects studied in the present paper, i.e., Tg production and total protein levels, are not inhibited by wortmannin, we suggest the non-involvement of phosphatidylinositol-3-kinase in HMBA mode of action. PMID:10650339

  2. Phosphatidylinositol 3-kinase and the actin network are not required for the stimulation of glucose transport caused by mitochondrial uncoupling: comparison with insulin action.

    PubMed Central

    Tsakiridis, T; Vranic, M; Klip, A

    1995-01-01

    In L6 myotubes insulin stimulates glucose transport through the translocation of glucose transporters GLUT1, GLUT3 and GLUT4 from intracellular stores to the plasma membrane. An intact actin network and phosphatidylinositol 3-kinase activity are required for this process. Glucose transport is also stimulated by the mitochondrial ATP-production uncoupler dinitrophenol. We show here that, in serum-depleted myotubes, dinitrophenol induced translocation of GLUT1 and GLUT4, but not GLUT3. This response was not affected by inhibiting phosphatidylinositol 3-kinase or disassembling the actin network. Insulin, but not dinitrophenol, caused tyrosine phosphorylation of several polypeptides, including the insulin-receptor substrate-1 and mitogen-activated protein kinase. Similarly, insulin, but not dinitrophenol, caused actin reorganization, which was inhibited by wortmannin. We conclude that insulin and dinitrophenol stimulate glucose transport by different mechanisms. Images Figure 2 Figure 3 Figure 4 PMID:7619042

  3. Agent Based Modelling Helps in Understanding the Rules by Which Fibroblasts Support Keratinocyte Colony Formation

    PubMed Central

    Sun, Tao; McMinn, Phil; Holcombe, Mike; Smallwood, Rod; MacNeil, Sheila

    2008-01-01

    Background Autologous keratincoytes are routinely expanded using irradiated mouse fibroblasts and bovine serum for clinical use. With growing concerns about the safety of these xenobiotic materials, it is desirable to culture keratinocytes in media without animal derived products. An improved understanding of epithelial/mesenchymal interactions could assist in this. Methodology/Principal Findings A keratincyte/fibroblast o-culture model was developed by extending an agent-based keratinocyte colony formation model to include the response of keratinocytes to both fibroblasts and serum. The model was validated by comparison of the in virtuo and in vitro multicellular behaviour of keratinocytes and fibroblasts in single and co-culture in Greens medium. To test the robustness of the model, several properties of the fibroblasts were changed to investigate their influence on the multicellular morphogenesis of keratinocyes and fibroblasts. The model was then used to generate hypotheses to explore the interactions of both proliferative and growth arrested fibroblasts with keratinocytes. The key predictions arising from the model which were confirmed by in vitro experiments were that 1) the ratio of fibroblasts to keratinocytes would critically influence keratinocyte colony expansion, 2) this ratio needed to be optimum at the beginning of the co-culture, 3) proliferative fibroblasts would be more effective than irradiated cells in expanding keratinocytes and 4) in the presence of an adequate number of fibroblasts, keratinocyte expansion would be independent of serum. Conclusions A closely associated computational and biological approach is a powerful tool for understanding complex biological systems such as the interactions between keratinocytes and fibroblasts. The key outcome of this study is the finding that the early addition of a critical ratio of proliferative fibroblasts can give rapid keratinocyte expansion without the use of irradiated mouse fibroblasts and bovine

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed

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

    2002-01-15

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

  6. Sorafenib/regorafenib and phosphatidyl inositol 3 kinase/thymoma viral proto-oncogene inhibition interact to kill tumor cells.

    PubMed

    Sajithlal, Gangadharan B; Hamed, Hossein A; Cruickshanks, Nichola; Booth, Laurence; Tavallai, Seyedmehrad; Syed, Jahangir; Grant, Steven; Poklepovic, Andrew; Dent, Paul

    2013-10-01

    The present studies were undertaken to determine whether the multikinase inhibitors sorafenib/regorafenib cooperated with clinically relevant , phosphatidyl inositol 3 kinase (PI3K)-thymoma viral proto-oncogene (AKT) inhibitors to kill tumor cells. In liver, colorectal, lung, breast, kidney, and brain cancer cells, at clinically achievable doses, sorafenib/regorafenib and the PI3K inhibitor acetic acid (1S,4E,10R,11R,13S,14R)-[4-diallylaminomethylene-6-hydroxy-1-methoxymethyl-10,13-dimethyl-3,7,17-trioxo-1,3,4,7,10,11,12,13,14,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenanthren-11-yl ester (PX-866) cooperated in a greater than additive fashion to kill tumor cells. Cells lacking phosphatase and tensin homolog were as sensitive to the drug combination as cells expressing the protein. Similar data were obtained using the AKT inhibitors perifosine and 8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4-f] [1,6]naphthyridin-3(2H)-one hydrochloride (MK2206). PX-866 treatment abolished AKT/glycogen synthase kinase 3 (GSK3) phosphorylation, and cell killing correlated with reduced activity of AKT and mammalian target of rapamycin (mTOR). Expression of activated AKT and to a lesser extent activated mTOR reduced drug combination lethality. Expression of B-cell lymphoma-extra large or dominant negative caspase 9, but not cellular FLICE (FADD-like IL-1b-converting enzyme)-inhibitory protein short, protected cells from the drug combination. Treatment of cells with PX-866 increased protein levels of p62, lysosome-associated membrane protein 2 (LAMP2), and microtubule-associated protein light chain (LC) 3 and LC3II that correlated with a large increase in LC3-green fluorescent protein (GFP) vesicle numbers. Exposure of PX-866 treated cells to sorafenib reduced p62 and LAMP2 levels, decreased the ratio of LC3 to LC3II, and reduced LC3-GFP vesicle levels. Knockdown of Beclin1 or autophagy-related 5 suppressed drug toxicity by ∼40%. In vivo, sorafenib and PX-866 or

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

    SciTech Connect

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

    2012-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  10. Different inhibition of Gβγ-stimulated class IB phosphoinositide 3-kinase (PI3K) variants by a monoclonal antibody

    PubMed Central

    Shymanets, Aliaksei; Prajwal; Vadas, Oscar; Czupalla, Cornelia; LoPiccolo, Jaclyn; Brenowitz, Michael; Ghigo, Alessandra; Hirsch, Emilio; Krause, Eberhard; Wetzker, Reinhard; Williams, Roger L.; Harteneck, Christian; Nürnberg, Bernd

    2015-01-01

    Class IB phosphoinositide 3-kinases (PI3Kγ) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled-receptors (GPCRs). PI3Kγ variants have one catalytic p110γ subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110γ and p101-p110γ allowing integration into distinct signalling pathways. Pharmacological tools enabling distinct modulation of the two variants are missing. The ability of an anti-p110γ monoclonal antibody (mAb(A)p110γ) to block PI3Kγ enzymatic activity attracted us to characterize this tool in detail using purified proteins. In order to get insight into the antibody-p110γ-interface, hydrogen-deuterium exchange coupled to mass spectrometry measurements were performed demonstrating binding of the monoclonal antibody to the C2 domain in p110γ, which was accompanied by conformational changes in the helical domain harbouring the Gβγ-binding site. We then studied the modulation of phospholipid vesicles association of PI3Kγ by the antibody. p87-p110γ showed a significantly reduced Gβγ-mediated phospholipid recruitment as compared with p101-p110γ. Concomitantly, in the presence of mAb(A)p110γ Gβγ did not bind to p87-p110γ. These data correlated with the ability of the antibody to block Gβγ-stimulated lipid kinase activity of p87-p110γ 30 times more potently than p101-p110γ. Our data argue for differential regulatory functions of the non-catalytic subunits and a specific Gβγ-dependent regulation of p101 in PI3Kγ activation. In this scenario, we consider the antibody as a valuable tool to dissect the distinct roles of the two PI3Kγ variants downstream of GPCRs. PMID:26173259

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

    PubMed

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

    2015-01-01

    The phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways mediate β cell growth, proliferation, survival and death. We investigated whether protein restriction during pregnancy alters islet morphometry or the expression and phosphorylation of several proteins involved in the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways. As controls, adult pregnant and non-pregnant rats were fed a normal-protein diet (17%). Pregnant and non-pregnant rats in the experimental groups were fed a low-protein diet (6%) for 15 days. Low protein diet during pregnancy increased serum prolactin level, reduced serum corticosterone concentration and the expression of both protein kinase B/AKT1 (AKT1) and p70 ribosomal protein S6 kinase (p70S6K), as well as the islets area, but did not alter the insulin content of pancreatic islets. Pregnancy increased the expression of the Src homology/collagen (SHC) protein and the extracellular signal-regulated kinases 1/2 (ERK1/2) independent of diet. ERK1/2 phosphorylation (pERK1/2) was similar in islets from pregnant and non-pregnant rats fed a low-protein diet, and was higher in islets from pregnant rats than in islets from non-pregnant rats fed a normal-protein diet. Thus, a short-term, low-protein diet during pregnancy was sufficient to reduce the levels of proteins in the phosphatidylinositol 3-kinase pathway and affect islet morphometry. PMID:25860970

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2013-04-01

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

  14. Acute regulation by insulin of phosphatidylinositol-3-kinase, Rad, Glut 4, and lipoprotein lipase mRNA levels in human muscle.

    PubMed

    Laville, M; Auboeuf, D; Khalfallah, Y; Vega, N; Riou, J P; Vidal, H

    1996-07-01

    We have investigated the acute regulation by insulin of the mRNA levels of nine genes involved in insulin action, in muscle biopsies obtained before and at the end of a 3-h euglycemic hyperinsulinemic clamp. Using reverse transcription-competitive PCR, we have measured the mRNAs encoding the two insulin receptor variants, the insulin receptor substrate-1, the p85alpha subunit of phosphatidylinositol-3-kinase, Ras associated to diabetes (Rad), the glucose transporter Glut 4, glycogen synthase, 6-phosphofructo-l-kinase, lipoprotein lipase, and the hormone-sensitive lipase. Insulin infusion induced a significant increase in the mRNA level of Glut 4 (+56 +/- 13%), Rad (+96 +/- 25%), the p85alpha subunit of phosphatidylinositol-3-kinase (+92 +/- 18%) and a decrease in the lipoprotein lipase mRNA level (-49 +/- 5%), while the abundance of the other mRNAs was unaffected. The relative expression of the two insulin receptor variants was not modified. These results demonstrate an acute coordinated regulation by insulin of the expression of genes coding key proteins involved in its action in human skeletal muscle and suggest that Rad and the p85alpha regulatory subunit of phosphatidylinositol-3-kinase can be added to the list of the genes controlled by insulin. PMID:8690802

  15. Involvement of the phosphoinositide 3-kinase/Akt pathway in apoptosis induced by capsaicin in the human pancreatic cancer cell line PANC-1

    PubMed Central

    ZHANG, JIAN-HONG; LAI, FU-JI; CHEN, HUI; LUO, JIANG; ZHANG, RI-YUAN; BU, HE-QI; WANG, ZHAO-HONG; LIN, HONG-HAI; LIN, SHENG-ZHANG

    2013-01-01

    Capsaicin, one of the major pungent ingredients found in red peppers, has been recently demonstrated to induce apoptosis in various malignant cell lines through an unclear mechanism. In this study, the effect of capsaicin on proliferation and apoptosis in the human pancreatic cancer cell line PANC-1 and its possible mechanism(s) of action were investigated. The results of a Cell Counting Kit-8 (CCK-8) assay revealed that capsaicin significantly decreased the viability of PANC-1 cells in a dose-dependent manner. Capsaicin induced G0/G1 phase cell cycle arrest and apoptosis in PANC-1 cells as demonstrated by a flow cytometric assessment. Caspase-3 expression at both the protein and mRNA level was promoted following capsaicin treatment. Furthermore, we revealed that phospho-PI3 Kinase p85 (Tyr458) and phospho-Akt (Ser473) in PANC-1 cells were downregulated in response to capsaicin. Moreover, capsaicin gavage significantly inhibited the growth of pancreatic cancer PANC-1 cell xenografts in athymic nude mice. An increased number of TUNEL-positive cells and cleaved caspase-3 were observed in capsaicin-treated mice. In vivo, capsaicin downregulated the expression of phospho-PI3 Kinase p85 (Tyr458) and phospho-Akt (Ser473). In conclusion, we have demonstrated that capsaicin is an inhibitor of growth of PANC-1 cells, and downregulation of the phosphoinositide 3-kinase/Akt pathway may be involved in capsaicin-induced apoptosis in vitro and in vivo. PMID:23255891

  16. Expression and modulation of nerve growth factor in murine keratinocytes (PAM 212)

    SciTech Connect

    Tron, V.A.; Coughlin, M.D.; Jang, D.E.; Stanisz, J.; Sauder, D.N. )

    1990-04-01

    Nerve growth factor (NGF) is a polypeptide that is required for normal development and maintenance of the sympathetic and sensory nervous systems. Skin has been shown to contain relatively high amounts of NGF, which is in keeping with the finding that the quantity of NGF in a tissue is proportional to the extent of sympathetic innervation of that organ. Since the keratinocyte, a major cellular constituent of the skin, is known to produce other growth factors and cytokines, our experiments were designed to determine whether keratinocytes are a source of NGF. Keratinocyte-conditioned media from the keratinocyte cell line PAM 212 contained NGF-like activity, approximately 2-3 ng/ml, as detected by the neurite outgrowth assay. Freshly isolated BALB/c keratinocytes contained approximately 0.1 ng/ml. Using a cDNA probe directed against NGF, we demonstrated the presence of a 1.3-kb NGF mRNA in both PAM 212 and BALB/c keratinocytes. Since ultraviolet radiation (UV) is a potentially important modulating factor for cytokines in skin, we examined the effect of UV on NGF mRNA expression. Although UV initially inhibited the expression of keratinocyte NGF mRNA (4 h), by 24 h an induction of NGF mRNA was seen. The NGF signal could also be induced by phorbol esters. Thus, keratinocytes synthesize and express NGF, and its expression is modulated by UVB and phorbol esters.

  17. CORONETTE KERATINOCYTE COLONY FORMATION IS SUPPORTED BY EPIDERMAL-DERMAL CELL INTERACTIONS IN THE BOVINE CLAW

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Delineating factors that orchestrate keratinocyte growth and differentiation in the claw is pivotal to understanding the quality of hoof horn production in health and disease. The specific objectives of this investigation were to establish an in vitro culture system for bovine coronette keratinocyt...

  18. Use of cyanoacrylate glue to prepare cultured keratinocyte sheets for grafting.

    PubMed

    Meana, A; Iglesias, J; Madrigal, B; Sanchez, J

    1997-01-01

    We describe a method to prepare keratinocyte cultured sheets for grafting based on the use of an organic glue (n-butyl-2-cyanoacrylate). It is a rapid and easy method in which the cultured layers of keratinocytes remain with a surface area similar to the original cultures.

  19. Genetic studies on the functional relevance of the protein prenyltransferases in skin keratinocytes.

    PubMed

    Lee, Roger; Chang, Sandy Y; Trinh, Hung; Tu, Yiping; White, Andrew C; Davies, Brandon S J; Bergo, Martin O; Fong, Loren G; Lowry, William E; Young, Stephen G

    2010-04-15

    The modification of proteins with farnesyl or geranylgeranyl lipids, a process called protein prenylation, facilitates interactions of proteins with membrane surfaces. Protein prenylation is carried out by a pair of cytosolic enzymes, protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase-I). FTase and GGTase-I have attracted interest as therapeutic targets for both cancer and progeria, but very little information exists on the importance of these enzymes for homeostasis of normal tissues. One study actually suggested that FTase is entirely dispensable. To explore the importance of the protein prenyltransferases for normal tissues, we used conditional knockout alleles for Fntb and Pggt1b (which encode the beta-subunits of FTase and GGTase-I, respectively) and a keratin 14-Cre transgene to create mice lacking FTase or GGTase-I in skin keratinocytes. Keratinocyte-specific Fntb knockout mice were viable but developed severe alopecia. Although hair follicles appeared normal during development, they were morphologically abnormal after birth, and ultrastructural and immunohistochemical studies revealed many apoptotic cells. The interfollicular epidermis of Fntb-deficient mice appeared normal; however, keratinocytes from these mice could not proliferate in culture. As expected, non-farnesylated prelamin A and non-farnesylated DNAJA1 accumulated in Fntb-deficient keratinocytes. Keratinocyte-specific Pggt1b knockout mice survived development but died shortly after birth. Like Fntb-deficient keratinocytes, Pggt1b-deficient keratinocytes did not proliferate in culture. Thus, both FTase and GGTase-I are required for the homeostasis of skin keratinocytes.

  20. Human melanocytes mitigate keratinocyte-dependent contraction in an in vitro collagen contraction assay.

    PubMed

    Rakar, Jonathan; Krammer, Markus P; Kratz, Gunnar

    2015-08-01

    Scarring is an extensive problem in burn care, and treatment can be especially complicated in cases of hypertrophic scarring. Contraction is an important factor in scarring but the contribution of different cell types remains unclear. We have investigated the contractile behavior of keratinocytes, melanocytes and fibroblasts by using an in vitro collagen gel assay aimed at identifying a modulating role of melanocytes in keratinocyte-mediated contraction. Cells were seeded on a collagen type I gel substrate and the change in gel dimensions were measured over time. Hematoxylin & Eosin-staining and immunohistochemistry against pan-cytokeratin and microphthalmia-associated transcription factor showed that melanocytes integrated between keratinocytes and remained there throughout the experiments. Keratinocyte- and fibroblast-seeded gels contracted significantly over time, whereas melanocyte-seeded gels did not. Co-culture assays showed that melanocytes mitigate the keratinocyte-dependent contraction (significantly slower and 18-32% less). Fibroblasts augmented the contraction in most assays (approximately 6% more). Non-contact co-cultures showed some influence on the keratinocyte-dependent contraction. Results show that mechanisms attributable to melanocytes, but not fibroblasts, can mitigate keratinocyte contractile behavior. Contact-dependent mechanisms are stronger modulators than non-contact dependent mechanisms, but both modes carry significance to the contraction modulation of keratinocytes. Further investigations are required to determine the mechanisms involved and to determine the utility of melanocytes beyond hypopigmentation in improved clinical regimes of burn wounds and wound healing.

  1. Integrins of the beta1 family influence keratinocyte-lymphocyte interaction.

    PubMed

    Boukhelifa, M; Paulin, Y; Font, J; Pichon, J; Giner, M; Wantyghem, J; Aubery, M; Braut-Boucher, F

    1998-10-01

    Data from the literature indicate that ICAM-1 molecules play an important role in keratinocyte interactions with lymphocytes via the lymphocyte function-associated-1 lymphocyte-adhesion molecule. We examined the role of beta1 integrins in keratinocyte-lymphocyte adhesion under different activation conditions. Among the beta1 integrins expressed on keratinocytes and lymphocytes detected by indirect immunofluorescence microscopy and flow cytofluorometry, primarily the alpha2 and the alpha3 subunits on both cell types were involved in keratinocyte-lymphocyte adhesion. Moreover, the highest adhesion level was observed when both cell types were activated by IFN-gamma for keratinocytes and phorbol 12-myristate 13-acetate for lymphocytes, suggesting that the former involved the protein kinase C pathway. Keratinocyte activation, characterized by the expression of ICAM-1, a decrease of beta1 integrins, and the absence of alpha5beta1 integrin, was required for optimal lymphocyte adhesion. Thus, beta1 integrins remaining at the surface of IFN-gamma-treated keratinocytes could be activated by this cytokine, and could synergize with ICAM-1 and lymphocyte function-associated-1 molecules to consolidate keratinocyte-lymphocyte adhesion.

  2. Arsenite suppression of BMP signaling in human keratinocytes

    SciTech Connect

    Phillips, Marjorie A.; Qin, Qin; Hu, Qin; Zhao, Bin; Rice, Robert H.

    2013-06-15

    Arsenic, a human skin carcinogen, suppresses differentiation of cultured keratinocytes. Exploring the mechanism of this suppression revealed that BMP-6 greatly increased levels of mRNA for keratins 1 and 10, two of the earliest differentiation markers expressed, a process prevented by co-treatment with arsenite. BMP also stimulated, and arsenite suppressed, mRNA for FOXN1, an important transcription factor driving early keratinocyte differentiation. Keratin mRNAs increased slowly after BMP-6 addition, suggesting they are indirect transcriptional targets. Inhibition of Notch1 activation blocked BMP induction of keratins 1 and 10, while FOXN1 induction was largely unaffected. Supporting a requirement for Notch1 signaling in keratin induction, BMP increased levels of activated Notch1, which was blocked by arsenite. BMP also greatly decreased active ERK, while co-treatment with arsenite maintained active ERK. Inhibition of ERK signaling mimicked BMP by inducing keratin and FOXN1 mRNAs and by increasing active Notch1, effects blocked by arsenite. Of 6 dual-specificity phosphatases (DUSPs) targeting ERK, two were induced by BMP unless prevented by simultaneous exposure to arsenite and EGF. Knockdown of DUSP2 or DUSP14 using shRNAs greatly reduced FOXN1 and keratins 1 and 10 mRNA levels and their induction by BMP. Knockdown also decreased activated Notch1, keratin 1 and keratin 10 protein levels, both in the presence and absence of BMP. Thus, one of the earliest effects of BMP is induction of DUSPs, which increases FOXN1 transcription factor and activates Notch1, both required for keratin gene expression. Arsenite prevents this cascade by maintaining ERK signaling, at least in part by suppressing DUSP expression. - Highlights: • BMP induces FOXN1 transcription. • BMP induces DUSP2 and DUSP14, suppressing ERK activation. • Arsenite suppresses levels of phosphorylated Smad1/5 and FOXN1 and DUSP mRNA. • These actions rationalize arsenite suppression of keratinocyte

  3. Effects triggered by platinum nanoparticles on primary keratinocytes

    PubMed Central

    Konieczny, Piotr; Goralczyk, Anna Grazyna; Szmyd, Radoslaw; Skalniak, Lukasz; Koziel, Joanna; Filon, Francesca Larese; Crosera, Matteo; Cierniak, Agnieszka; Zuba-Surma, Ewa K; Borowczyk, Julia; Laczna, Eliza; Drukala, Justyna; Pyza, Elzbieta; Semik, Danuta; Woznicka, Olga; Klein, Andrzej; Jura, Jolanta

    2013-01-01

    The platinum (Pt)-group elements (PGEs) represent a new kind of environmental pollutant and a new hazard for human health. Since their introduction as vehicle-exhaust catalysts, their emissions into the environment have grown considerably compared with their low natural concentration in the earth crust. PGE emissions from vehicle catalysts can be also in the form of nanometer-sized particles (Pt nanoparticles [PtNPs]). These elements, both in their metallic form or as ions solubilized in biological media, are now recognized as potent allergens and sensitizers. Human skin is always exposed to toxic particles; therefore, in the present study we addressed the question of whether polyvinylpyrrolidone-coated PtNPs may have any negative effects on skin cells, including predominantly epidermal keratinocytes. In this study, PtNPs of two sizes were used: 5.8 nm and 57 nm, in concentrations of 6.25, 12.5, and 25 μg/mL. Both types of NPs were protected with polyvinylpyrrolidone. Primary keratinocytes were treated for 24 and 48 hours, then cytotoxicity, genotoxicity, morphology, metabolic activity, and changes in the activation of signaling pathways were investigated in PtNP-treated cells. We found that PtNPs trigger toxic effects on primary keratinocytes, decreasing cell metabolism, but these changes have no effects on cell viability or migration. Moreover, smaller NPs exhibited more deleterious effect on DNA stability than the big ones. Analyzing activation of caspases, we found changes in activity of caspase 9 and caspase 3/7 triggered mainly by smaller NPs. Changes were not so significant in the case of larger nanoparticles. Importantly, we found that PtNPs have antibacterial properties, as is the case with silver NPs (AgNPs). In comparison to our previous study regarding the effects of AgNPs on cell biology, we found that PtNPs do not exhibit such deleterious effects on primary keratinocytes as AgNPs and that they also can be used as potential antibacterial agents

  4. Effects triggered by platinum nanoparticles on primary keratinocytes.

    PubMed

    Konieczny, Piotr; Goralczyk, Anna Grazyna; Szmyd, Radoslaw; Skalniak, Lukasz; Koziel, Joanna; Filon, Francesca Larese; Crosera, Matteo; Cierniak, Agnieszka; Zuba-Surma, Ewa K; Borowczyk, Julia; Laczna, Eliza; Drukala, Justyna; Pyza, Elzbieta; Semik, Danuta; Woznicka, Olga; Klein, Andrzej; Jura, Jolanta

    2013-01-01

    The platinum (Pt)-group elements (PGEs) represent a new kind of environmental pollutant and a new hazard for human health. Since their introduction as vehicle-exhaust catalysts, their emissions into the environment have grown considerably compared with their low natural concentration in the earth crust. PGE emissions from vehicle catalysts can be also in the form of nanometer-sized particles (Pt nanoparticles [PtNPs]). These elements, both in their metallic form or as ions solubilized in biological media, are now recognized as potent allergens and sensitizers. Human skin is always exposed to toxic particles; therefore, in the present study we addressed the question of whether polyvinylpyrrolidone-coated PtNPs may have any negative effects on skin cells, including predominantly epidermal keratinocytes. In this study, PtNPs of two sizes were used: 5.8 nm and 57 nm, in concentrations of 6.25, 12.5, and 25 μg/mL. Both types of NPs were protected with polyvinylpyrrolidone. Primary keratinocytes were treated for 24 and 48 hours, then cytotoxicity, genotoxicity, morphology, metabolic activity, and changes in the activation of signaling pathways were investigated in PtNP-treated cells. We found that PtNPs trigger toxic effects on primary keratinocytes, decreasing cell metabolism, but these changes have no effects on cell viability or migration. Moreover, smaller NPs exhibited more deleterious effect on DNA stability than the big ones. Analyzing activation of caspases, we found changes in activity of caspase 9 and caspase 3/7 triggered mainly by smaller NPs. Changes were not so significant in the case of larger nanoparticles. Importantly, we found that PtNPs have antibacterial properties, as is the case with silver NPs (AgNPs). In comparison to our previous study regarding the effects of AgNPs on cell biology, we found that PtNPs do not exhibit such deleterious effects on primary keratinocytes as AgNPs and that they also can be used as potential antibacterial agents

  5. A Heat-Sensitive TRP Channel Expressed in Keratinocytes

    NASA Astrophysics Data System (ADS)

    Peier, Andrea M.; Reeve, Alison J.; Andersson, David A.; Moqrich, Aziz; Earley, Taryn J.; Hergarden, Anne C.; Story, Gina M.; Colley, Sian; Hogenesch, John B.; McIntyre, Peter; Bevan, Stuart; Patapoutian, Ardem

    2002-06-01

    Mechanical and thermal cues stimulate a specialized group of sensory neurons that terminate in the skin. Three members of the transient receptor potential (TRP) family of channels are expressed in subsets of these neurons and are activated at distinct physiological temperatures. Here, we describe the cloning and characterization of a novel thermosensitive TRP channel. TRPV3 has a unique threshold: It is activated at innocuous (warm) temperatures and shows an increased response at noxious temperatures. TRPV3 is specifically expressed in keratinocytes; hence, skin cells are capable of detecting heat via molecules similar to those in heat-sensing neurons.

  6. Steroid synthesis by primary human keratinocytes; implications for skin disease

    SciTech Connect

    Hannen, Rosalind F.; Michael, Anthony E.; Jaulim, Adil; Bhogal, Ranjit; Burrin, Jacky M.; Philpott, Michael P.

    2011-01-07

    Research highlights: {yields} Primary keratinocytes express the steroid enzymes required for cortisol synthesis. {yields} Normal primary human keratinocytes can synthesise cortisol. {yields} Steroidogenic regulators, StAR and MLN64, are expressed in normal epidermis. {yields} StAR expression is down regulated in eczema and psoriatic epidermis. -- Abstract: Cortisol-based therapy is one of the most potent anti-inflammatory treatments available for skin conditions including psoriasis and atopic dermatitis. Previous studies have investigated the steroidogenic capabilities of keratinocytes, though none have demonstrated that these skin cells, which form up to 90% of the epidermis are able to synthesise cortisol. Here we demonstrate that primary human keratinocytes (PHK) express all the elements required for cortisol steroidogenesis and metabolise pregnenolone through each intermediate steroid to cortisol. We show that normal epidermis and cultured PHK express each of the enzymes (CYP11A1, CYP17A1, 3{beta}HSD1, CYP21 and CYP11B1) that are required for cortisol synthesis. These enzymes were shown to be metabolically active for cortisol synthesis since radiometric conversion assays traced the metabolism of [7-{sup 3}H]-pregnenolone through each steroid intermediate to [7-{sup 3}H]-cortisol in cultured PHK. Trilostane (a 3{beta}HSD1 inhibitor) and ketoconazole (a CYP17A1 inhibitor) blocked the metabolism of both pregnenolone and progesterone. Finally, we show that normal skin expresses two cholesterol transporters, steroidogenic acute regulatory protein (StAR), regarded as the rate-determining protein for steroid synthesis, and metastatic lymph node 64 (MLN64) whose function has been linked to cholesterol transport in steroidogenesis. The expression of StAR and MLN64 was aberrant in two skin disorders, psoriasis and atopic dermatitis, that are commonly treated with cortisol, suggesting dysregulation of epidermal steroid synthesis in these patients. Collectively these data

  7. Systemic suppression of delayed-type hypersensitivity by supernatants from UV-irradiated keratinocytes. An essential role for keratinocyte-derived IL-10.

    PubMed

    Rivas, J M; Ullrich, S E

    1992-12-15

    Exposing murine keratinocyte cultures to UV radiation causes the release of a suppressive cytokine that mimics the immunosuppressive effects of total-body UV exposure. Injecting supernatants from UV-irradiated keratinocyte cultures into mice inhibits their ability to generate a delayed-type hypersensitivity reaction against allogeneic histocompatibility Ag, and spleen cells from mice injected with supernatant do not respond to alloantigen in the in vitro MLR. A unique feature of the immunosuppression induced by either total-body UV-exposure or injecting the suppressive cytokine from UV-irradiated keratinocytes is the selectivity of suppression. Although cellular immune reactions such as delayed-type hypersensitivity are suppressed antibody production is unaffected. Because the selective nature to the UV-induced immunosuppression is similar to the biologic activity of IL-10, we examined the hypothesis that UV exposure of keratinocytes causes the release of IL-10. Keratinocyte monolayers were exposed to UV radiation and at specific times after exposure mRNA was isolated or the culture supernatant from the cells was collected. IL-10 mRNA expression was enhanced in UV-irradiated keratinocytes. The secretion of IL-10 by the irradiated keratinocytes was determined by Western blot analysis. A band reactive with anti-IL-10 mAb was found in supernatants from the UV-irradiated but not the mock-irradiated cells. IL-10 biologic activity was determined by the ability of the supernatants from the UV-irradiated keratinocytes to suppress IFN-gamma production by Ag-activated Th 1 cell clones. Anti-IL-10 mAb neutralized the ability of supernatants from UV-irradiated keratinocytes to suppress the induction of delayed-type hypersensitivity in vivo. Furthermore, injecting UV-irradiated mice with antibodies against IL-10 partially inhibited in vivo immunosuppression. These data indicate that activated keratinocytes are capable of secreting IL-10 and suggest that the release of IL-10 by

  8. Keratinocyte-derived laminin-332 protein promotes melanin synthesis via regulation of tyrosine uptake.

    PubMed

    Chung, Heesung; Jung, Hyejung; Lee, Jung-Hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

    2014-08-01

    Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake.

  9. Characterisation of human fibroblasts as keratinocyte feeder layer using p63 isoforms status.

    PubMed

    Auxenfans, Céline; Thépot, Amélie; Justin, Virginie; Hautefeuille, Agnès; Shahabeddin, Lili; Damour, Odile; Hainaut, Pierre

    2009-01-01

    Large-scale culture of primary keratinocytes allows the production of large epidermal sheet surfaces for the treatment of extensive skin burns. This method is dependent upon the capacity to establish cultures of proliferating keratinocytes in conditions compatible with their clonal expansion while maintaining their capacity to differentiate into the typical squamous pattern of human epidermis. Feeder layers are critical in this process because the fibroblasts that compose this layer serve as a source of adhesion, growth and differentiation factors. In this report, we have characterise the expression patterns of p63 isoforms in primary keratinocytes cultured on two different feeder layer systems, murine 3T3 and human fibroblasts. We show that with the latter, keratinocytes express a higher ratio of Delta N to TAp63 isoform, in relation with higher clonogenic potential. These results indicate that human fibroblasts represent an adequate feeder layer system to support the culture of primary human keratinocytes. PMID:20042803

  10. Stimulatory effect of Brazilian propolis on hair growth through proliferation of keratinocytes in mice.

    PubMed

    Miyata, Shota; Oda, Yozo; Matsuo, Chika; Kumura, Haruto; Kobayashi, Ken

    2014-12-10

    Propolis is a natural honeybee hive product with the potential for use in the treatment of dermatological conditions, such as cutaneous abrasions, burns, and acne. In this study, we investigated whether propolis stimulates hair growth in mice. Ethanol-extracted propolis, which contains various physiologically active substances such as caffeic acid and kaempferol, stimulated anagen induction in shaved back skin. Anagen induction occurred without any detectable abnormalities in the shape of the hair follicles (HFs), hair stem cells in the bulge, proliferating hair matrix keratinocytes in the hair bulb, or localization of versican in the dermal papilla. Propolis treatment also stimulated migration of hair matrix keratinocytes into the hair shaft in HFs during late anagen in the depilated back skin. Organotypic culture of skin containing anagen stage HFs revealed significant stimulation of hair matrix keratinocyte proliferation by propolis. Furthermore, propolis facilitated the proliferation of epidermal keratinocytes. These results indicate that propolis stimulates hair growth by inducing hair keratinocyte proliferation.

  11. Stimulatory effect of Brazilian propolis on hair growth through proliferation of keratinocytes in mice.

    PubMed

    Miyata, Shota; Oda, Yozo; Matsuo, Chika; Kumura, Haruto; Kobayashi, Ken

    2014-12-10

    Propolis is a natural honeybee hive product with the potential for use in the treatment of dermatological conditions, such as cutaneous abrasions, burns, and acne. In this study, we investigated whether propolis stimulates hair growth in mice. Ethanol-extracted propolis, which contains various physiologically active substances such as caffeic acid and kaempferol, stimulated anagen induction in shaved back skin. Anagen induction occurred without any detectable abnormalities in the shape of the hair follicles (HFs), hair stem cells in the bulge, proliferating hair matrix keratinocytes in the hair bulb, or localization of versican in the dermal papilla. Propolis treatment also stimulated migration of hair matrix keratinocytes into the hair shaft in HFs during late anagen in the depilated back skin. Organotypic culture of skin containing anagen stage HFs revealed significant stimulation of hair matrix keratinocyte proliferation by propolis. Furthermore, propolis facilitated the proliferation of epidermal keratinocytes. These results indicate that propolis stimulates hair growth by inducing hair keratinocyte proliferation. PMID:25418897

  12. Impairment of human keratinocyte mobility and proliferation by advanced glycation end products-modified BSA.

    PubMed

    Zhu, Ping; Yang, Chuan; Chen, Li-Hong; Ren, Meng; Lao, Guo-Juan; Yan, Li

    2011-07-01

    The migration and proliferation of keratinocytes is critical to wound re-epithelialization and defects in this function are associated with the clinical phenomenon of chronic non-healing wounds. Advanced glycation end products (AGEs) occur through non-enzymatic glycation of long-lived proteins in diabetes and play important roles in diabetic complications. However, specific roles for AGEs in keratinocyte migration and proliferation, and the underlying molecular mechanisms, have not been fully established. The aim of the current study was to elucidate the interaction between AGE-modified bovine serum albumin (AGE-BSA) and keratinocytes. As a result, we found that AGE-BSA had no effect on the viability of keratinocytes for up to 48 h of incubation with 50 μg/ml of AGE-BSA. AGE-BSA (but not non-glycated BSA) exerted a concentration-dependent suppression of keratinocyte migration at a range of concentrations. The expression of matrix metalloproteinase-9 (MMP-9) was significantly up-regulated in keratinocytes incubated with increasing AGE-BSA, but tissue inhibitor of metalloproteinases-1 (TIMP-1) expression was down-regulated. AGE-BSA also profoundly depressed phospho-focal adhesion kinase-Tyr397 (p-FAK) and α2β1 integrin expression, while total-FAK expression levels remained constant, in keratinocytes. The proliferative capacity of keratinocytes was diminished after 72 h AGE-BSA incubation. Taken together, these findings suggested that in the presence of AGE-BSA, keratinocytes lose their migratory and proliferation abilities. These data also indicated that, in the context of the chronic hyperglycemia in diabetes, the effects of AGE-BSA on keratinocyte migration might be mediated through MMP-9/TIMP-1, p-FAK and α2β1 integrin.

  13. Cortactin involvement in the keratinocyte growth factor and fibroblast growth factor 10 promotion of migration and cortical actin assembly in human keratinocytes

    SciTech Connect

    Ceccarelli, Simona; Cardinali, Giorgia; Aspite, Nicaela; Picardo, Mauro; Marchese, Cinzia; Torrisi, Maria Rosaria; Mancini, Patrizia . E-mail: patrizia.mancini@uniroma1.it

    2007-05-15

    Keratinocyte growth factor (KGF/FGF7) and fibroblast growth factor 10 (FGF10/KGF2) regulate keratinocyte proliferation and differentiation by binding to the tyrosine kinase KGF receptor (KGFR). KGF induces keratinocyte motility and cytoskeletal rearrangement, whereas a direct role of FGF10 on keratinocyte migration is not clearly established. Here we analyzed the motogenic activity of FGF10 and KGF on human keratinocytes. Migration assays and immunofluorescence of actin cytoskeleton revealed that FGF10 is less efficient than KGF in promoting migration and exerts a delayed effect in inducing lamellipodia and ruffles formation. Both growth factors promoted phosphorylation and subsequent membrane translocation of cortactin, an F-actin binding protein involved in cell migration; however, FGF10-induced cortactin phosphorylation was reduced, more transient and delayed with respect to that promoted by KGF. Cortactin phosphorylation induced by both growth factors was Src-dependent, while its membrane translocation and cell migration were blocked by either Src and PI3K inhibitors, suggesting that both pathways are involved in KGF- and FGF10-dependent motility. Furthermore, siRNA-mediated downregulation of cortactin inhibited KGF- and FGF10-induced migration. These results indicate that cortactin is involved in keratinocyte migration promoted by both KGF and FGF10.

  14. Overexpression of CRABPI in suprabasal keratinocytes enhances the proliferation of epidermal basal keratinocytes in mouse skin topically treated with all-trans retinoic acid

    SciTech Connect

    Tang, X.-H.; Vivero, Marina; Gudas, Lorraine J.

    2008-01-01

    We investigated whether ectopic expression of CRABPI, a cellular retinoic acid binding protein, influenced the actions of all-trans retinoic acid (ATRA) in transgenic (TG) mice. We targeted CRABPI to the basal vs. suprabasal layers of mouse epidermis by using the keratin 14 (K14) and keratin 10 (K10) promoters, respectively. Greater CRABPI protein levels were detected in the epidermis of adult transgenic(+) mice than in transgenic(-) mice for both transgenes. In adult mouse skin CRABPI overexpression in the basal or suprabasal keratinocytes did not cause morphological abnormalities, but did result in decreased CRABPII mRNA levels. Ectopically overexpressed CRABPI in suprabasal keratinocytes, but not in basal keratinocytes, enhanced the thickening of the epidermis induced by topical ATRA treatments (10 {mu}M, 400 {mu}l for 4 days) by 1.59 {+-} 0.2-fold (p < 0.05). ATRA treatment (10 {mu}M) resulted in a 59.9 {+-} 9.8% increase (p < 0.05) in the BrdU labeling index in K10/FLAG-CRABPI TG(+) mice vs. TG(-) mice. Retinoid topical treatments reduced p27 and CYP26A1 mRNA levels in TG(+) and TG(-) mouse skin in K14 and K10/FLAG-CRABPI transgenic mice. As epidermal basal keratinocyte proliferation is stimulated by paracrine growth factors secreted by ATRA activated suprabasal keratinocytes, our results indicate that CRABPI overexpression in suprabasal keratinocytes enhances the physiological functions of ATRA.

  15. Effects of small interfering RNA inhibit Class I phosphoinositide 3-kinase on human gastric cancer cells

    PubMed Central

    Zhu, Bao-Song; Yu, Li-Yan; Zhao, Kui; Wu, Yong-You; Cheng, Xiao-Li; Wu, Yong; Zhong, Feng-Yun; Gong, Wei; Chen, Qiang; Xing, Chun-Gen

    2013-01-01

    AIM: To investigate the effects of small interfering RNA (siRNA)-mediated inhibition of Class I phosphoinositide 3-kinase (Class I PI3K) signal transduction on the proliferation, apoptosis, and autophagy of gastric cancer SGC7901 and MGC803 cells. METHODS: We constructed the recombinant replication adenovirus PI3K(I)-RNA interference (RNAi)-green fluorescent protein (GFP) and control adenovirus NC-RNAi-GFP, and infected it into human gastric cancer cells. MTT assay was used to determine the growth rate of the gastric cancer cells. Activation of autophagy was monitored with monodansylcadaverine (MDC) staining after adenovirus PI3K(I)-RNAi-GFP and control adenovirus NC-RNAi-GFP treatment. Immunofluorescence staining was used to detect the expression of microtubule-associated protein 1 light chain 3 (LC3). Mitochondrial membrane potential was measured using the fluorescent probe JC-1. The expression of autophagy was monitored with MDC, LC3 staining, and transmission electron microscopy. Western blotting was used to detect p53, Beclin-1, Bcl-2, and LC3 protein expression in the culture supernatant. RESULTS: The viability of gastric cancer cells was inhibited after siRNA targeting to the Class I PI3K blocked Class I PI3K signal pathway. MTT assays revealed that, after SGC7901 cancer cells were treated with adenovirus PI3K(I)-RNAi-GFP, the rate of inhibition reached 27.48% ± 2.71% at 24 h, 41.92% ± 2.02% at 48 h, and 50.85% ± 0.91% at 72 h. After MGC803 cancer cells were treated with adenovirus PI3K(I)-RNAi-GFP, the rate of inhibition reached 24.39% ± 0.93% at 24 h, 47.00% ± 0.87% at 48 h, and 70.30% ± 0.86% at 72 h (P < 0.05 compared to control group). It was determined that when 50 MOI, the transfection efficiency was 95% ± 2.4%. Adenovirus PI3K(I)-RNAi-GFP (50 MOI) induced mitochondrial dysfunction and activated cell apoptosis in SGC7901 cells, and the results described here prove that RNAi of Class I PI3K induced apoptosis in SGC7901 cells

  16. Migration of keratinocytes through tunnels of digested fibrin

    NASA Astrophysics Data System (ADS)

    Ronfard, Vincent; Barrandon, Yann

    2001-04-01

    We report here a hitherto undescribed form of cell migration. When a suspension of human keratinocytes is plated on a fibrin matrix, single cells invade the matrix and progress through it as rounded cells by dissolving the fibrin and thereby creating tunnels. These tunnels are cylindrical or helical, the latter being the result of constant change in the path of cellular advance around the helical axis. Helical tunnel formation is strongly promoted by epidermal growth factor. The rate of migration of the cell through the track of a helical tunnel (up to 2.1 mm per day) is about 7-fold greater than through a cylindrical tunnel. Pericellular fibrinolysis leading to tunnel formation depends on the presence of plasminogen in the medium and its conversion to plasmin by a cellular activator. Formation of tunnels requires that plasminogen activator be localized on the advancing surface of the keratinocyte; we propose that the tunnel is cylindrical when the site of release of plasmin is located at a fixed point on the cell surface and helical when the site of release precesses.

  17. Potential applications of keratinocytes derived from human embryonic stem cells.

    PubMed

    Movahednia, Mohammad M; Kidwai, Fahad K; Jokhun, Doorgesh S; Squier, Christopher A; Toh, Wei Seong; Cao, Tong

    2016-01-01

    Although skin grafting is one of the most advanced cell therapy technique, wide application of skin substitutes is hampered by the difficulty in securing sufficient amount of epidermal substitute. Additionally, in understanding the progression of skin aging and disease, and in screening the cosmetic and pharmaceutical products, there is lack of a satisfactory human skin-specific in vitro model. Recently, human embryonic stem cells (hESCs) have been proposed as an unlimited and reliable cell source to obtain almost all cell types present in the human body. This review focuses on the potential off-the-shelf use of hESC-derived keratinocytes for future clinical applications as well as a powerful in vitro skin model to study skin function and integrity, host-pathogen interactions and disease pathogenesis. Furthermore, we discuss the industrial applications of hESC-derived keratinized multi-layer epithelium which provides a human-like test platform for understanding disease pathogenesis, evaluation of new therapeutic modalities and assessment of the safety and efficacy of skin cosmetics and therapeutics. Overall, we conclude that the hESC-derived keratinocytes have great potential for clinical, research and industrial applications. PMID:26663861

  18. TOXICITY OF AMORPHOUS SILICA NANOPARTICLES IN MOUSE KERATINOCYTES

    SciTech Connect

    Yu, Kyung; Wang, Wei; Gu, Baohua; Hussain, Saber

    2009-01-01

    The present study was designed to examine the uptake, localization and the cytotoxic effects of well-dispersed amorphous silica nanoparticles in mouse keratinocytes (HEL-30). Mouse keratinocytes were exposed for 24h to various concentrations of amorphous silica nanoparticles in homogeneous suspensions of average size distribution (30, 48, 118 and 535 nm SiO2) then assessed for uptake and biochemical changes. Results of transmission electron microscopy revealed all sizes of silica were taken up into the cells and localized into the cytoplasm. The lactate dehydrogenase (LDH) assay shows LDH leakage was dose- and size-dependent with exposure to 30 and 48 nm nanoparticles. However, no LDH leakage was observed for either 118 or 535 nm nanoparticles. The mitochondrial viability assay (MTT) showed significant toxicity for 30 and 48 nm at high concentrations (100 g/mL) compare to the 118 and 535 nm particles. Further studies were carried out to investigate if cellular reduced GSH and mitochondria membrane potential are involved in the mechanism of SiO2 toxicity. The redox potential of cells (GSH) was reduced significantly at concentrations of 50, 100 and 200 g/mL at 30 nm nanoparticle exposures. However, silica nanoparticles larger than 30 nm showed no changes in GSH levels. Reactive oxygen species (ROS) formation did not show any significant change between controls and the exposed cells. In summary, amorphous silica nanoparticles below 100 nm induced cytotoxicity suggest size-of the particles is critical to produce biological effects.

  19. Epithelial expression of keratinocytes growth factor in oral precancer lesions

    PubMed Central

    Jimson, Sudha; Murali, S.; Zunt, Susan L.; Goldblatt, Lawrence I.; Srinivasan, Mythily

    2016-01-01

    Background: Keratinocyte growth factor (KGF) is a potent epithelial mitogen that acts by binding the KGF receptors (KGFRs) expressed on epithelial cells and regulates proliferation and differentiation. The objective of this study was to investigate the expression of KGF in the epithelium in oral precancer. Materials and Methods: Archival tissues of oral submucous fibrosis (SMF) and leukoplakia were assessed for epithelial KGF expression by immunohistochemistry and real-time quantitative polymerase chain reaction. Results: KGF was predominantly expressed in the basal and parabasal cells in the epithelium of SMF tissues. KGF transcript in the epithelial cells increased with increasing severity of epithelial dysplasia in oral leukoplakia. Conclusion: Although widely reported as a product secreted by the mesenchymal cells, our data suggest that the KGF is also expressed in oral epithelial cells much like the expression in ovarian epithelial cells. Based on the localization of KGF in cells at the epithelial mesenchymal junction and that of the reported presence of KGFR in oral keratinocytes, a potential mechanism involving paracrine and autocrine interactions of KGF and KGFR in early stages of oral precancer is postulated. PMID:27274338

  20. Possible role of epidermal keratinocytes in the construction of acupuncture meridians.

    PubMed

    Denda, Mitsuhiro; Tsutsumi, Moe

    2014-04-01

    Acupuncture meridians consist of a network of acupuncture points on the skin, stimulation of which is well established to have a variety of physiological effects. We have previously demonstrated that epidermal keratinocytes contain multiple sensory systems for temperature, mechanical stimuli, electric potentials and other stimuli. These sensory systems generate changes in the calcium-ion concentration in the epidermis, so epidermal keratinocytes can generate spatially-localized electro-physiological patterns in the skin. We have previously demonstrated signaling between epidermal keratinocytes and peripheral nerve systems. Therefore, stimuli sensed by epidermal keratinocytes might be transferred to the unmyelinated nerve fibers that are known to exist in the epidermis and, thence, to the spinal cord and brain. We propose that epidermal keratinocytes form an information-gathering network in the skin and that this network plays a key role in whole-body homeostasis in response to the changing environment. We also hypothesize that this network corresponds to the acupuncture meridians. As supporting examples, we present some striking calcium propagation patterns observed in cultured human keratinocytes after adenosine-triphosphate (ATP) stimulation. These results support the ideas that keratinocytes can generate spatially-restricted signaling patterns after environmental stimulation and that the cultures might be in-vitro models of meridians as an information-gathering network in skin. PMID:24745868

  1. Systemic suppression of delayed-type hypersensitivity by supernatants from UV-irradiated keratinocytes

    SciTech Connect

    Rivas, J.M.; Ullrich, S.E. )

    1992-12-15

    Exposing murine keratinocyte cultures to UV radiation causes the release of a suppressive cytokine that mimics the immunosuppressive effects of total-body UV exposure. Injecting supernatants from UV-irradiated keratinocyte cultures into mice inhibits their ability to generate a delayed-type hypersensitivity reaction against allogeneic histocompatibility Ag, and spleen cells from mice injected with supernatant do not respond to alloantigen in the in vitro MLR. A unique feature of the immunosuppression induced by either total-body UV-exposure or injecting the suppressive cytokine from UV-irradiated keratinocytes is the selectivity of suppression. Although cellular immune reactions such as delayed-type hypersensitivity are suppressed antibody production is unaffected. Because the selective nature to the UV-induced immunosuppression is similar to the biologic activity of IL-10, the authors examined the hypothesis that UV exposure of keratinocytes causes the release of IL-10. Keratinocyte monolayers were exposed to UV radiation and at specific times after exposure mRNA was isolated or the culture supernatant from the cells was collected. These data indicate that activated keratinocytes are capable of secreting IL-10 and suggest that the release of IL-10 by UV-irradiated keratinocytes plays an essential role in the induction of systemic immunosuppression after total-body UV exposure. 44 refs., 3 figs., 2 tabs.

  2. Galectin-7 regulates keratinocyte proliferation and differentiation through JNK-miR-203-p63 signaling

    PubMed Central

    Chen, Hung-Lin; Chiang, Po-Cheng; Lo, Chia-Hui; Lo, Yuan-Hsin; Hsu, Daniel K.; Chen, Huan-Yuan; Liu, Fu-Tong

    2015-01-01

    Galectin-7, a member of the β-galactoside-binding protein family, is primarily expressed in stratified epithelial cells, including keratinocytes. There is information in the literature suggesting a role for this protein in regulation of keratinocyte survival and growth, but the underlying mechanism remains relatively unknown. Moreover, its expression pattern in the epidermis suggests that it is also involved in the regulation of keratinocyte differentiation. Here, we demonstrate that galectin-7 knockdown results in reduced differentiation and increased proliferation of keratinocytes. Using microarray and deep-sequencing analyses, we found that galectin-7 positively and negatively regulates microRNA (miR)-203 and miR-146a expression, respectively. We show that galectin-7 regulates keratinocyte differentiation and proliferation through miR-203 but not miR-146a. A knockdown of either galectin-7 or miR-203 in keratinocytes increases expression of p63, an essential transcription factor involved in skin development. Rescue of miR-203 expression in a galectin-7 knockdown model reduces p63 expression to baseline. Increased galectin-7 expression up-regulates c-Jun N-terminal kinase (JNK) protein levels, which is required for miR-203 expression. Finally, we establish that galectin-7 can be associated with JNK1 and protect it from ubiquitination and degradation. Thus, our data suggest an intracellular function of galectin-7: regulation of keratinocyte proliferation and differentiation through the JNK1-miR-203-p63 pathway. PMID:26763438

  3. Vitiligo patient-derived keratinocytes exhibit characteristics of normal wound healing via epithelial to mesenchymal transition.

    PubMed

    Banerjee, Poulomi; Venkatachalam, Sandhyaa; Mamidi, Murali Krishna; Bhonde, Ramesh; Shankar, Krupa; Pal, Rajarshi

    2015-05-01

    Vitiligo is an autoimmune disorder that leads to depigmentation of skin via melanocyte dysfunction. Keratinocyte-induced toxicity is one among the several etiological factors implicated for vitiligo, and hence, autologous keratinocyte grafting is projected as one of the primary mode of treatment for vitiligo. However, reports indicate that perilesional keratinocytes not only display signatures of apoptosis but also could secrete cytokines and mediators which have antagonistic effect on proliferation or survival. Therefore, we investigated how vitiligo patients' derived keratinocytes respond to surplus amounts of inflammatory cytokines and whether they recapitulate events that take place during conventional wound healing. The primary objective of our study was to determine whether keratinocytes isolated from a vitiligo patient would undergo epithelial-mesenchymal transition similar to their normal counterparts upon induction with inflammatory cytokines such as TGF-b1 and EGF. We found that these keratinocytes undergo EMT during wound repair accompanied with increase in the levels of mesenchymal markers and ECM proteins; decrease in the levels of epithelial markers and enhanced migratory ability. Besides, we also demonstrated that EMT induction leads to activation of SMAD and MAPK pathways via Ras, Raf, PAI 1, Snail, Slug and ZO1. To our knowledge, this is the first report on the characterization of primary keratinocytes isolated from vitiligo patients with respect to their wound healing capacity.

  4. The E5 oncoprotein of human papillomavirus type 16 enhances endothelin-1-induced keratinocyte growth.

    PubMed

    Venuti, A; Salani, D; Poggiali, F; Manni, V; Bagnato, A

    1998-08-15

    Human keratinocytes express ETA receptors and produce endothelin-1 (ET-1), which stimulates growth response. Previously, we reported that a twofold increase in ETA receptors is present in human papillomavirus type 16 (HPV16) immortalized keratinocytes and that ET-1 induces enhanced proliferative response in these cell lines compared to normal cells. The present studies examine whether the E5 gene of HPV16 is responsible for the enhanced activity of ET-1 in HPV-transfected keratinocytes. The presence of the E5 gene in growth factor-starved keratinocytes induced the DNA synthesis and enhanced the mitogenic activity of ET-1 or epidermal growth factor. The selection of primary keratinocytes in growth factor-free medium with the addition of ET-1 as a growth factor showed that E5-transfected keratinocytes were able to grow and to form a higher number of larger colonies with respect to untransfected cells. This effect seems to be related to the interaction of E5 with the mitogenic signaling pathway of ET-1 rather than to an increase in the expression of the receptors for ET-1. In conclusion, our data demonstrate that E5 enhances ligand signaling in keratinocytes outside the EGF pathway by the amplification of the proliferative effect of ET-1/ETA receptor signaling.

  5. Regulation of adrenomedullin secretion in cultured human skin and oral keratinocytes.

    PubMed

    Kapas, S; Tenchini, M L; Farthing, P M

    2001-08-01

    Adrenomedullin, a potent vasoactive peptide, is actively secreted from primary cultures of human oral and skin keratinocytes, but nothing is known of the regulation of its release. This study describes the effects of a range of substances on adrenomedullin production from cultures of oral and skin keratinocytes. We have established that keratinocytes do not store adrenomedullin but secrete it constitutively. Cytokines interleukin-1alpha and -1beta, tumor necrosis factor-alpha and -beta, and the bacterial product, lipopolysaccharide, significantly stimulate adrenomedullin secretion from oral but not skin keratinocytes. Both transforming growth factor-beta1 and interferon-gamma are potent suppressors of adrenomedullin secretion from both cell types, as are forskolin, di-butyryl cyclic adenosine monophosphate, and adrenocorticotropin. The peptides thrombin and endothelin-1 increase adrenomedullin production, particularly from skin keratinocytes. These findings indicate that there are differences in the regulation of adrenomedullin production between oral and skin keratinocytes and that oral keratinocytes are particularly responsive to the action of inflammatory cytokines. This raises the possibility that adrenomedullin may serve a different functions in oral mucosa and skin.

  6. Galectin-7 Regulates Keratinocyte Proliferation and Differentiation through JNK-miR-203-p63 Signaling.

    PubMed

    Chen, Hung-Lin; Chiang, Po-Cheng; Lo, Chia-Hui; Lo, Yuan-Hsin; Hsu, Daniel K; Chen, Huan-Yuan; Liu, Fu-Tong

    2016-01-01

    Galectin-7, a member of the β-galactoside-binding protein family, is primarily expressed in stratified epithelial cells, including keratinocytes. There is information in the literature suggesting a role for this protein in regulation of keratinocyte survival and growth, but the underlying mechanism remains relatively unknown. Moreover, its expression pattern in the epidermis suggests that it is also involved in the regulation of keratinocyte differentiation. Here, we demonstrate that galectin-7 knockdown results in reduced differentiation and increased proliferation of keratinocytes. Using microarray and deep-sequencing analyses, we found that galectin-7 positively and negatively regulates microRNA (miR)-203 and miR-146a expression, respectively. We show that galectin-7 regulates keratinocyte differentiation and proliferation through miR-203 but not miR-146a. A knockdown of either galectin-7 or miR-203 in keratinocytes increases expression of p63, an essential transcription factor involved in skin development. Rescue of miR-203 expression in a galectin-7 knockdown model reduces p63 expression to baseline. Increased galectin-7 expression upregulates c-Jun N-terminal kinase (JNK) protein levels, which is required for miR-203 expression. Finally, we establish that galectin-7 can be associated with JNK1 and protect it from ubiquitination and degradation. Thus, our data suggest an intracellular function of galectin-7: regulation of keratinocyte proliferation and differentiation through the JNK1-miR-203-p63 pathway.

  7. Lactobacillus rhamnosus GG Lysate Increases Re-Epithelialization of Keratinocyte Scratch Assays by Promoting Migration.

    PubMed

    Mohammedsaeed, Walaa; Cruickshank, Sheena; McBain, Andrew J; O'Neill, Catherine A

    2015-11-05

    A limited number of studies have investigated the potential of probiotics to promote wound healing in the digestive tract. The aim of the current investigation was to determine whether probiotic bacteria or their extracts could be beneficial in cutaneous wound healing. A keratinocyte monolayer scratch assay was used to assess re-epithelialization; which comprises keratinocyte proliferation and migration. Primary human keratinocyte monolayers were scratched then exposed to lysates of Lactobacillus (L) rhamnosus GG, L. reuteri, L. plantarum or L. fermentum. Re-epithelialization of treated monolayers was compared to that of untreated controls. Lysates of L. rhamnosus GG and L. reuteri significantly increased the rate of re-epithelialization, with L. rhamnosus GG being the most efficacious. L. reuteri increased keratinocyte proliferation while L. rhamnosus GG lysate significantly increased proliferation and migration. Microarray analysis of L. rhamnosus GG treated scratches showed increased expression of multiple genes including the chemokine CXCL2 and its receptor CXCR2. These are involved in normal wound healing where they stimulate keratinocyte proliferation and/or migration. Increased protein expression of both CXCL2 and CXCR2 were confirmed by ELISA and immunoblotting. These data demonstrate that L. rhamnosus GG lysate accelerates re-epithelialization of keratinocyte scratch assays, potentially via chemokine receptor pairs that induce keratinocyte migration.

  8. Conjugation of extracellular matrix proteins to basal lamina analogs enhances keratinocyte attachment.

    PubMed

    Bush, Katie A; Downing, Brett R; Walsh, Sarah E; Pins, George D

    2007-02-01

    The dermal-epidermal junction of skin contains extracellular matrix proteins that are involved in initiating and controlling keratinocyte signaling events such as attachment, proliferation, and terminal differentiation. To characterize the relationship between extracellular matrix proteins and keratinocyte attachment, a biomimetic design approach was used to precisely tailor the surface of basal lamina analogs with biochemistries that emulate the native biochemical composition found at the dermal-epidermal junction. A high-throughput screening device was developed by our laboratory that allows for the simultaneous investigation of the conjugation of individual extracellular matrix proteins (e.g. collagen type I, collagen type IV, laminin, or fibronectin) as well as their effect on keratinocyte attachment, on the surface of an implantable collagen membrane. Fluorescence microscopy coupled with quantitative digital image analyses indicated that the extracellular matrix proteins adsorbed to the collagen-GAG membranes in a dose-dependent manner. To determine the relationship between extracellular matrix protein signaling cues and keratinocyte attachment, cells were seeded on protein-conjugated collagen-GAG membranes and a tetrazolium-based colorimetric assay was used to quantify viable keratinocyte attachment. Our results indicate that keratinocyte attachment was significantly enhanced on the surfaces of collagen membranes that were conjugated with fibronectin and type IV collagen. These findings define a set of design parameters that will enhance keratinocyte binding efficiency on the surface of collagen membranes and ultimately improve the rate of epithelialization for dermal equivalents.

  9. Keratinocyte differentiation is regulated by the Rho and ROCK signaling pathway.

    PubMed

    McMullan, Rachel; Lax, Siân; Robertson, Vicki H; Radford, David J; Broad, Simon; Watt, Fiona M; Rowles, Alison; Croft, Daniel R; Olson, Michael F; Hotchin, Neil A

    2003-12-16

    The epidermis comprises multiple layers of specialized epithelial cells called keratinocytes. As cells are lost from the outermost epidermal layers, they are replaced through terminal differentiation, in which keratinocytes of the basal layer cease proliferating, migrate upwards, and eventually reach the outermost cornified layers. Normal homeostasis of the epidermis requires that the balance between proliferation and differentiation be tightly regulated. The GTP binding protein RhoA plays a fundamental role in the regulation of the actin cytoskeleton and in the adhesion events that are critically important to normal tissue homeostasis. Two central mediators of the signals from RhoA are the ROCK serine/threonine kinases ROCK-I and ROCK-II. We have analyzed ROCK's role in the regulation of epidermal keratinocyte function by using a pharmacological inhibitor and expressing conditionally active or inactive forms of ROCK-II in primary human keratinocytes. We report that blocking ROCK function results in inhibition of keratinocyte terminal differentiation and an increase in cell proliferation. In contrast, activation of ROCK-II in keratinocytes results in cell cycle arrest and an increase in the expression of a number of genes associated with terminal differentiation. Thus, these results indicate that ROCK plays a critical role in regulating the balance between proliferation and differentiation in human keratinocytes.

  10. Dynamic and physical clustering of gene expression during epidermal barrier formation in differentiating keratinocytes.

    PubMed

    Taylor, Jennifer M; Street, Teresa L; Hao, Lizhong; Copley, Richard; Taylor, Martin S; Hayden, Patrick J; Stolper, Gina; Mott, Richard; Hein, Jotun; Moffatt, Miriam F; Cookson, William O C M

    2009-01-01

    The mammalian epidermis is a continually renewing structure that provides the interface between the organism and an innately hostile environment. The keratinocyte is its principal cell. Keratinocyte proteins form a physical epithelial barrier, protect against microbial damage, and prepare immune responses to danger. Epithelial immunity is disordered in many common diseases and disordered epithelial differentiation underlies many cancers. In order to identify the genes that mediate epithelial development we used a tissue model of the skin derived from primary human keratinocytes. We measured global gene expression in triplicate at five times over the ten days that the keratinocytes took to fully differentiate. We identified 1282 gene transcripts that significantly changed during differentiation (false discovery rate <0.01%). We robustly grouped these transcripts by K-means clustering into modules with distinct temporal expression patterns, shared regulatory motifs, and biological functions. We found a striking cluster of late expressed genes that form the structural and innate immune defences of the epithelial barrier. Gene Ontology analyses showed that undifferentiated keratinocytes were characterised by genes for motility and the adaptive immune response. We systematically identified calcium-binding genes, which may operate with the epidermal calcium gradient to control keratinocyte division during skin repair. The results provide multiple novel insights into keratinocyte biology, in particular providing a comprehensive list of known and previously unrecognised major components of the epidermal barrier. The findings provide a reference for subsequent understanding of how the barrier functions in health and disease. PMID:19888454

  11. Comparison of the enzymatic and explant methods for the culture of keratinocytes isolated from human foreskin

    PubMed Central

    ORAZIZADEH, MAHMOUD; HASHEMITABAR, MAHMOUD; BAHRAMZADEH, SOMAYEH; DEHBASHI, FRESHTEH NEJAD; SAREMY, SADEGH

    2015-01-01

    Currently, culture and growth keratinocytes are important stages in achieving a reliable and reproducible skin tissue. In the present study, two different methods, enzymatic and explant methods, for keratinocytes isolation from human foreskin were compared. Foreskins were cut into 2–3 mm pieces and placed in trypsin at 4°C overnight for separation of the epidermis from the dermis. Subsequently, these samples were divided into two groups: i) Keratinocytes separated from the epidermis by trypsin and ii) by the explant method. These keratinocytes were divided into two groups: i) With no feeder layer and ii) onto a type I collagen scaffold. The cells were evaluated using immunocytochemistry and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) staining. In the enzymatic treatment, after 7–10 days no attached cells were found in the cell culture dishes. In the explant method, keratinocytes were separated after ~24 h, attached rapidly and formed big colonies into a collagen scaffold. In the absence of a feeder layer, small colonies were developed with rapid loss of proliferation within 2–3 days. Keratinocytes showed positive immunoreactivity for the pan-cytokeratin marker and keratinocytes' nuclei were clearly observed. This method could be applied and developed as a component of skin substitutes to treat burns and wounds and also in laboratory testing. PMID:26137227

  12. The activation of PI 3-kinase/Akt pathway is involved in the acute effects of simvastatin against ischaemia and reperfusion-induced arrhythmias in anaesthetised dogs.

    PubMed

    Kisvári, Gábor; Kovács, Mária; Seprényi, György; Végh, Ágnes

    2015-12-15

    The objective of this study was to examine whether the PI3-kinase/Akt pathway is involved in the activation of endothelial nitric oxide synthase (eNOS) and in the subsequent increase of nitric oxide (NO) production that has been proved to play a role in the antiarrhythmic effect of acute simvastatin treatment in anaesthetised dogs, subjected to a 25min occlusion and reperfusion of the left anterior descending coronary artery. Using the same model, 12 dogs out of the 26 controls (given the solvent of simvastatin) and 11 dogs out of the 23 animals treated with intracoronary administered simvastatin (0.1mg/kg), were now received wortmannin (1.5mg/kg, ic.), a selective inhibitor of PI3-kinase. In another 13 dogs the effects of DMSO (0.1%), the vehicle of wortmannin, were examined. Compared to the controls, simvastatin markedly reduced the severity of ischaemia (epicardial ST-segment, inhomogeneity) and ventricular arrhythmias that were reversed (except the occlusion-induced ventricular fibrillation [VF; 50%, 0%, 0%]) by the administration of wortmannin. Thus in these groups there were 310±45, 62±14, 307±59 ectopic beats, 7.1±1.4, 0.3± 0.2, 4.3±1.3 tachycardiac episodes that occurred 93%, 17% and 73% of the dogs during occlusion, whereas survival following reperfusion was 0%, 67% and 0%, respectively. Simvastatin also increased the phosphorylation of eNOS and the plasma nitrate/nitrite levels, but reduced myocardial superoxide production on reperfusion. These effects of simvastatin were also abolished in the presence of wortmannin. We conclude that the NO-dependent antiarrhythmic effect of simvastatin involves the rapid activation of eNOS through the stimulation of the PI3-kinase/Akt pathway.

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

    PubMed

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

    2009-09-01

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

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

    SciTech Connect

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

    2009-02-01

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

  15. Differential effects of detergents on keratinocyte gene expression.

    PubMed

    van Ruissen, F; Le, M; Carroll, J M; van der Valk, P G; Schalkwijk, J

    1998-04-01

    We have studied the effect of various detergents on keratinocyte gene expression in vitro, using an anionic detergent (sodium dodecyl sulfate), a cationic detergent cetyltrimethylammoniumbromide (CTAB), and two nonionic detergents, Nonidet P-40 and Tween-20. We measured the effect of these detergents on direct cellular toxicity (lactate dehydrogenase release), on the expression of markers for normal differentiation (cytokeratin 1 and involucrin expression), and on disturbed keratinocyte differentiation (SKALP) by northern blot analysis. As reported in other studies, large differences were noted in direct cellular toxicity. In a culture model that mimics normal epidermal differentiation we found that low concentrations of sodium dodecyl sulfate could induce the expression of SKALP, a proteinase inhibitor that is not normally expressed in human epidermis but is found in hyperproliferative skin. Sodium dodecyl sulfate caused upregulation of involucrin and downregulation of cytokeratin 1 expression, which is associated with the hyperproliferative/inflammatory epidermal phenotype found in psoriasis, wound healing, and skin irritation. These changes were not induced after treatment of cultures with CTAB, Triton X-100, and Nonidet-P40. This effect appeared to be specific for the class of anionic detergents because sodium dodecyl benzene sulfonate and sodium laurate also induced SKALP expression. These in vitro findings showed only a partial correlation with the potential of different detergents to induce clinical, biophysical, and cell biologic changes in vivo in human skin. Both sodium dodecyl sulfate and CTAB were found to cause induction and upregulation of SKALP and involucrin at low doses following a 24 h patch test, whereas high concentrations of Triton X-100 did not. Sodium dodecyl sulfate induced higher rates of transepidermal water loss, whereas CTAB treated skin showed more signs of cellular toxicity. We conclude that the action of anionic detergents on

  16. Ultraviolet B irradiation increases endothelin-1 and endothelin receptor expression in cultured human keratinocytes.

    PubMed

    Tsuboi, R; Sato, C; Oshita, Y; Hama, H; Sakurai, T; Goto, K; Ogawa, H

    1995-09-01

    The effect of ultraviolet B (UVB) irradiation on endothelin-1 (ET-1) and ET receptor expression was examined using cultured normal human keratinocytes. Keratinocytes secreted ET-1 in the medium at a level of 2.1 pg/day/10(5) cells. UVB irradiation up to 10 mJ/cm2 increased ET-1 secretion 3-fold, and potentiated expression of mRNA for ET-1. Both ETA and ETB receptor mRNAs were detected in keratinocytes, and their expression was up-regulated by 5 mJ/cm2 UVB irradiation.

  17. Grape Cells (Multinucleated Keratinocytes) in Noninfectious Dermatoses: Case Series and Review of the Literature.

    PubMed

    Sweeney, Sarah A; Sulit, Daryl J; Adams, Erin G; Shvartsman, Katerina R; Rapini, Ronald P

    2015-12-01

    Multinucleated keratinocytes (also known as multinucleated epidermal giant cells) are a frequently overlooked histological finding in noninfectious inflammatory dermatoses. They are sometimes found in conditions characterized by chronic rubbing and pruritus, such as lichen simplex chronicus or prurigo nodularis, and may be a helpful clue in making the clinical diagnosis. This finding must be differentiated from other conditions characterized by multinucleated keratinocytes on histopathology, specifically herpes simplex, varicella zoster, or measles viral infections. The authors present a case series of 2 patients with unique clinical noninfectious diagnoses but similar histopathologic findings on biopsy. The histopathologic findings on both cases demonstrated multinucleated keratinocytes, which were related to manipulation of the epidermis.

  18. Grape Cells (Multinucleated Keratinocytes) in Noninfectious Dermatoses: Case Series and Review of the Literature

    PubMed Central

    Sulit, Daryl J.; Adams, Erin G.; Shvartsman, Katerina R.; Rapini, Ronald P.

    2015-01-01

    Abstract: Multinucleated keratinocytes (also known as multinucleated epidermal giant cells) are a frequently overlooked histological finding in noninfectious inflammatory dermatoses. They are sometimes found in conditions characterized by chronic rubbing and pruritus, such as lichen simplex chronicus or prurigo nodularis, and may be a helpful clue in making the clinical diagnosis. This finding must be differentiated from other conditions characterized by multinucleated keratinocytes on histopathology, specifically herpes simplex, varicella zoster, or measles viral infections. The authors present a case series of 2 patients with unique clinical noninfectious diagnoses but similar histopathologic findings on biopsy. The histopathologic findings on both cases demonstrated multinucleated keratinocytes, which were related to manipulation of the epidermis. PMID:26588345

  19. Mathematical model for wound healing following autologous keratinocyte transplantation.

    PubMed

    Renner, Regina; Teuwen, Isabell; Gebhardt, Carl; Simon, Jan C

    2008-06-01

    In times of increasing economical pressure on the health care systems, it is important to optimise the outpatient treatment of chronic wounds. Another aim of wound healing research is to discover agents to accelerate healing. Wound healing trajectories or healing velocities can provide information to demonstrate the endpoints for wound healing. A great problem in clinical trials is to specify these parameters. Therefore, we developed a mathematical model for more transparency. In this initial project, we observed 19 wounds to construct the wound healing trajectories after transplantation of autologous keratinocytes, and the results are so encouraging that investigation in this area will continue. The developed mathematical model describes the clinical observed healing process. It was possible to find parameters to distinguish between old and young patients, retrospectively or prospectively calculate the healing rates and to determine exactly the endpoint of healing. Therefore, our model might be very useful in practices or for studies.

  20. Effects of ozone in normal human epidermal keratinocytes.

    PubMed

    McCarthy, James T; Pelle, Edward; Dong, Kelly; Brahmbhatt, Krupa; Yarosh, Dan; Pernodet, Nadine

    2013-05-01

    Ozone is a tropospheric pollutant that can form at ground level as a result of an interaction between sunlight and hydrocarbon engine emissions. As ozone is an extremely oxidative reaction product, epidermal cells are in the outer layer of defense against ozone. We exposed normal human epidermal keratinocytes (NHEK) to concentrations of ozone that have been measured in cities and assayed for its effects. Hydrogen peroxide and IL-1α levels both increased while ATP levels decreased. We found a decrease in the NAD-dependent histone deacetylase, sirtuin 3. Lastly, we found that ozone increased DNA damage as evaluated by Comet assay. Taken together, our results show increased damage to NHEK that will ultimately impair normal cellular function as a result of an environmentally relevant ozone exposure.

  1. Human keratinocytes have two interconvertible modes of proliferation.

    PubMed

    Roshan, Amit; Murai, Kasumi; Fowler, Joanna; Simons, Benjamin D; Nikolaidou-Neokosmidou, Varvara; Jones, Philip H

    2016-02-01

    Single stem cells, including those in human epidermis, have a remarkable ability to reconstitute tissues in vitro, but the cellular mechanisms that enable this are ill-defined. Here we used live imaging to track the outcome of thousands of divisions in clonal cultures of primary human epidermal keratinocytes. Two modes of proliferation were seen. In 'balanced' mode, similar proportions of proliferating and differentiating cells were generated, achieving the 'population asymmetry' that sustains epidermal homeostasis in vivo. In 'expanding' mode, an excess of cycling cells was produced, generating large expanding colonies. Cells in expanding mode switched their behaviour to balanced mode once local confluence was attained. However, when a confluent area was wounded in a scratch assay, cells near the scratch switched back to expanding mode until the defect was closed. We conclude that the ability of a single epidermal stem cell to reconstitute an epithelium is explained by two interconvertible modes of proliferation regulated by confluence. PMID:26641719

  2. Human keratinocytes have two interconvertible modes of proliferation.

    PubMed

    Roshan, Amit; Murai, Kasumi; Fowler, Joanna; Simons, Benjamin D; Nikolaidou-Neokosmidou, Varvara; Jones, Philip H

    2016-02-01

    Single stem cells, including those in human epidermis, have a remarkable ability to reconstitute tissues in vitro, but the cellular mechanisms that enable this are ill-defined. Here we used live imaging to track the outcome of thousands of divisions in clonal cultures of primary human epidermal keratinocytes. Two modes of proliferation were seen. In 'balanced' mode, similar proportions of proliferating and differentiating cells were generated, achieving the 'population asymmetry' that sustains epidermal homeostasis in vivo. In 'expanding' mode, an excess of cycling cells was produced, generating large expanding colonies. Cells in expanding mode switched their behaviour to balanced mode once local confluence was attained. However, when a confluent area was wounded in a scratch assay, cells near the scratch switched back to expanding mode until the defect was closed. We conclude that the ability of a single epidermal stem cell to reconstitute an epithelium is explained by two interconvertible modes of proliferation regulated by confluence.

  3. Ratite oils promote keratinocyte cell growth and inhibit leukocyte activation

    PubMed Central

    Bennett, Darin C.; Leung, Gigi; Wang, Eddy; Ma, Sam; Lo, Blanche K. K.; McElwee, Kevin J.; Cheng, Kimberly M.

    2015-01-01

    Traditionally, native Australian aborigines have used emu oil for the treatment of inflammation and to accelerate wound healing. Studies on mice suggest that topically applied emu oil may have anti-inflammatory properties and may promote wound healing. We investigated the effects of ratite oils (6 emu, 3 ostrich, 1 rhea) on immortalized human keratinocytes (HaCaT cells) in vitro by culturing the cells in media with oil concentrations of 0%, 0.5%, and 1.0%. Peking duck, tea tree, and olive oils were used as comparative controls. The same oils at 0.5% concentration were evaluated for their influence on peripheral blood mononuclear cell (PBMC) survival over 48 hr and their ability to inhibit IFNγ production in PBMCs activated by phytohemagglutinin (PHA) in ELISpot assays. Compared to no oil control, significantly shorter population doubling time durations were observed for HaCaT cells cultured in emu oil (1.51 × faster), ostrich oil (1.46 × faster), and rhea oil (1.64 × faster). Tea tree oil demonstrated significant antiproliferative activity and olive oil significantly prolonged (1.35 × slower) cell population doubling time. In contrast, almost all oils, particularly tea tree oil, significantly reduced PBMC viability. Different oils had different levels of inhibitory effect on IFNγ production with individual emu, ostrich, rhea, and duck oil samples conferring full inhibition. This preliminary investigation suggests that emu oil might promote wound healing by accelerating the growth rate of keratinocytes. Combined with anti-inflammatory properties, ratite oil may serve as a useful component in bandages and ointments for the treatment of wounds and inflammatory skin conditions. PMID:26217022

  4. Ultraviolet radiation induction of ornithine decarboxylase in rat keratinocytes

    SciTech Connect

    Rosen, C.F.; Gajic, D.; Drucker, D.J. )

    1990-05-01

    UV radiation plays an important role in the induction of cutaneous malignancy, including basal cell and squamous cell carcinomas and malignant melanoma. In addition to its effects on DNA damage and repair mechanisms, UV radiation has been shown to modulate the expression of specific genes, altering the levels of their mRNAs and the synthesis of their corresponding proteins. In order to gain further information about the molecular effects of UV radiation, we have studied the regulation of ornithine decarboxylase (ODC) gene expression in response to UVB radiation. ODC is the rate-limiting enzyme in polyamine biosynthesis, is involved in growth and differentiation, and has been implicated in carcinogenesis. Keratinocytes grown in culture were either sham-irradiated or exposed to increasing doses of UVB (1-5 mJ/cm2). Northern blot analysis of keratinocyte RNA under basal conditions demonstrated the presence of two ODC mRNA transcripts. Increasing exposure to UVB resulted in a dose-dependent increase in the levels of both ODC mRNA transcripts. The induction of ODC gene expression following UVB was noted 2 h after UVB exposure, and ODC mRNA levels continued to increase up to 24 h after UVB exposure. The UVB-induced increase in ODC gene expression was not serum dependent, despite the ability of serum alone to induce ODC gene expression. The mRNA transcripts for actin and hexosaminidase A were not induced after UVB exposure. These studies show that the UVB-induced increase in ODC activity is due, at least in part, to an increase in ODC gene expression and they provide a useful model for the analysis of the molecular effects of UVB radiation.

  5. Ratite oils promote keratinocyte cell growth and inhibit leukocyte activation.

    PubMed

    Bennett, Darin C; Leung, Gigi; Wang, Eddy; Ma, Sam; Lo, Blanche K K; McElwee, Kevin J; Cheng, Kimberly M

    2015-09-01

    Traditionally, native Australian aborigines have used emu oil for the treatment of inflammation and to accelerate wound healing. Studies on mice suggest that topically applied emu oil may have anti-inflammatory properties and may promote wound healing. We investigated the effects of ratite oils (6 emu, 3 ostrich, 1 rhea) on immortalized human keratinocytes (HaCaT cells) in vitro by culturing the cells in media with oil concentrations of 0%, 0.5%, and 1.0%. Peking duck, tea tree, and olive oils were used as comparative controls. The same oils at 0.5% concentration were evaluated for their influence on peripheral blood mononuclear cell (PBMC) survival over 48 hr and their ability to inhibit IFNγ production in PBMCs activated by phytohemagglutinin (PHA) in ELISpot assays. Compared to no oil control, significantly shorter population doubling time durations were observed for HaCaT cells cultured in emu oil (1.51×faster), ostrich oil (1.46×faster), and rhea oil (1.64×faster). Tea tree oil demonstrated significant antiproliferative activity and olive oil significantly prolonged (1.35×slower) cell population doubling time. In contrast, almost all oils, particularly tea tree oil, significantly reduced PBMC viability. Different oils had different levels of inhibitory effect on IFNγ production with individual emu, ostrich, rhea, and duck oil samples conferring full inhibition. This preliminary investigation suggests that emu oil might promote wound healing by accelerating the growth rate of keratinocytes. Combined with anti-inflammatory properties, ratite oil may serve as a useful component in bandages and ointments for the treatment of wounds and inflammatory skin conditions. PMID:26217022

  6. Regulation of p53 during senescence in normal human keratinocytes

    PubMed Central

    Kim, Reuben H; Kang, Mo K; Kim, Terresa; Yang, Paul; Bae, Susan; Williams, Drake W; Phung, Samantha; Shin, Ki-Hyuk; Hong, Christine; Park, No-Hee

    2015-01-01

    p53, the guardian of the genome, is a tumor suppressor protein and critical for the genomic integrity of the cells. Many studies have shown that intracellular level of p53 is enhanced during replicative senescence in normal fibroblasts, and the enhanced level of p53 is viewed as the cause of senescence. Here, we report that, unlike in normal fibroblasts, the level of intracellular p53 reduces during replicative senescence and oncogene-induced senescence (OIS) in normal human keratinocytes (NHKs). We found that the intracellular p53 level was also decreased in age-dependent manner in normal human epithelial tissues. Senescent NHKs exhibited an enhanced level of p16INK4A, induced G2 cell cycle arrest, and lowered the p53 expression and transactivation activity. We found that low level of p53 in senescent NHKs was due to reduced transcription of p53. The methylation status at the p53 promoter was not altered during senescence, but senescent NHKs exhibited notably lower level of acetylated histone 3 (H3) at the p53 promoter in comparison with rapidly proliferating cells. Moreover, p53 knockdown in rapidly proliferating NHKs resulted in the disruption of fidelity in repaired DNA. Taken together, our study demonstrates that p53 level is diminished during replicative senescence and OIS and that such diminution is associated with H3 deacetylation at the p53 promoter. The reduced intracellular p53 level in keratinocytes of the elderly could be a contributing factor for more frequent development of epithelial cancer in the elderly because of the loss of genomic integrity of cells. PMID:26138448

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

    PubMed

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

    2006-09-01

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

  8. Dihydrotestosterone induces SREBP-1 expression and lipogenesis through the phosphoinositide 3-kinase/Akt pathway in HaCaT cells

    PubMed Central

    2012-01-01

    Background The purpose of this study was to investigate the effects and mechanisms of dihydrotestosterone (DHT)-induced expression of sterol regulatory element binding protein-1 (SREBP-1), and the synthesis and secretion of lipids, in HaCaT cells. HaCaT cells were treated with DHT and either the phosphoinositide 3-kinase inhibitor LY294002 or the extracellular-signal-regulated kinase (ERK) inhibitor PD98059. Real time-PCR, Western blot, Oil Red staining and flow cytometry were employed to examine the mRNA and protein expressions of SREBP-1, the gene transcription of lipid synthesis, and lipid secretion in HaCaT cells. Findings We found that DHT upregulated mRNA and protein expressions of SREBP-1. DHT also significantly upregulated the transcription of lipid synthesis-related genes and increased lipid secretion, which can be inhibited by the addition of LY294002. Conclusions Collectively, these results indicate that DHT induces SREBP-1 expression and lipogenesis in HaCaT cells via activation of the phosphoinositide 3-kinase/Akt Pathway. PMID:23153363

  9. Dysfunction of the PI3 kinase/Rap1/integrin α(IIb)β(3) pathway underlies ex vivo platelet hypoactivity in essential thrombocythemia.

    PubMed

    Moore, Samantha F; Hunter, Roger W; Harper, Matthew T; Savage, Joshua S; Siddiq, Samreen; Westbury, Sarah K; Poole, Alastair W; Mumford, Andrew D; Hers, Ingeborg

    2013-02-14

    Patients with myeloproliferative disorders (MPDs), such as essential thrombocythemia (ET) have increased risk of thrombosis and bleeding, which are major sources of morbidity and mortality. Most MPD patients have a gain of function mutation in Janus kinase 2 (JAK2V617F), but little is known how JAK2V617F affects platelet function. Here, we demonstrate that platelets from ET patients have impaired SFLLRN-mediated fibrinogen binding and have lost the potentiating effect of thrombopoietin (which couples to JAK2) on this pathway. In contrast, SFLLRN-mediated P-selectin expression, ATP secretion, phosphorylation of the PKC substrate pleckstrin, and Ca(2+) mobilization were unaffected in JAK2V617F positive platelets. In addition, thrombopoietin-mediated JAK2 phosphorylation was unchanged, suggesting that signaling pathways activated downstream of JAK2 are impaired. Indeed, we found that platelets from JAK2V617F positive ET patients have significantly reduced phosphorylation of the PI3 kinase substrate Akt, and have reduced activation of Rap1 in response to thrombopoietin, IGF-1,ADP, SFLLRN, and thrombin. This effect was independent of Giα P2Y12 purinergic receptor function as ADP-mediated inhibition of VASP phosphorylation was unchanged. These results demonstrate that the PI3 kinase/Rap1 pathway is intrinsically impaired in platelets from JAK2V617F-positive ET patients, resulting in diminished thrombin and thrombopoietin-mediated integrin α(IIb)β(3) activation. PMID:23243278

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

    PubMed

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

    2013-01-01

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

  11. Transcriptional regulation of human mucin MUC4 by bile acids in oesophageal cancer cells is promoter-dependent and involves activation of the phosphatidylinositol 3-kinase signalling pathway.

    PubMed Central

    Mariette, Christophe; Perrais, Michaël; Leteurtre, Emmanuelle; Jonckheere, Nicolas; Hémon, Brigitte; Pigny, Pascal; Batra, Surinder; Aubert, Jean-Pierre; Triboulet, Jean-Pierre; Van Seuningen, Isabelle

    2004-01-01

    Abnormal gastro-oesophageal reflux and bile acids have been linked to the presence of Barrett's oesophageal premalignant lesion associated with an increase in mucin-producing goblet cells and MUC4 mucin gene overexpression. However, the molecular mechanisms underlying the regulation of MUC4 by bile acids are unknown. Since total bile is a complex mixture, we undertook to identify which bile acids are responsible for MUC4 up-regulation by using a wide panel of bile acids and their conjugates. MUC4 apomucin expression was studied by immunohistochemistry both in patient biopsies and OE33 oesophageal cancer cell line. MUC4 mRNA levels and promoter regulation were studied by reverse transcriptase-PCR and transient transfection assays respectively. We show that among the bile acids tested, taurocholic, taurodeoxycholic, taurochenodeoxycholic and glycocholic acids and sodium glycocholate are strong activators of MUC4 expression and that this regulation occurs at the transcriptional level. By using specific pharmacological inhibitors of mitogen-activated protein kinase, phosphatidylinositol 3-kinase, protein kinase A and protein kinase C, we demonstrate that bile acid-mediated up-regulation of MUC4 is promoter-specific and mainly involves activation of phosphatidylinositol 3-kinase. This new mechanism of regulation of MUC4 mucin gene points out an important role for bile acids as key molecules in targeting MUC4 overexpression in early stages of oesophageal carcinogenesis. PMID:14583090

  12. ADP-ribosylation factor 1 controls the activation of the phosphatidylinositol 3-kinase pathway to regulate epidermal growth factor-dependent growth and migration of breast cancer cells.

    PubMed

    Boulay, Pierre-Luc; Cotton, Mathieu; Melançon, Paul; Claing, Audrey

    2008-12-26

    Activation of intracellular signaling pathways by growth factors is one of the major causes of cancer development and progression. Recent studies have demonstrated that monomeric G proteins of the Ras family are key regulators of cell proliferation, migration, and invasion. Using an invasive breast cancer cell lines, we demonstrate that the ADP-ribosylation factor 1 (ARF1), a small GTPase classically associated with the Golgi, is an important regulator of the biological effects induced by epidermal growth factor. Here, we show that this ARF isoform is activated following epidermal growth factor stimulation and that, in MDA-MB-231 cells, ARF1 is found in dynamic plasma membrane ruffles. Inhibition of endogenous ARF1 expression results in the inhibition of breast cancer cell migration and proliferation. The underlying mechanism involves the activation of the phosphatidylinositol 3-kinase pathway. Our data demonstrate that depletion of ARF1 markedly impairs the recruitment of the phosphatidylinositol 3-kinase catalytic subunit (p110alpha) to the plasma membrane, and the association of the regulatory subunit (p85alpha) to the activated receptor. These results uncover a novel molecular mechanism by which ARF1 regulates breast cancer cell growth and invasion during cancer progression.

  13. H(+)/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport.

    PubMed

    Kudo, Michiko; Katayoshi, Takeshi; Kobayashi-Nakamura, Kumiko; Akagawa, Mitsugu; Tsuji-Naito, Kentaro

    2016-07-01

    Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalization analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H(+) gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake. PMID:27216463

  14. Single Low-Dose Radiation Induced Regulation of Keratinocyte Differentiation in Calcium-Induced HaCaT Cells

    PubMed Central

    Hahn, Hyung Jin; Youn, Hae Jeong; Cha, Hwa Jun; Kim, Karam; An, Sungkwan

    2016-01-01

    Background We are continually exposed to low-dose radiation (LDR) in the range 0.1 Gy from natural sources, medical devices, nuclear energy plants, and other industrial sources of ionizing radiation. There are three models for the biological mechanism of LDR: the linear no-threshold model, the hormetic model, and the threshold model. Objective We used keratinocytes as a model system to investigate the molecular genetic effects of LDR on epidermal cell differentiation. Methods To identify keratinocyte differentiation, we performed western blots using a specific antibody for involucrin, which is a precursor protein of the keratinocyte cornified envelope and a marker for keratinocyte terminal differentiation. We also performed quantitative polymerase chain reaction. We examined whether LDR induces changes in involucrin messenger RNA (mRNA) and protein levels in calcium-induced keratinocyte differentiation. Results Exposure of HaCaT cells to LDR (0.1 Gy) induced p21 expression. p21 is a key regulator that induces growth arrest and represses stemness, which accelerates keratinocyte differentiation. We correlated involucrin expression with keratinocyte differentiation, and examined the effects of LDR on involucrin levels and keratinocyte development. LDR significantly increased involucrin mRNA and protein levels during calcium-induced keratinocyte differentiation. Conclusion These studies provide new evidence for the biological role of LDR, and identify the potential to utilize LDR to regulate or induce keratinocyte differentiation. PMID:27489424

  15. Differential behaviors toward ultraviolet A and B radiation of fibroblasts and keratinocytes from normal and DNA-repair-deficient patients.

    PubMed

    Otto, A I; Riou, L; Marionnet, C; Mori, T; Sarasin, A; Magnaldo, T

    1999-03-15

    Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are rare genodermatoses transmitted as recessive and autosomal traits that result in reduced capacity to repair UV-induced DNA lesions. Although XP, but not TTD, patients are prone to basal and squamous cell carcinomas, to date no comparative studies of the XP and TTD phenotypes have included epidermal keratinocytes. We compared the DNA repair capacity (by unscheduled DNA synthesis) and cell survival (by clonal analysis) of epidermal keratinocytes and dermal fibroblasts grown from normal individuals and patients with xeroderma pigmentosum and trichothiodystrophy following UVA and UVB irradiation. The same dose of UVB (1000 J/m2) induced twice as many DNA lesions in normal fibroblasts as in normal keratinocytes. UV survival rates were always higher in keratinocytes than in fibroblasts. Normal and TTD keratinocytes survived better following UVA and UVB irradiation than XP-C and XP-D keratinocytes. XP-C keratinocytes exhibited exacerbated sensitivity toward UVA radiation. Unscheduled DNA synthesis at UV doses leading to 50% cell survival indicated that the ratio of DNA repair capacity to cell survival is higher in keratinocytes than in fibroblasts. In addition, UVA and UVB irradiation induced a transition from proliferative to abortive keratinocyte colonies. This transition varied between donors and was in part correlated with their cancer susceptibility. Altogether these data provide the first evidence of the differential behaviors of normal, XP, and TTD keratinocytes toward UV radiation. PMID:10096550

  16. Failure of extracts from Malassezia pachydermatis to stimulate canine keratinocyte proliferation in vitro.

    PubMed

    Chen, Tai-An; Halliwell, Richard E W; Hill, Peter B

    2002-12-01

    Epidermal hyperplasia is one of the major histopathological features seen in dogs with Malassezia dermatitis. The aim of this study was to investigate the effects of extracts and culture supernatants from Malassezia pachydermatis on the proliferation of canine keratinocytes. Keratinocyte cultures were established from normal dog skin, and cell monolayers were co-cultured with Malassezia extracts (prepared either with or without protease inhibitors) and supernatants derived from organisms grown in liquid culture. The proliferation of keratinocytes was measured using a colourimetric assay. Neither the culture supernatants nor the Malassezia extracts had significant effects on the proliferation rate of canine keratinocytes, regardless of whether protease inhibitors were present or not. The results indicate that the epidermal hyperplasia seen in Malassezia dermatitis is unlikely to be caused directly by secretion of products from the organism. PMID:12464065

  17. Expression of human beta defensin 4 in genetically modified keratinocytes enhances antimicrobial activity.

    PubMed

    Smiley, Andrea K; Gardner, Jason; Klingenberg, Jennifer M; Neely, Alice N; Supp, Dorothy M

    2007-01-01

    Defensins are cationic peptides of the innate host defense system with antimicrobial activity against many of the microorganisms commonly found in burn units. Beta defensins are variably expressed in the epithelia of skin and other organs. Human beta defensin 4 reportedly has antimicrobial activity against Pseudomonas aeruginosa and is not normally expressed in intact skin. Genetic modification was used to ectopically express human beta defensin 4 in cultured primary epidermal keratinocytes. Keratinocytes expressing human beta defensin 4 showed significantly elevated antimicrobial activity against clinically-isolated P. aeruginosa compared with controls. These results suggest that genetic modification of keratinocytes can increase their resistance to microbial contamination. Bioengineered skin replacements containing human beta defensin 4-modified keratinocytes may be useful for transplantation to contaminated burn wounds.

  18. Expression of membrane glycoproteins in normal keratinocytes and squamous carcinoma cell lines

    SciTech Connect

    Rayter, Z. ); McIlhinney, R. ); Gusterson, B. )

    1989-08-01

    Con A acceptor glycoproteins were analyzed by 2D-PAGE and {sup 125}I-Con A overlay in three squamous carcinoma cell lines and compared with those in the simian virus (SV40)-transformed keratinocyte cell line SVK-14 and in normal keratinocytes. The majority of the glycoproteins identified by this technique were expressed at similar levels in all of the cells examined, independent of the culture conditions used. A cell surface glycoprotein gp34 was increased in the tumor cells compared with normal keratinocytes and expression varied with the culture density. Another glycoprotein, gp21, was found to be increased in expression in normal keratinocytes and stratified hyperconfluent cultures of squamous carcinoma cell lines. This paper describes the potential of this technique to identify membrane glycoproteins which may be expressed as a function of proliferation or differentiation.

  19. Human epidermal keratinocyte cell response on integrin-specific artificial extracellular matrix proteins.

    PubMed

    Tjin, Monica Suryana; Chua, Alvin Wen Choong; Ma, Dong Rui; Lee, Seng Teik; Fong, Eileen

    2014-08-01

    Cell-matrix interactions play critical roles in regulating cellular behavior in wound repair and regeneration of the human skin. In particular, human skin keratinocytes express several key integrins such as alpha5beta1, alpha3beta1, and alpha2beta1 for binding to the extracellular matrix (ECM) present in the basement membrane in uninjured skin. To mimic these key integrin-ECM interactions, artificial ECM (aECM) proteins containing functional domains derived from laminin 5, type IV collagen, fibronectin, and elastin are prepared. Human skin keratinocyte cell responses on the aECM proteins are specific to the cell-binding domain present in each construct. Keratinocyte attachment to the aECM protein substrates is also mediated by specific integrin-material interactions. In addition, the aECM proteins are able to support the proliferation of keratinocyte stem cells, demonstrating their promise for use in skin tissue engineering.

  20. Retinol and retinal metabolism. Relationship to the state of differentiation of cultured human keratinocytes.

    PubMed Central

    Siegenthaler, G; Saurat, J H; Ponec, M

    1990-01-01

    Cultured keratinocytes offer an attractive model for studying the metabolism of retinol in relation to cell differentiation, since the extent of keratinocyte differentiation can be modulated experimentally. The metabolism of retinol and retinal was studied in cytosol fractions prepared from two distinct keratinocyte populations, differentiating and non-differentiated. The enzymic activities were analysed using physiological concentrations of [3H]retinol and [3H]retinal in the presence of cofactors. The products formed were quantified by h.p.l.c. In the population of differentiating keratinocytes, the formation of retinoic acid from retinol occurred at a rate of 4.49 +/- 0.17 pmol/h per mg of protein, but no such conversion was observed in the population of non-differentiated cells. However, when retinal was used as substrate, retinoic acid was formed in both cell populations, at rates of 14.4 pmol/h per mg of protein in non-differentiated and 51.6 pmol/h per mg of protein in differentiating keratinocytes. Using PAGE/radiobinding assay, we demonstrated that retinoic acid formed from retinol was bound in differentiating keratinocytes to endogenous cellular retinoic acid-binding protein (CRABP). Furthermore, retinal was reduced to retinol in the presence of NADH in both differentiating and non-differentiated keratinocytes at a similar rate (8 pmol/h per mg of protein). Although retinal could not be detected under physiological conditions, it was found in significant amounts at pH 8.5-9, which is optimal for enzymic activity. This indicates that in keratinocytes retinal is an intermediate metabolite in retinoic acid formation from retinol. The enzymes catalysing the conversion of retinol into retinoic acid were found to differ from other alcohol and aldehyde dehydrogenases, since the formation of retinoic acid was not significantly affected by specific inhibitors of alcohol metabolism, such as 4-methylpyrazole and disulfiram. Moreover, the cytosol of non

  1. Keratinocytes derived from psoriatic plaques are resistant to apoptosis compared with normal skin.

    PubMed Central

    Wrone-Smith, T.; Mitra, R. S.; Thompson, C. B.; Jasty, R.; Castle, V. P.; Nickoloff, B. J.

    1997-01-01

    Previously we observed that hyperplastic epidermal keratinocytes characteristic of psoriasis had abundant amounts of the cell survival protein Bcl-xL; however, whether this overexpression correlated with enhanced survival was unclear because the majority of epidermal cells possess nuclei that are positively labeled by an assay typically regarded as indicative of cells undergoing apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining). To clarify this apparent discrepancy, we explored the propensity of keratinocytes derived from psoriatic plaques to undergo apoptosis and also determined the reliability of TUNEL staining as an indicator of apoptosis in keratinocytes in vitro and in vivo. First, a keratinocyte cell line, HaCat, was examined before and after being suspended in semisolid medium (methylcellulose) using flow cytometry to detect TUNEL-positive cells, and the percentage of positive cells was correlated to the presence or absence of double-stranded DNA fragmentation using pulsed field gel electrophoresis. After 18 hours in methylcellulose suspension, apoptosis was detected in HaCat cells when at least 5% of the cell population was undergoing programmed cell death. Second, we examined 23 clinical specimens of skin (13 from psoriatic patients and 10 from healthy control subjects) and observed that no double-stranded DNA fragmentation was present in any of the freshly isolated keratinocytes from either normal or psoriatic patients. Keratinocytes from 9 of 12 normal skin samples underwent double-stranded DNA fragmentation after being in methylcellulose for 18 to 24 hours, which contrasts with keratinocytes from lesions of psoriasis where only 1 of 13 of the skin samples had these changes. Third, two-color immunofluorescence staining of psoriatic plaques revealed that numerous TUNEL-positive keratinocytes were also positive for proliferating cell nuclear antigen and Ki-67 antigens and that by flow cytometry TUNEL

  2. Fetal fibroblasts and keratinocytes with immunosuppressive properties for allogeneic cell-based wound therapy.

    PubMed

    Zuliani, Thomas; Saiagh, Soraya; Knol, Anne-Chantal; Esbelin, Julie; Dréno, Brigitte

    2013-01-01

    Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with in vitro scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO) activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the in vitro scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors.

  3. Effects of epidermal Langerhans cell's conditioned medium on keratinocytes: a role of Langerhans cells in cholesteatoma.

    PubMed

    Kamide, Y; Sasaki, H; Abramson, M; Huang, C C

    1991-01-01

    Langerhans cells (LCs) are known to play an important role in the immunosurveillance system. In this study, as in others, numerous LCs were detected in the epithelial layer of acquired cholesteatoma by immunohistochemical staining. This finding suggests that cell-mediated immune responses are initiated by LCs in cholesteatoma; however, documentation concerning the microenvironment of LCs-keratinocytes in cholesteatoma is limited. Therefore, we investigated the effects of LCs on keratinocytes in vitro. To study these effects it was necessary to isolate and purify LCs. Our present study revealed that good enrichment and a high degree of purity (95%) of LCs could be obtained from neonatal rat skin using the immunomagnetic beads (Dynabeads M-450) sorting technique. These isolated LCs have the biologic activity of LCs, and Langerhans cells' conditioned medium (LCCM) stimulates DNA synthesis in thymocytes. The effect of LCCM on keratinocytes was then studied. We found that (1) LCCM stimulated DNA synthesis in keratinocytes was then studied. We found that (1) LCCM stimulated DNA synthesis in keratinocytes, but not protein synthesis, and (2) LCCM stimulated the incorporation of 3H-putrescine into keratinocytes by the activation of transglutaminase. Transglutaminase is a known marker of terminal differentiation in keratinocytes. By Western blot analysis, we identified a 17-kd immunoreactive mouse interleukin-1 alpha in LCCM. Our results imply that LCs found in cholesteatoma tissue may play an important role in stimulating both hyper-proliferation and cornification of keratinocytes; two characteristic features of cholesteatoma formation. These stimulatory effects may be due to the release of interleukin-1 or other factors by LCs.

  4. Development of an inducible gene expression system for primary murine keratinocytes

    PubMed Central

    Nagarajan, Priyadharsini

    2008-01-01

    Background The tetracycline (Tet) responsive system is a valuable tool that is routinely used in a wide variety of mammalian cells for regulatable expression of gene products. However, technical difficulties such as harsh selection conditions and extensive screening processes to identify suitably responsive clones limit the generation of stable cell lines. Hence, application of this system in mammalian cells with relatively slow growth rates and / or the capacity to undergo terminal differentiation such as primary mouse keratinocytes is particularly challenging. Objective To our knowledge, no Tet-responsive stable cell lines have been generated from mouse keratinocytes, presumably due to their sensitivity to selection conditions. Our goal was to utilize a modified and robust Tet-expression system to generate a stable primary mouse keratinocyte cell line. These cells could be then utilized for conditional expression of potentially toxic proteins in an inducible fashion. Methods We utilized a eukaryotic promoter instead of a viral promoter to express a modified reverse tetracycline transactivator in mouse keratinocytes and optimized the selection process for generating stable cell lines. Results Here, we report the generation of a stable mouse keratinocyte cell line for Tet-regulated gene expression with minimal leakiness and high degree of Tet responsivity. This mouse keratinocyte cell line was further engineered for generation of a double stable cell line, which expresses the transcription factor AP-2α in an inducible manner. Importantly, the selected cells retain their inherent keratinocyte morphology, respond to differentiation signals and exhibit a persistent and highly tunable Tet inducibility upon continuous culturing. Conclusion We have generated a tetracycline inducible gene expression model system in mouse epidermal keratinocytes. Such inducible cell lines will serve as valuable in vitro models for future gain-of-function and loss-of-function studies. PMID

  5. In vitro investigations on the effect of dermal fibroblasts on keratinocyte responses to ultraviolet B radiation.

    PubMed

    Fernandez, Tara L; Van Lonkhuyzen, Derek R; Dawson, Rebecca A; Kimlin, Michael G; Upton, Zee

    2014-01-01

    Exposure to ultraviolet radiation is closely linked to the development of skin cancers in humans. The ultraviolet B (UVB) radiation wavelength (280-320 nm), in particular, causes DNA damage in epidermal keratinocytes, which are linked to the generation of signature premalignant mutations. Interactions between dermal fibroblasts and keratinocytes play a role in epidermal repair and regeneration after UVB-induced damage. To investigate these processes, established two and three-dimensional culture models were utilized to study the impact of fibroblast-keratinocyte crosstalk during the acute UVB response. Using a coculture system it was observed that fibroblasts enhanced keratinocyte survival and the repair of cyclobutane pyrimidine dimers (CPDs) after UVB radiation exposure. These findings were also mirrored in irradiated human skin coculture models employed in this study. Fibroblast coculture was shown to play a role in the expression and activation of members of the apoptotic cascade, including caspase-3 and Bad. Interestingly, the expression and phosphorylation of p53, a key player in the regulation of keratinocyte cell fate postirradiation, was also shown to be influenced by fibroblast-produced factors. This study highlights the importance of synergistic interactions between fibroblasts and keratinocytes in maintaining a functional epidermis while promoting repair and regeneration following UVB radiation-induced damage.

  6. Alpha actinin-1 regulates cell-matrix adhesion organization in keratinocytes: consequences for skin cell motility.

    PubMed

    Hamill, Kevin J; Hiroyasu, Sho; Colburn, Zachary T; Ventrella, Rosa V; Hopkinson, Susan B; Skalli, Omar; Jones, Jonathan C R

    2015-04-01

    The migration of keratinocytes in wound healing requires coordinated activities of the motility machinery of a cell, the cytoskeleton, and matrix adhesions. In this study, we assessed the role of alpha actinin-1 (ACTN1), one of the two alpha actinin isoforms expressed in keratinocytes, in skin cell migration via a small hairpin RNA-mediated knockdown approach. Keratinocytes deficient in ACTN1 exhibit changes in their actin cytoskeleton organization, a loss in front-rear polarity, and impaired lamellipodial dynamics. They also display aberrant directed motility and move slower compared with their wild-type counterparts. Moreover, they have abnormally arranged matrix adhesion sites. Specifically, the focal adhesions in ACTN1 knockdown keratinocytes are not organized as distinct entities. Rather, focal adhesion proteins are arranged in a circle subjacent to cortical fibers of actin. In the same cells, hemidesmosome proteins arrange in cat paw patterns, more typical of confluent, stationary cells, and β4 integrin dynamics are reduced in knockdown cells compared with control keratinocytes. In summary, our data suggest a mechanism by which ACTN1 determines the motility of keratinocytes by regulating the organization of the actin cytoskeleton, focal adhesion, and hemidesmosome proteins complexes, thereby modulating cell speed, lamellipodial dynamics, and directed migration. PMID:25431851

  7. Endothelin-1 of keratinocyte origin is a mediator of melanocyte dendricity.

    PubMed

    Hara, M; Yaar, M; Gilchrest, B A

    1995-12-01

    Melanocytes synthesize melanin and transfer it to keratinocytes via dendritic processes. Keratinocytes are known to produce constitutively several factors, including endothelin-1 (ET-1), that together affect melanocyte proliferation, migration, melanogenesis, and dendrite formation. After ultraviolet (UV) irradiation, synthesis and secretion of ET-1 are up-regulated in keratinocytes. Because UV irradiation of skin is known to be associated with increased melanocyte dendricity, and because medium conditioned by UV-irradiated keratinocytes (UV-KCM) induces melanocyte dendricity to a greater degree than does baseline keratinocyte-conditioned medium (KCM), we investigated whether ET-1 promotes melanocyte dendricity. ET-1, originally recognized as a vasoconstrictive peptide, has recently been shown to stimulate melanocyte proliferation and tyrosinase activity. We now report that ET-1 supplementation of cultured melanocytes significantly increases the percentage of dendritic melanocytes, as well as dendrite length, in a dose-dependent manner. Moreover, UV-KCM was found to contain over 25-fold more ET-1 than KCM, and ET-1 supplementation of KCM induced melanocyte dendricity comparable to that induced by UV-KCM. Further, melanocyte dendricity induced by UV-KCM was significantly inhibited by the addition of anti-ET-1 monoclonal antibody to the medium, suggesting that the UV-KCM effect on melanocyte dendricity is mediated largely through ET-1. Our findings suggest that in the skin, ET-1 of keratinocyte origin promotes melanocyte dendricity in response to UV irradiation.

  8. FOXM1 regulates proliferation, senescence and oxidative stress in keratinocytes and cancer cells

    PubMed Central

    Smirnov, Artem; Panatta, Emanuele; Lena, AnnaMaria; Castiglia, Daniele; Di Daniele, Nicola; Melino, Gerry; Candi, Eleonora

    2016-01-01

    Several transcription factors, including the master regulator of the epidermis, p63, are involved in controlling human keratinocyte proliferation and differentiation. Here, we report that in normal keratinocytes, the expression of FOXM1, a member of the Forkhead superfamily of transcription factors, is controlled by p63. We observe that, together with p63, FOXM1 strongly contributes to the maintenance of high proliferative potential in keratinocytes, whereas its expression decreases during differentiation, as well as during replicative-induced senescence. Depletion of FOXM1 is sufficient to induce keratinocyte senescence, paralleled by an increased ROS production and an inhibition of ROS-scavenger genes (SOD2, CAT, GPX2, PRDX). Interestingly, FOXM1 expression is strongly reduced in keratinocytes isolated from old human subjects compared with young subjects. FOXM1 depletion sensitizes both normal keratinocytes and squamous carcinoma cells to apoptosis and ROS-induced apoptosis. Together, these data identify FOXM1 as a key regulator of ROS in normal dividing epithelial cells and suggest that squamous carcinoma cells may also use FOXM1 to control oxidative stress to escape premature senescence and apoptosis. PMID:27385468

  9. A Two-Stepped Culture Method for Efficient Production of Trichogenic Keratinocytes

    PubMed Central

    Chan, Chih-Chieh; Fan, Sabrina Mai-Yi; Wang, Wei-Hung; Mu, Yi-Fen

    2015-01-01

    Successful hair follicle (HF) neogenesis in adult life depends on the existence of both capable dermal cells and competent epidermal keratinocytes that recapitulate embryonic organogenesis through epithelial–mesenchymal interaction. In tissue engineering, the maintenance of trichogenic potential of adult epidermal cells, while expanding them remains a challenging issue. We found that although HF outer root sheath keratinocytes could be expanded for more than 100 passages as clonogenic cells without losing the proliferative potential with a 3T3J2 fibroblast feeder layer, these keratinocytes were unable to form new HFs when combined with inductive HF dermal papilla (DP) cells. However, when these high-passage keratinocytes were cocultured with HF DP cells for 4 days in vitro, they regained the trichogenic ability to form new HFs after transplantation. We found that the short-term coculture with DP cells enhanced both Wnt/β-catenin signaling, a signaling cascade key to HF development, and upregulated the expression of HF-specific genes, including K6, K16, K17, and K75, in keratinocytes, indicating that these cells were poised toward a HF fate. Hence, efficient production of trichogenic keratinocytes can be obtained by a two-stepped procedure with initial cell expansion with a 3T3J2 fibroblast feeder followed by short-term coculture with DP cells. PMID:25951188

  10. The Retinoid-Related Orphan Receptor RORα Promotes Keratinocyte Differentiation via FOXN1

    PubMed Central

    Dai, Jun; Brooks, Yang; Lefort, Karine; Getsios, Spiro; Dotto, G. Paolo

    2013-01-01

    RORα is a retinoid-related orphan nuclear receptor that regulates inflammation, lipid metabolism, and cellular differentiation of several non-epithelial tissues. In spite of its high expression in skin epithelium, its functions in this tissue remain unclear. Using gain- and loss-of-function approaches to alter RORα gene expression in human keratinocytes (HKCs), we have found that this transcription factor functions as a regulator of epidermal differentiation. Among the 4 RORα isoforms, RORα4 is prominently expressed by keratinocytes in a manner that increases with differentiation. In contrast, RORα levels are significantly lower in skin squamous cell carcinoma tumors (SCCs) and cell lines. Increasing the levels of RORα4 in HKCs enhanced the expression of structural proteins associated with early and late differentiation, as well as genes involved in lipid barrier formation. Gene silencing of RORα impaired the ability of keratinocytes to differentiate in an in vivo epidermal cyst model. The pro-differentiation function of RORα is mediated at least in part by FOXN1, a well-known pro-differentiation transcription factor that we establish as a novel direct target of RORα in keratinocytes. Our results point to RORα as a novel node in the keratinocyte differentiation network and further suggest that the identification of RORα ligands may prove useful for treating skin disorders that are associated with abnormal keratinocyte differentiation, including cancer. PMID:23922987

  11. Crucial role of vinexin for keratinocyte migration in vitro and epidermal wound healing in vivo

    SciTech Connect

    Kioka, Noriyuki; Ito, Takuya; Yamashita, Hiroshi; Uekawa, Natsuko; Umemoto, Tsutomu; Motoyoshi, Soh; Imai, Hiroshi; Takahashi, Kenzo; Watanabe, Hideto; Yamada, Masayasu; Ueda, Kazumitsu

    2010-06-10

    In the process of tissue injury and repair, epithelial cells rapidly migrate and form epithelial sheets. Vinexin is a cytoplasmic molecule of the integrin-containing cell adhesion complex localized at focal contacts in vitro. Here, we investigated the roles of vinexin in keratinocyte migration in vitro and wound healing in vivo. Vinexin knockdown using siRNA delayed migration of both HaCaT human keratinocytes and A431 epidermoid carcinoma cells in scratch assay but did not affect cell proliferation. Induction of cell migration by scratching the confluent monolayer culture of these cells activated both EGFR and ERK, and their inhibitors AG1478 and U0126 substantially suppressed scratch-induced keratinocyte migration. Vinexin knockdown in these cells inhibited the scratch-induced activation of EGFR, but not that of ERK, suggesting that vinexin promotes cell migration via activation of EGFR. We further generated vinexin (-/-) mice and isolated their keratinocytes. They similarly showed slow migration in scratch assay. Furthermore, vinexin (-/-) mice exhibited a delay in cutaneous wound healing in both the back skin and tail without affecting the proliferation of keratinocytes. Together, these results strongly suggest a crucial role of vinexin in keratinocyte migration in vitro and cutaneous wound healing in vivo.

  12. Actin filaments participate in the relocalization of phosphatidylinositol3-kinase to glucose transporter-containing compartments and in the stimulation of glucose uptake in 3T3-L1 adipocytes.

    PubMed Central

    Wang, Q; Bilan, P J; Tsakiridis, T; Hinek, A; Klip, A

    1998-01-01

    Insulin stimulates the rate of glucose uptake into muscle and adipose cells by translocation of glucose transporters from an intracellular storage pool to the plasma membrane. This event requires the prior activation of phosphatidylinositol 3-kinase (PI 3-kinase). Here we report that insulin causes an increase in wortmannin-sensitive PI 3-kinase activity and a gain in the enzyme's regulatory and catalytic subunits p85alpha and p110beta (but not p110alpha) in the intracellular compartments containing glucose transporters. The hormone also caused a marked reorganization of actin filaments, which was prevented by cytochalasin D. Cytochalasin D also decreased significantly the insulin-dependent association of PI 3-kinase activity and the levels of insulin receptor substrate (IRS)-1, p85alpha and p110beta with immunopurified GLUT4-containing compartments. In contrast, the drug did not alter the insulin-induced tyrosine phosphorylation of IRS-1, the association of PI 3-kinase with IRS-1, or the stimulation of PI 3-kinase by insulin in anti-(IRS-1) or anti-p85 immunoprecipitates from whole cell lysates. Cytochalasin D, and the chemically unrelated latrunculin B, which also inhibits actin filament reassembly, prevented the insulin stimulation of glucose transport by approx. 50%. Cytochalasin D decreased by about one-half the insulin-dependent translocation to the plasma membrane of the GLUT1 and GLUT4 glucose transporters. The results suggest that the existence of intact actin filament is correlated with the full recruitment of glucose transporters by insulin. The underlying function of the actin filaments might be to facilitate the insulin-mediated association of the p85-p110 PI 3-kinase with glucose-transporter-containing compartments. PMID:9560323

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

    SciTech Connect

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

    2014-06-01

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

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

    PubMed

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

    2015-12-01

    Rumex acetosa L. (RA) (Polygonaceae) is an important traditional Chinese medicine (TCM) commonly used in clinic for a long history in China and the aerial parts of RA has a wide variety of pharmacological actions such as diuretic, anti-hypertensive, anti-oxidative, and anti-cancer effects. However, the mechanisms involved are to be defined. The purpose of the present study was to evaluate the vasorelaxant effect and define the mechanism of action of the ethanol extract of Rumex acetosa L. (ERA) in rat aorta. ERA was examined for its vascular relaxant effect in isolated phenylephrine-precontracted rat thoracic aorta and its acute effects on arterial blood pressure. In addition, the roles of the nitric oxide synthase (NOS)-nitric oxide (NO) signaling in the ERA-induced effects were tested in human umbilical vein endothelial cells (HUVECs). The phosphorylation levels of Akt and eNOS were assessed by Western blot analysis in the cultured HUVECs. ERA induced endothelium-dependent vasorelaxation. The ERA-induced vasorelaxation was abolished by L-NAME (an NOS inhibitor) or ODQ (a sGC inhibitor), but not by indomethacin. Inhibition of PI3-kinase/Akt signaling pathway markedly reduced the ERA-induced vasorelaxation. In HUVECs, ERA increased NO formation in a dose-dependent manner, which was inhibited by L-NAME and by removing extracellular Ca(2+). In addition, ERA promoted phosphorylation of Akt and eNOS, which was prevented by wortmannin and LY294002, indicating that ERA induces eNOS phosphorylation through the PI3-kinase/Akt pathway. Further, in anesthetized rats, intravenously administered ERA decreased arterial blood pressure in a dose-dependent manner through an activation of the NOS-NO system. In summary, the ERA- induced vasorelaxation was dependent on endothelial integrity and NO production, and was mediated by activation of both the endothelial PI3-kinase/Akt- and Ca(2+)-eNOS-NO signaling and muscular NO-sGC-cGMP signaling. PMID:26769840

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

    PubMed

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

    2015-12-01

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

  16. Loss of nuclear receptor RXRα in epidermal keratinocytes promotes the formation of Cdk4-activated invasive melanomas.

    PubMed

    Hyter, Stephen; Bajaj, Gaurav; Liang, Xiaobo; Barbacid, Mariano; Ganguli-Indra, Gitali; Indra, Arup Kumar

    2010-10-01

    Keratinocytes contribute to melanocyte transformation by affecting their microenvironment, in part through the secretion of paracrine factors. Here we report a loss of expression of nuclear receptor RXRα in epidermal keratinocytes during human melanoma progression. In the absence of keratinocytic RXRα, in combination with mutant Cdk4, cutaneous melanoma was generated that metastasized to lymph nodes in a bigenic mouse model. Expression of several keratinocyte-derived mitogenic growth factors (Et-1, Hgf, Scf, α-MSH and Fgf 2 ) was elevated in skin of bigenic mice, whereas Fas, E-cadherin and Pten, implicated in apoptosis, cellular invasion and melanomagenesis, respectively, were downregulated within the microdissected melanocytic tumors. We demonstrated that RXRα is recruited on the proximal promoter of both Et-1 and Hgf, possibly directly regulating their transcription in keratinocytes. These studies demonstrate the contribution of keratinocytic paracrine signaling during the cellular transformation and malignant conversion of melanocytes.

  17. Loss of nuclear receptor RXRα in epidermal keratinocytes promotes the formation of Cdk4-activated invasive melanomas.

    PubMed

    Hyter, Stephen; Bajaj, Gaurav; Liang, Xiaobo; Barbacid, Mariano; Ganguli-Indra, Gitali; Indra, Arup Kumar

    2010-10-01

    Keratinocytes contribute to melanocyte transformation by affecting their microenvironment, in part through the secretion of paracrine factors. Here we report a loss of expression of nuclear receptor RXRα in epidermal keratinocytes during human melanoma progression. In the absence of keratinocytic RXRα, in combination with mutant Cdk4, cutaneous melanoma was generated that metastasized to lymph nodes in a bigenic mouse model. Expression of several keratinocyte-derived mitogenic growth factors (Et-1, Hgf, Scf, α-MSH and Fgf 2 ) was elevated in skin of bigenic mice, whereas Fas, E-cadherin and Pten, implicated in apoptosis, cellular invasion and melanomagenesis, respectively, were downregulated within the microdissected melanocytic tumors. We demonstrated that RXRα is recruited on the proximal promoter of both Et-1 and Hgf, possibly directly regulating their transcription in keratinocytes. These studies demonstrate the contribution of keratinocytic paracrine signaling during the cellular transformation and malignant conversion of melanocytes. PMID:20629968

  18. Transient Receptor Potential Vanilloid 4 Ion Channel Functions as a Pruriceptor in Epidermal Keratinocytes to Evoke Histaminergic Itch.

    PubMed

    Chen, Yong; Fang, Quan; Wang, Zilong; Zhang, Jennifer Y; MacLeod, Amanda S; Hall, Russell P; Liedtke, Wolfgang B

    2016-05-01

    TRPV4 ion channels function in epidermal keratinocytes and in innervating sensory neurons; however, the contribution of the channel in either cell to neurosensory function remains to be elucidated. We recently reported TRPV4 as a critical component of the keratinocyte machinery that responds to ultraviolet B (UVB) and functions critically to convert the keratinocyte into a pain-generator cell after excess UVB exposure. One key mechanism in keratinocytes was increased expression and secretion of endothelin-1, which is also a known pruritogen. Here we address the question of whether TRPV4 in skin keratinocytes functions in itch, as a particular form of "forefront" signaling in non-neural cells. Our results support this novel concept based on attenuated scratching behavior in response to histaminergic (histamine, compound 48/80, endothelin-1), not non-histaminergic (chloroquine) pruritogens in Trpv4 keratinocyte-specific and inducible knock-out mice. We demonstrate that keratinocytes rely on TRPV4 for calcium influx in response to histaminergic pruritogens. TRPV4 activation in keratinocytes evokes phosphorylation of mitogen-activated protein kinase, ERK, for histaminergic pruritogens. This finding is relevant because we observed robust anti-pruritic effects with topical applications of selective inhibitors for TRPV4 and also for MEK, the kinase upstream of ERK, suggesting that calcium influx via TRPV4 in keratinocytes leads to ERK-phosphorylation, which in turn rapidly converts the keratinocyte into an organismal itch-generator cell. In support of this concept we found that scratching behavior, evoked by direct intradermal activation of TRPV4, was critically dependent on TRPV4 expression in keratinocytes. Thus, TRPV4 functions as a pruriceptor-TRP in skin keratinocytes in histaminergic itch, a novel basic concept with translational-medical relevance.

  19. Transient Receptor Potential Vanilloid 4 Ion Channel Functions as a Pruriceptor in Epidermal Keratinocytes to Evoke Histaminergic Itch*

    PubMed Central

    Chen, Yong; Fang, Quan; Wang, Zilong; Zhang, Jennifer Y.; MacLeod, Amanda S.; Hall, Russell P.; Liedtke, Wolfgang B.

    2016-01-01

    TRPV4 ion channels function in epidermal keratinocytes and in innervating sensory neurons; however, the contribution of the channel in either cell to neurosensory function remains to be elucidated. We recently reported TRPV4 as a critical component of the keratinocyte machinery that responds to ultraviolet B (UVB) and functions critically to convert the keratinocyte into a pain-generator cell after excess UVB exposure. One key mechanism in keratinocytes was increased expression and secretion of endothelin-1, which is also a known pruritogen. Here we address the question of whether TRPV4 in skin keratinocytes functions in itch, as a particular form of “forefront” signaling in non-neural cells. Our results support this novel concept based on attenuated scratching behavior in response to histaminergic (histamine, compound 48/80, endothelin-1), not non-histaminergic (chloroquine) pruritogens in Trpv4 keratinocyte-specific and inducible knock-out mice. We demonstrate that keratinocytes rely on TRPV4 for calcium influx in response to histaminergic pruritogens. TRPV4 activation in keratinocytes evokes phosphorylation of mitogen-activated protein kinase, ERK, for histaminergic pruritogens. This finding is relevant because we observed robust anti-pruritic effects with topical applications of selective inhibitors for TRPV4 and also for MEK, the kinase upstream of ERK, suggesting that calcium influx via TRPV4 in keratinocytes leads to ERK-phosphorylation, which in turn rapidly converts the keratinocyte into an organismal itch-generator cell. In support of this concept we found that scratching behavior, evoked by direct intradermal activation of TRPV4, was critically dependent on TRPV4 expression in keratinocytes. Thus, TRPV4 functions as a pruriceptor-TRP in skin keratinocytes in histaminergic itch, a novel basic concept with translational-medical relevance. PMID:26961876

  20. Activation of phosphatidylinositol 3-kinase is required for transcriptional activity of F-type 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: assessment of the role of protein kinase B and p70 S6 kinase.

    PubMed Central

    Fernández de Mattos , S; de los Pinos E, E; Joaquin, M; Tauler, A

    2000-01-01

    Previous studies have demonstrated that the F isoform of6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase(6PF2K/Fru-2,6-BPase) is transcriptionally regulated by growth factors. The aim of this study was to investigate the importance of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway in the regulation of 6PF2K/Fru-2,6-BPase gene expression. We have completed studies using chemical inhibitors and expression vectors for the proteins involved in this signalling cascade. Treatment of cells with LY 294002, an inhibitor of PI 3-kinase, blocked the epidermal growth factor (EGF)-dependent stimulation of 6PF2K/Fru-2,6-BPase gene transcription. Transient transfection of a constitutively active PI 3-kinase was sufficient to activate transcription from the F-type 6PF2K/Fru-2,6-BPase promoter. In contrast, co-transfection with a dominant-negative form of PI 3-kinase completely abrogated the stimulation by EGF, and down-regulated the basal promoter activity. In an attempt to determine downstream proteins that lie between PI 3-kinase and 6PF2K/Fru-2,6-BPase gene expression, the overexpression of a constitutively active form of protein kinase B (PKB) was sufficient to activate 6PF2K/Fru-2,6-BPase gene expression, even in the presence of either a dominant-negative form of PI 3-kinase or LY 294002. The over-expression of p70/p85 ribosomal S6 kinase or the treatment with its inhibitor rapamycin did not affect 6PF2K/Fru-2,6-BPase transcription. We conclude that PI 3-kinase is necessary for the transcriptional activity of F-type 6PF2K/Fru-2,6-BPase, and that PKB is a downstream effector of PI 3-kinase directly involved in the regulation of 6PF2K/Fru-2,6-BPase gene expression. PMID:10861211

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

    PubMed

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

    2014-12-20

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  4. Targeted Inhibition of Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Sensitizes Pancreatic Cancer Cells to Doxorubicin without Exacerbating Cardiac Toxicity

    PubMed Central

    Durrant, David E.; Das, Anindita; Dyer, Samya; Tavallai, Seyedmehrad; Dent, Paul

    2015-01-01

    Pancreatic cancer has the lowest 5-year survival rate of all major cancers despite decades of effort to design and implement novel, more effective treatment options. In this study, we tested whether the dual phosphoinositide 3-kinase/mechanistic target of rapamycin inhibitor BEZ235 (BEZ) potentiates the antitumor effects of doxorubicin (DOX) against pancreatic cancer. Cotreatment of BEZ235 with DOX resulted in dose-dependent inhibition of the phosphoinositide 3-kinase/mechanistic target of rapamycin survival pathway, which corresponded with an increase in poly ADP ribose polymerase cleavage. Moreover, BEZ cotreatment significantly improved the effects of DOX toward both cell viability and cell death in part through reduced Bcl-2 expression and increased expression of the shorter, more cytotoxic forms of BIM. BEZ also facilitated intracellular accumulation of DOX, which led to enhanced DNA damage and reactive oxygen species generation. Furthermore, BEZ in combination with gemcitabine reduced MiaPaca2 cell proliferation but failed to increase reactive oxygen species generation or BIM expression, resulting in reduced necrosis and apoptosis. Treatment with BEZ and DOX in mice bearing tumor xenographs significantly repressed tumor growth as compared with BEZ, DOX, or gemcitabine. Additionally, in contrast to the enhanced expression seen in MiaPaca2 cells, BEZ and DOX cotreatment reduced BIM expression in H9C2 cardiomyocytes. Also, the Bcl-2/Bax ratio was increased, which was associated with a reduction in cell death. In vivo echocardiography showed decreased cardiac function with DOX treatment, which was not improved by combination treatment with BEZ. Thus, we propose that combining BEZ with DOX would be a better option for patients than current standard of care by providing a more effective tumor response without the associated increase in toxicity. PMID:26101222

  5. Us3 kinase encoded by herpes simplex virus 1 mediates downregulation of cell surface major histocompatibility complex class I and evasion of CD8+ T cells.

    PubMed

    Imai, Takahiko; Koyanagi, Naoto; Ogawa, Ryo; Shindo, Keiko; Suenaga, Tadahiro; Sato, Ayuko; Arii, Jun; Kato, Akihisa; Kiyono, Hiroshi; Arase, Hisashi; Kawaguchi, Yasushi

    2013-01-01

    Detection and elimination of virus-infected cells by CD8(+) cytotoxic T lymphocytes (CTLs) depends on recognition of virus-derived peptides presented by major histocompatibility complex class I (MHC-I) molecules on the surface of infected cells. In the present study, we showed that inactivation of the activity of viral kinase Us3 encoded by herpes simplex virus 1 (HSV-1), the etiologic agent of several human diseases and a member of the alphaherpesvirinae, significantly increased cell surface expression of MHC-I, thereby augmenting CTL recognition of infected cells in vitro. Overexpression of Us3 by itself had no effect on cell surface expression of MHC-I and Us3 was not able to phosphorylate MHC-I in vitro, suggesting that Us3 indirectly downregulated cell surface expression of MHC-I in infected cells. We also showed that inactivation of Us3 kinase activity induced significantly more HSV-1-specific CD8(+) T cells in mice. Interestingly, depletion of CD8(+) T cells in mice significantly increased replication of a recombinant virus encoding a kinase-dead mutant of Us3, but had no effect on replication of a recombinant virus in which the kinase-dead mutation was repaired. These results indicated that Us3 kinase activity is required for efficient downregulation of cell surface expression of MHC-I and mediates evasion of HSV-1-specific CD8(+) T cells. Our results also raised the possibility that evasion of HSV-1-specific CD8(+) T cells by HSV-1 Us3-mediated inhibition of MHC-I antigen presentation might in part contribute to viral replication in vivo.

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

    SciTech Connect

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

    2011-11-15

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

  7. Development of an immunohistochemical protein quantification system in conjunction with tissue microarray technology for identifying predictive biomarkers for phosphatidylinositol 3-kinase inhibitors.

    PubMed

    Isoyama, Sho; Yoshimi, Hisashi; Dan, Shingo; Okamura, Mutsumi; Seki, Mariko; Irimura, Tatsuro; Yamori, Takao

    2012-01-01

    The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in human cancers by gain-of-function mutations of phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) or dysfunction of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Therefore PI3K is thought to be a promising target for cancer therapy. Many agents targeting PI3K have been developed and some of them have been evaluated in clinical trials. In recent years, development of predictive biomarkers as companion diagnostics for molecular targeted drugs has become an important requirement for clinical development; however, no clinically established biomarkers that predict the efficacy of PI3K inhibitors have been found. We previously reported that expression of phosphorylated Akt determined by immunoblot analysis correlated with the antitumor efficacy of a PI3K inhibitor ZSTK474 in vitro and in vivo, suggesting that it might be used as a predictive biomarker. In this study, to evaluate biomarker candidates in in vivo tumor samples, we developed an immunohistochemical protein detection/quantification system in conjunction with the tissue microarray technology using a panel of 24 human tumor xenografts (JFCR24). We have clearly demonstrated that expression levels of phosphorylated v-akt murine thymoma viral oncogene homolog (Akt) and mitogen-activated protein kinase (MAPK) determined by this system significantly correlated with those determined by immunoblot analysis. As expected, PTEN status correlated with expression of phosphorylated Akt but not MAPK. Finally, we confirmed that phosphorylated Akt levels determined using this system correlated with the in vivo efficacy of ZSTK474. The present results indicate that the immunohistochemical protein detection/quantification system could be used to quantify expression of biomarker proteins in xenografted tumor tissues as well as in human tumor specimens to predict drug efficacy in future clinical trials. PMID:22975517

  8. Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis.

    PubMed

    Tarafder, Abul K; Bolasco, Giulia; Correia, Maria S; Pereira, Francisco J C; Iannone, Lucio; Hume, Alistair N; Kirkpatrick, Niall; Picardo, Mauro; Torrisi, Maria R; Rodrigues, Inês P; Ramalho, José S; Futter, Clare E; Barral, Duarte C; Seabra, Miguel C

    2014-04-01

    The transfer of melanin from melanocytes to keratinocytes is a crucial process underlying maintenance of skin pigmentation and photoprotection against UV damage. Here, we present evidence supporting coupled exocytosis of the melanin core, or melanocore, by melanocytes and subsequent endocytosis by keratinocytes as a predominant mechanism of melanin transfer. Electron microscopy analysis of human skin samples revealed three lines of evidence supporting this: (1) the presence of melanocores in the extracellular space; (2) within keratinocytes, melanin was surrounded by a single membrane; and (3) this membrane lacked the melanosomal membrane protein tyrosinase-related protein 1 (TYRP1). Moreover, co-culture of melanocytes and keratinocytes suggests that melanin exocytosis is specifically induced by keratinocytes. Furthermore, depletion of Rab11b, but not Rab27a, caused a marked decrease in both keratinocyte-stimulated melanin exocytosis and transfer to keratinocytes. Thus, we propose that the predominant mechanism of melanin transfer is keratinocyte-induced exocytosis, mediated by Rab11b through remodeling of the melanosome membrane, followed by subsequent endocytosis by keratinocytes.

  9. Overexpressed TRPV3 ion channels in skin keratinocytes modulate pain sensitivity via prostaglandin E2

    PubMed Central

    Huang, Susan M.; Lee, Hyosang; Chung, Man-Kyo; Park, Una; Yu, Yin Yin; Bradshaw, Heather B.; Coulombe, Pierre A.; Walker, J. Michael; Caterina, Michael J.

    2009-01-01

    The ability to sense changes in the environment is essential for survival because it permits responses such as withdrawal from noxious stimuli and regulation of body temperature. Keratinocytes, which occupy much of the skin epidermis, are situated at the interface between the external environment and the body's internal milieu, and have long been appreciated for their barrier function against external insults. The recent discovery of temperature-sensitive TRPV ion channels in keratinocytes has raised the possibility that these cells also actively participate in acute temperature and pain sensation. To address this notion, we generated and characterized transgenic mice that overexpress TRPV3 in epidermal keratinocytes under the control of the keratin 14 promoter. Compared to wild-type controls, keratinocytes overexpressing TRPV3 exhibited larger currents as well as augmented prostaglandin E2 (PGE2) release in response to two TRPV3 agonists, 2-aminoethoxydiphenyl borate (2APB) and heat. Thermal selection behavior and heat-evoked withdrawal behavior of naïve mice overexpressing TRPV3 were not consistently altered. Upon selective pharmacological inhibition of TRPV1 with JNJ-7203212, however, the keratinocyte-specific TRPV3 transgenic mice showed increased escape responses to noxious heat relative to their wild-type littermates. Co-administration of the cyclooxygenase inhibitor, ibuprofen, with the TRPV1 antagonist decreased inflammatory thermal hyperalgesia in transgenic but not wild-type animals. Our results reveal a previously undescribed mechanism for keratinocyte participation in thermal pain transduction through keratinocyte TRPV3 ion channels and the intercellular messenger PGE2. PMID:19091963

  10. MiR-146a negatively regulates TLR2-induced inflammatory responses in keratinocytes.

    PubMed

    Meisgen, Florian; Xu Landén, Ning; Wang, Aoxue; Réthi, Bence; Bouez, Charbel; Zuccolo, Michela; Gueniche, Audrey; Ståhle, Mona; Sonkoly, Enikö; Breton, Lionel; Pivarcsi, Andor

    2014-07-01

    Keratinocytes represent the first line of defense against pathogens in the skin and have important roles in initiating and regulating inflammation during infection and autoimmunity. Here we investigated the role of miR-146a in the regulation of the innate immune response of keratinocytes. Toll-like receptor 2 (TLR2) stimulation of primary human keratinocytes resulted in an NF-κB- and mitogen-activated protein kinase-dependent upregulation of miR-146a expression, which was surprisingly long lasting, contrasting with the rapid and transient induction of inflammatory mediators. Overexpression of miR-146a significantly suppressed the production of IL-8, CCL20, and tumor necrosis factor-α, which functionally suppressed the chemotactic attraction of neutrophils by keratinocytes. Inhibition of endogenous miR-146a induced the production of inflammatory mediators even in nonstimulated keratinocytes, and potentiated the effect of TLR2 stimulation. Transcriptomic profiling revealed that miR-146a suppresses the expression of a large number of immune-related genes in keratinocytes. MiR-146a downregulated interleukin-1 receptor-associated kinase 1 and TNF receptor-associated factor 6, two key adapter molecules downstream of TLR signaling, and suppressed NF-κB promoter-binding activity as shown by promoter luciferase experiments. Together, these data identify miR-146a as a regulatory element in keratinocyte innate immunity, which prevents the production of inflammatory mediators under homeostatic conditions and serves as a potent negative feedback regulator after TLR2 stimulation. PMID:24670381

  11. Low-energy helium-neon laser irradiation increases the motility of cultured human keratinocytes

    SciTech Connect

    Haas, A.F.; Isseroff, R.R.; Wheeland, R.G.; Rood, P.A.; Graves, P.J. )

    1990-06-01

    Helium-neon (HeNe) laser irradiation is known to stimulate wound healing. We investigated whether the biostimulatory effects of HeNe irradiation result from enhancement of keratinocyte proliferation or motility. HeNe effects on keratinocyte motility were evaluated by irradiating a wounded culture with 0.8 J/cm2 3 times over a 20-h period. At 20 h post-irradiation, videocinemicroscopy and sequential quantitative measurements of the leading edge were taken over a 6-h period. There was a significant difference in migration of the leading edge in irradiated wounds compared to non-irradiated wounded controls (12.0 microns/h vs 4.0 microns/h, p less than 0.0001). To determine if the increase in migration observed in irradiated cultures resulted from a proliferative effect of HeNe irradiation, subconfluent human keratinocyte cultures were irradiated with single or multiple doses of different fluences of HeNe irradiation (0.4 to 7.2 J/cm2) and evaluated 72 h post-irradiation. Irradiated and non-irradiated keratinocyte cultures grown on a microporous membrane surface were co-cultured with irradiated and non-irradiated fibroblasts to determine if HeNe irradiation induced a paracrine effect on keratinocyte proliferation. No significant increase in keratinocyte proliferation was demonstrated in any of these treatments. The biostimulatory effects of HeNe irradiation may now be extended to include enhancement of keratinocyte motility in vitro; this may contribute to the efficacy of HeNe irradiation in wound healing.

  12. Transcription factor 7-like 1 dysregulates keratinocyte differentiation through upregulating lipocalin 2

    PubMed Central

    Xu, M; Zhang, Y; Cheng, H; Liu, Y; Zou, X; Zhan, N; Xiao, S; Xia, Y

    2016-01-01

    Recent studies strongly suggested that transcription factor 7-like 1 (Tcf7l1, also known as Tcf3) is involved in the differentiation of several types of cells, and demonstrated that Tcf7l1 modulates keratinocytes physiologically through regulating lipocalin 2 (LCN2), a key regulator of cell differentiation. To reveal the potential role of Tcf7l1 in the dysregulation of keratinocyte differentiation, both Tcf7l1 and LCN2 were determined in a variety of skin disorders. The in vitro effect of Tcf7l1 on keratinocyte differentiation was studied by culturing SCC-13 cells, and the human foreskin keratinocytes (HFKs) that were transfected with vectors for overexpressing human papillomavirus E6/E7 or Tcf7l1 genes. We found that both Tcf7l1 and LCN2 were highly expressed in those diseases characterized by defective keratinocyte differentiation (especially psoriasis vulgaris, condyloma acuminatum, squamous cell carcinoma, etc). Moreover, compared with control HFKs, SCC-13 cells and E6/E7-harboring HFKs expressed more Tcf7l1 and LCN2. Tcf7l1 siRNA transfection decreased LCN2 but increased involucrin and loricrin in HFKs under calcium stimuli. Conversely, Tcf7l1 overexpression in SCC-13 cells or vector-transfected HFKs induced lower involucrin and loricrin expression and less keratinocyte apoptosis, both of which, however, were partially abrogated by LCN2 siRNA or neutralizing anti-LCN2 antibody. Interestingly, the Tcf7l1 expression in HFKs correlated positively with the MMP-2 level, and the inhibition of MMP-2 decreased the LCN2 level and even attenuated the effect of Tcf7l1 on LCN2 expression. Therefore, Tcf7l1 dysregulates keratinocyte differentiation, possibly through upregulating the LCN2 pathway in an MMP-2 mediated manner. Elucidating the interaction between Tcf7l1 and LCN2 may help understand disordered cell differentiation in some skin diseases. PMID:27551519

  13. Platelet-activating factor biosynthesis induced by various stimuli in human HaCaT keratinocytes.

    PubMed

    Travers, J B; Harrison, K A; Johnson, C A; Clay, K L; Morelli, J G

    1996-07-01

    Platelet-activating factor (PAF) is a potent inflammatory mediator that is thought to play a role in cutaneous inflammation. These studies used mass spectrometry to examine the molecular species of PAF precursor glycerophosphocholine lipids (GPC) as well as the biosynthesis of PAF and other sn-2 acetyl-GPC in a human keratinocyte-derived cell line (HaCaT keratinocytes). Approximately 28% of HaCaT keratinocyte GPC consisted of 1-alkyl species, and the relative amounts of the sn-1 alkyl constituents of the PAF precursor 1-alkyl-2-acyl-GPC were as follows: hexadecyl > octadecenyl > octadecyl. Ionophore (A23187)-stimulated HaCaT keratinocytes synthesized both PAF (1-hexadecyl, 1-octadecenyl, and 1-octadecyl species) and less potent 1-acyl analogs (1-palmitoyl, 1-oleoyl, and 1-stearoyl species). PAF production was rapid and maximal by 10 min. The major species of sn-2acetyl-GPC at 2.5 min were 1-hexadecyl-2-acetyl-GPC (2.2 ng/10(6) cells) and 1-palmitoyl-2-acetyl-GPC (2.4 ng/10(6) cells). HaCaT keratinocytes also synthesized PAF and 1-acyl PAF analogs when stimulated with the peptide growth factor endothelin-1 and the nonhydrolyzable PAF receptor agonist carbamyl-PAF. Both 1-hexadecyl-2- acetyl-GPC and 1-palmitoyl-2-acetyl-GPC stimulated intracellular calcium mobilization in HaCaT cells, indicating that these sn-2 acetyl-GPC act in autocrine fashion. These studies revealed that the human keratinocyte-derived cell line HaCaT can synthesize significant amounts of PAF and 1-acyl analogs in vitro from both nonspecific (A23187) and specific (endothelin-1, carbamyl-PAF) stimulation, suggesting a role for this inflammatory lipid mediator in keratinocyte pathophysiology.

  14. Endothelins secreted from human keratinocytes are intrinsic mitogens for human melanocytes.

    PubMed

    Imokawa, G; Yada, Y; Miyagishi, M

    1992-12-01

    We recently demonstrated that human melanocyte proliferation and differentiation could be stimulated by endothelin (ET) derivatives via a receptor-mediated signal transduction pathway (Yada, Y., Higuchi, K., and Imokawa, G. (1991) J. Biol. Chem. 266, 18352-18357). We show here that the growth factors for human melanocytes are produced and secreted by the surrounding cells, namely human keratinocytes for ET-1 and Big-ET-1. Northern blots have revealed the presence of ET-1 gene transcripts in proliferating human keratinocytes. The ET-1 production by human keratinocytes increased after irradiation with ultraviolet B (UVB) in a dose-dependent manner, accompanied by the significant secretion of interleukin 1 alpha (IL-1 alpha). Among the cytokines related to UVB-induced cellular reactions and keratinocyte growth, only IL-1 alpha and -1 beta stimulated the secretion of ET-1 and Big-ET-1 but not of ET-3 and Big-ET-3 in a time-dependent manner. Northern blots for IL-1 alpha-stimulated or UVB-exposed human keratinocytes revealed that production of ET-1 gene transcripts markedly increased (by about 300 or 1,200%) with constant levels of beta-actin gene transcripts. In a parallel study, the medium conditioned by UVB-exposed human keratinocytes elicited a significant anti-ET-1 antibody-suppressible increase in DNA synthesis by cultured human melanocytes in a UV dose-dependent manner, which was associated with a marked and rapid (80 s) increase in the intracellular calcium level upon incubation with human melanocytes. These studies suggest that ETs produced and secreted by keratinocytes play an essential role in the maintenance of melanocyte proliferation and UV hyperpigmentation in the epidermis.

  15. Lactobacillus rhamnosus GG inhibits the toxic effects of Staphylococcus aureus on epidermal keratinocytes.

    PubMed

    Mohammedsaeed, Walaa; McBain, Andrew J; Cruickshank, Sheena M; O'Neill, Catherine A

    2014-09-01

    Few studies have evaluated the potential benefits of the topical application of probiotic bacteria or material derived from them. We have investigated whether a probiotic bacterium, Lactobacillus rhamnosus GG, can inhibit Staphylococcus aureus infection of human primary keratinocytes in culture. When primary human keratinocytes were exposed to S. aureus, only 25% of the keratinocytes remained viable following 24 h of incubation. However, in the presence of 10(8) CFU/ml of live L. rhamnosus GG, the viability of the infected keratinocytes increased to 57% (P = 0.01). L. rhamnosus GG lysates and spent culture fluid also provided significant protection to keratinocytes, with 65% (P = 0.006) and 57% (P = 0.01) of cells, respectively, being viable following 24 h of incubation. Keratinocyte survival was significantly enhanced regardless of whether the probiotic was applied in the viable form or as cell lysates 2 h before or simultaneously with (P = 0.005) or 12 h after (P = 0.01) S. aureus infection. However, spent culture fluid was protective only if added before or simultaneously with S. aureus. With respect to mechanism, both L. rhamnosus GG lysate and spent culture fluid apparently inhibited adherence of S. aureus to keratinocytes by competitive exclusion, but only viable bacteria or the lysate could displace S. aureus (P = 0.04 and 0.01, respectively). Furthermore, growth of S. aureus was inhibited by either live bacteria or lysate but not spent culture fluid. Together, these data suggest at least two separate activities involved in the protective effects of L. rhamnosus GG against S. aureus, growth inhibition and reduction of bacterial adhesion.

  16. IL-1R–MyD88 signaling in keratinocyte transformation and carcinogenesis

    PubMed Central

    Cataisson, Christophe; Salcedo, Rosalba; Hakim, Shakeeb; Moffitt, B. Andrea; Wright, Lisa; Yi, Ming; Stephens, Robert; Dai, Ren-Ming; Lyakh, Lyudmila; Schenten, Dominik

    2012-01-01

    Constitutively active RAS plays a central role in the development of human cancer and is sufficient to induce tumors in two-stage skin carcinogenesis. RAS-mediated tumor formation is commonly associated with up-regulation of cytokines and chemokines that mediate an inflammatory response considered relevant to oncogenesis. In this study, we report that mice lacking IL-1R or MyD88 are less sensitive to topical skin carcinogenesis than their respective wild-type (WT) controls. MyD88−/− or IL-1R−/− keratinocytes expressing oncogenic RAS are hyperproliferative and fail to up-regulate proinflammatory genes or down-regulate differentiation markers characteristic of RAS-expressing WT keratinocytes. Although RAS-expressing MyD88−/− keratinocytes form only a few small tumors in orthotopic grafts, IL-1R–deficient RAS-expressing keratinocytes retain the ability to form tumors in orthotopic grafts. Using both genetic and pharmacological approaches, we find that the differentiation and proinflammatory effects of oncogenic RAS in keratinocytes require the establishment of an autocrine loop through IL-1α, IL-1R, and MyD88 leading to phosphorylation of IκBα and NF-κB activation. Blocking IL-1α–mediated NF-κB activation in RAS-expressing WT keratinocytes reverses the differentiation defect and inhibits proinflammatory gene expression. Collectively, these results demonstrate that MyD88 exerts a cell-intrinsic function in RAS-mediated transformation of keratinocytes. PMID:22908325

  17. Transcription factor 7-like 1 dysregulates keratinocyte differentiation through upregulating lipocalin 2.

    PubMed

    Xu, M; Zhang, Y; Cheng, H; Liu, Y; Zou, X; Zhan, N; Xiao, S; Xia, Y

    2016-01-01

    Recent studies strongly suggested that transcription factor 7-like 1 (Tcf7l1, also known as Tcf3) is involved in the differentiation of several types of cells, and demonstrated that Tcf7l1 modulates keratinocytes physiologically through regulating lipocalin 2 (LCN2), a key regulator of cell differentiation. To reveal the potential role of Tcf7l1 in the dysregulation of keratinocyte differentiation, both Tcf7l1 and LCN2 were determined in a variety of skin disorders. The in vitro effect of Tcf7l1 on keratinocyte differentiation was studied by culturing SCC-13 cells, and the human foreskin keratinocytes (HFKs) that were transfected with vectors for overexpressing human papillomavirus E6/E7 or Tcf7l1 genes. We found that both Tcf7l1 and LCN2 were highly expressed in those diseases characterized by defective keratinocyte differentiation (especially psoriasis vulgaris, condyloma acuminatum, squamous cell carcinoma, etc). Moreover, compared with control HFKs, SCC-13 cells and E6/E7-harboring HFKs expressed more Tcf7l1 and LCN2. Tcf7l1 siRNA transfection decreased LCN2 but increased involucrin and loricrin in HFKs under calcium stimuli. Conversely, Tcf7l1 overexpression in SCC-13 cells or vector-transfected HFKs induced lower involucrin and loricrin expression and less keratinocyte apoptosis, both of which, however, were partially abrogated by LCN2 siRNA or neutralizing anti-LCN2 antibody. Interestingly, the Tcf7l1 expression in HFKs correlated positively with the MMP-2 level, and the inhibition of MMP-2 decreased the LCN2 level and even attenuated the effect of Tcf7l1 on LCN2 expression. Therefore, Tcf7l1 dysregulates keratinocyte differentiation, possibly through upregulating the LCN2 pathway in an MMP-2 mediated manner. Elucidating the interaction between Tcf7l1 and LCN2 may help understand disordered cell differentiation in some skin diseases. PMID:27551519

  18. Modeling cell-matrix traction forces in Keratinocyte colonies

    NASA Astrophysics Data System (ADS)

    Banerjee, Shiladitya

    2013-03-01

    Crosstalk between cell-cell and cell-matrix adhesions plays an essential role in the mechanical function of tissues. The traction forces exerted by cohesive keratinocyte colonies with strong cell-cell adhesions are mostly concentrated at the colony periphery. In contrast, for weak cadherin-based intercellular adhesions, individual cells in a colony interact with their matrix independently, with a disorganized distribution of traction forces extending throughout the colony. In this talk I will present a minimal physical model of the colony as contractile elastic media linked by springs and coupled to an elastic substrate. The model captures the spatial distribution of traction forces seen in experiments. For cell colonies with strong cell-cell adhesions, the total traction force of the colony measured in experiments is found to scale with the colony's geometrical size. This scaling suggests the emergence of an effective surface tension of magnitude comparable to that measured for non-adherent, three-dimensional cell aggregates. The physical model supports the scaling and indicates that the surface tension may be controlled by acto-myosin contractility. Supported by the NSF through grant DMR-1004789. This work was done in collaboration with Aaron F. Mertz, Eric R. Dufresne and Valerie Horsley (Yale University) and M. Cristina Marchetti (Syracuse University).

  19. Cell wounding activates phospholipase D in primary mouse keratinocytes

    PubMed Central

    Arun, Senthil N.; Xie, Ding; Howard, Amber C.; Zhong, Quincy; Zhong, Xiaofeng; McNeil, Paul L.; Bollag, Wendy B.

    2013-01-01

    Plasma membrane disruptions occur in mechanically active tissues such as the epidermis and can lead to cell death if the damage remains unrepaired. Repair occurs through fusion of vesicle patches to the damaged membrane region. The enzyme phospholipase D (PLD) is involved in membrane traffickiing; therefore, the role of PLD in membrane repair was investigated. Generation of membrane disruptions by lifting epidermal keratinocytes from the substratum induced PLD activation, whereas removal of cells from the substratum via trypsinization had no effect. Pretreatment with 1,25-dihydroxyvitamin D3, previously shown to increase PLD1 expression and activity, had no effect on, and a PLD2-selective (but not a PLD1-selective) inhibitor decreased, cell lifting-induced PLD activation, suggesting PLD2 as the isoform activated. PLD2 interacts functionally with the glycerol channel aquaporin-3 (AQP3) to produce phosphatidylglycerol (PG); however, wounding resulted in decreased PG production, suggesting a potential PG deficiency in wounded cells. Cell lifting-induced PLD activation was transient, consistent with a possible role in membrane repair, and PLD inhibitors inhibited membrane resealing upon laser injury. In an in vivo full-thickness mouse skin wound model, PG accelerated wound healing. These results suggest that PLD and the PLD2/AQP3 signaling module may be involved in membrane repair and wound healing. PMID:23288946

  20. Human Beta-papillomavirus infection and keratinocyte carcinomas.

    PubMed

    Quint, Koen D; Genders, Roel E; de Koning, Maurits N C; Borgogna, Cinzia; Gariglio, Marisa; Bouwes Bavinck, Jan Nico; Doorbar, John; Feltkamp, Mariet C

    2015-01-01

    Although the role of oncogenic human Alpha-papillomaviruses (HPVs) in the development of mucosal carcinomas at different body sites (eg cervix, anus, oropharynx) is fully recognized, a role for HPV in keratinocyte carcinomas (KCs; basal and squamous cell carcinomas) of the skin is not yet clear. KCs are the most common cancers in Caucasians, with the major risk factor being ultraviolet (UV) light exposure. A possible role for Beta-HPV types (BetaPV) in the development of KC was suggested several decades ago, supported by a number of epidemiological studies. Our current review summarizes the recent molecular and histopathological evidence in support of a causal association between BetaPV and the development of KC, and outlines the suspected synergistic effect of viral gene expression with UV radiation and immune suppression. Further insights into the molecular pathways and protein interactions used by BetaPV and the host cell is likely to extend our understanding of the role of BetaPV in KC. PMID:25131163

  1. p63 regulates proliferation and differentiation of developmentally mature keratinocytes

    PubMed Central

    Truong, Amy B.; Kretz, Markus; Ridky, Todd W.; Kimmel, Robin; Khavari, Paul A.

    2006-01-01

    p63 is a multi-isoform p53 family member required for epidermal development. Contrasting roles for p63 in either the initial commitment to the stratified epithelial cell fate or in stem cell-based self-renewal have been proposed. To investigate p63 function in a post-developmental context, we used siRNAs directed against p63 to down-regulate p63 expression in regenerating human epidermis. Loss of p63 resulted in severe tissue hypoplasia and inhibited both stratification and differentiation in a cell-autonomous manner. Although p63-deficient cells exhibited hypoproliferation, differentiation defects were not due to tissue hypoplasia. Simultaneous p63 and p53 knockdown rescued the cell proliferation defect of p63 knockdown alone but failed to restore differentiation, suggesting that defects in epidermal proliferation and differentiation are mediated via p53-dependent and -independent mechanisms, respectively. Furthermore, ΔNp63 isoforms are the main mediators of p63 effects, although TAp63 isoforms may contribute to late differentiation. These data indicate that p63 is required for both the proliferative and differentiation potential of developmentally mature keratinocytes. PMID:17114587

  2. Vanillin protects human keratinocyte stem cells against ultraviolet B irradiation.

    PubMed

    Lee, Jienny; Cho, Jae Youl; Lee, Sang Yeol; Lee, Kyung-Woo; Lee, Jongsung; Song, Jae-Young

    2014-01-01

    Ultraviolet-B (UVB) irradiation is one of major factors which induce cellular damages in the epidermis. We investigated protective effects and mechanisms of vanillin, a main constituent of vanilla beans, against UVB-induced cellular damages in keratinocyte stem cells (KSC). Here, vanillin significantly attenuated UVB irradiation-induced cytotoxicity. The vanillin effects were also demonstrated by the results of the senescence-associated β-galactosidase and alkaline comet assays. In addition, vanillin induced production of pro-inflammatory cytokines. Attempts to elucidate a possible mechanism underlying the vanillin-mediated effects revealed that vanillin significantly reduced UVB-induced phosphorylation of ataxia telangiectasia mutated (ATM), serine threonine kinase checkpoint kinase 2 (Chk2), tumor suppressor protein 53 (p53), p38/mitogen-activated protein kinase (p38), c-Jun N-terminal kinase/stress-activated protein kinase (JNK), S6 ribosomal protein (S6RP), and histone 2A family member X (H2A.X). UVB-induced activation of p53 luciferase reporter was also significantly inhibited by vanillin. In addition, while ATM inhibitor had no effect on the vanillin effects, mouse double minute 2 homolog (MDM2) inhibitor significantly attenuated suppressive effects of vanillin on UVB-induced activation of p53 reporter in KSC. Taken together, these findings suggest that vanillin protects KSC from UVB irradiation and its effects may occur through the suppression of downstream step of MDM2 in UVB irradiation-induced p53 activation.

  3. Human Keratinocytes have two interconvertible modes of proliferation

    PubMed Central

    Roshan, Amit; Murai, Kasumi; Fowler, Joanna; Simons, Benjamin D; Nikolaidou-Neokosmidou, Varvara; Jones, Philip H

    2016-01-01

    Summary Single stem cells, including those in human epidermis, have a remarkable ability to reconstitute tissues in vitro, but the cellular mechanisms that enable this are ill defined. We used live imaging to track the outcome of thousands of divisions in clonal cultures of primary human epidermal keratinocytes. Two modes of proliferation were seen. In ‘balanced’ mode, similar proportions of proliferating and differentiating cells were generated, achieving the ‘population asymmetry’ that sustains epidermal homeostasis in vivo. In ‘expanding’ mode, an excess of cycling cells was produced, generating large expanding colonies. Cells in expanding mode switched their behaviour to balanced mode once local confluence was attained. However when a confluent area is wounded in a scratch assay, cells near the scratch switch back to expanding mode until the defect is closed. We conclude that the ability of a single epidermal stem cell to reconstitute an epithelium is explained by two interconvertible modes of proliferation regulated by confluence. PMID:26641719

  4. Microgravity influences circadian clock oscillation in human keratinocytes

    PubMed Central

    Ranieri, Danilo; Cucina, Alessandra; Bizzarri, Mariano; Alimandi, Maurizio; Torrisi, Maria Rosaria

    2015-01-01

    Microgravity and sudden changes of gravitational forces exert numerous effects on tissues, organs and apparatus. Responses to these forces variably applied to cells indicate the existence of mechanotransduction pathways able to modulate transcription. Oscillation of circadian clocks similarly influences many cellular and metabolic processes. Here we hypothesized that signals derived from changes of gravitational forces applied to epidermal cells might influence their physiology in harmony with the oscillation of the molecular clock. In this study, we describe amplified oscillations of Bmal1 circadian clock gene in human keratinocytes exposed to short simulated microgravity and to rapid variation of gravitational forces. We found that exposure to microgravity enhances the amplitude of the Bmal1 feedback loop sustained by an apparently lower variability of Rev-erbα transcription, while recovery from microgravity is characterized by increased amplitude of Bmal1 expression and elongation of the oscillatory periods of Bmal1 and Rev-erbα. These data highlight the existence of integrated signaling network connecting mechanosensitive pathways to circadian gene regulation. PMID:26448904

  5. Actin filament dynamics impacts keratinocyte stem cell maintenance

    PubMed Central

    Nanba, Daisuke; Toki, Fujio; Matsushita, Natsuki; Matsushita, Sachi; Higashiyama, Shigeki; Barrandon, Yann

    2013-01-01

    Cultured human epidermal keratinocyte stem cells (holoclones) are crucial for regenerative medicine for burns and genetic disorders. In serial culture, holoclones progressively lose their proliferative capacity to become transient amplifying cells with limited growth (paraclones), a phenomenon termed clonal conversion. Although it negatively impacts the culture lifespan and the success of cell transplantation, little is known on the molecular mechanism underlying clonal conversion. Here, we show that holoclones and paraclones differ in their actin filament organization, with actin bundles distributed radially in holoclones and circumferentially in paraclones. Moreover, actin organization sets the stage for a differing response to epidermal growth factor (EGF), since EGF signalling induces a rapid expansion of colony size in holoclones and a significant reduction in paraclones. Furthermore, inhibition of PI3K or Rac1 in holoclones results in the reorganization of actin filaments in a pattern that is similar to that of paraclones. Importantly, continuous Rac1 inhibition in holoclones results in clonal conversion and reduction of growth potential. Together, our data connect loss of stem cells to EGF-induced colony dynamics governed by Rac1. PMID:23554171

  6. Transformation of human epidermal keratinocytes with fission neutrons.

    PubMed

    Thraves, P J; Varghese, S; Jung, M; Grdina, D J; Rhim, J S; Dritschilo, A

    1994-12-01

    The biological effects of exposures to high LET radiations have particular relevance to radiation protection and risk assessment. Since most cancers are of epithelial origin, it is important to obtain a better understanding of radiation-induced oncogenic transformation in this cell type. Accordingly we have initiated studies to determine whether immortalized human epidermal keratinocytes (RHEK) can be transformed with high LET radiations. Exponentially growing RHEK cells were treated with single doses (1, 10, 25, 50 and 100 cGy) of 0.85 MeV fission neutrons from the Janus reactor. Neutron exposure led to the development of morphologically altered cells and foci formation after 6 weeks at confluence. These transformed cultures grew with an increased saturation density, exhibited anchorage-independent growth and formed tumors in athymic mice. Single-strand conformational polymorphism analysis and DNA sequencing demonstrated the absence of point mutations in codons 12/13 and 61 in the Ha-ras, Ki-ras, or N-ras genes and exons 4-9 of the p53 tumor suppressor gene. These studies demonstrate that high LET radiations (fission neutrons) can transform immortalized human epithelial cells to a malignant phenotype that does not appear to involve mutations in either the cellular p53 or ras genes.

  7. Identification of copper/zinc superoxide dismutase as a nitric oxide-regulated gene in human (HaCaT) keratinocytes: implications for keratinocyte proliferation.

    PubMed

    Frank, S; Kämpfer, H; Podda, M; Kaufmann, R; Pfeilschifter, J

    2000-03-15

    Recent studies have demonstrated an induction of expression of inducible nitric oxide synthase that is associated with several inflammatory diseases of the skin. To define the mechanisms of action of nitric oxide (NO) in the skin, we attempted to identify genes that are regulated by NO in keratinocytes. Using the human keratinocyte cell line HaCaT as a model system, we identified a Cu/Zn superoxide dismutase (SOD) that was strongly induced by high concentrations (500 microM) of NO-donating agents ¿S-nitrosoglutathione, sodium nitroprusside and (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino] diazen-1-ium-1,2 -diolate (DETA-NO)¿, but not by serum or by single recombinant growth factors and inflammatory cytokines or by treatment with superoxide anions. Furthermore, endogenously produced NO increased the expression of Cu/Zn SOD mRNA in keratinocytes. Moreover, treatment of HaCaT cells with NO was associated with a biphasic effect on cell proliferation, because low doses (100 microM) of different NO donors (S-nitrosoglutathione and DETA-NO) mediated a proliferative signal to the cells, whereas high concentrations (500 microM) were cytostatic. To determine a possible correlation between the close regulation of Cu/Zn SOD expression and proliferation by NO in keratinocytes, we established a cell line (psp1CZ1N) carrying a human Cu/Zn SOD cDNA under the control of a ponasterone-inducible promoter construct. Ponasterone-induced overexpression of Cu/Zn SOD caused a cytostatic effect in proliferating psp1CZ1N cells. We therefore suggest that the up-regulation of Cu/Zn SOD expression by NO establishes an inhibitory mechanism on keratinocyte proliferation. PMID:10698699

  8. Contrasting signaling pathways of alpha1A- and alpha1B-adrenergic receptor subtype activation of phosphatidylinositol 3-kinase and Ras in transfected NIH3T3 cells.

    PubMed

    Hu, Z W; Shi, X Y; Lin, R Z; Hoffman, B B

    1999-01-01

    Activation of protein kinases is an important intermediate step in signaling pathways of many G protein-coupled receptors including alpha1-adrenergic receptors. The present study was designed to investigate the capacity of the three cloned subtypes of human alpha1-receptors, namely, alpha1A, alpha1B and alpha1D to activate phosphatidylinositol 3-kinase (PI 3-kinase) and p21ras in transfected NIH3T3 cells. Norepinephrine activated PI 3-kinase in cells expressing human alpha1A and alpha1B via pertussis toxin-insensitive G proteins; alpha1D-receptors did not detectably activate this kinase. Transient transfection of NIH 3T3 cells with the alpha-subunit of the G protein transducin (alpha(t)) a scavenger of betagamma-subunits released from activated G proteins, inhibited alpha1B-receptor but not alpha1A-receptor-stimulated PI 3-kinase activity. Stimulation of both alpha1A- and alpha1B-receptors activated p21ras and stimulated guanine nucleotide exchange on Ras protein. Overexpression of a dominant negative mutant of p21ras attenuated alpha1B-receptor but not alpha1A-receptor activation of PI 3-kinase. Overexpression of a dominant negative mutant of PI 3-kinase attenuated alpha1A- but not alpha1B-receptor-stimulated mitogen-activated protein kinase activity. These results demonstrate the capacity for heterologous signaling of the alpha1-adrenergic receptor subtypes in promoting cellular responses in NIH3T3 cells.

  9. Improvement of human keratinocyte migration by a redox active bioelectric dressing.

    PubMed

    Banerjee, Jaideep; Das Ghatak, Piya; Roy, Sashwati; Khanna, Savita; Sequin, Emily K; Bellman, Karen; Dickinson, Bryan C; Suri, Prerna; Subramaniam, Vish V; Chang, Christopher J; Sen, Chandan K

    2014-01-01

    Exogenous application of an electric field can direct cell migration and improve wound healing; however clinical application of the therapy remains elusive due to lack of a suitable device and hence, limitations in understanding the molecular mechanisms. Here we report on a novel FDA approved redox-active Ag/Zn bioelectric dressing (BED) which generates electric fields. To develop a mechanistic understanding of how the BED may potentially influence wound re-epithelialization, we direct emphasis on understanding the influence of BED on human keratinocyte cell migration. Mapping of the electrical field generated by BED led to the observation that BED increases keratinocyte migration by three mechanisms: (i) generating hydrogen peroxide, known to be a potent driver of redox signaling, (ii) phosphorylation of redox-sensitive IGF1R directly implicated in cell migration, and (iii) reduction of protein thiols and increase in integrinαv expression, both of which are known to be drivers of cell migration. BED also increased keratinocyte mitochondrial membrane potential consistent with its ability to fuel an energy demanding migration process. Electric fields generated by a Ag/Zn BED can cross-talk with keratinocytes via redox-dependent processes improving keratinocyte migration, a critical event in wound re-epithelialization. PMID:24595050

  10. Unsaturated fatty acids induce calcium influx into keratinocytes and cause abnormal differentiation of epidermis.

    PubMed

    Katsuta, Yuji; Iida, Toshii; Inomata, Shinji; Denda, Mitsuhiro

    2005-05-01

    Abnormal follicular keratinization is involved in comedogenesis in acne vulgaris. We recently demonstrated that calcium influx into epidermal keratinocytes is associated with impaired skin barrier function and epidermal proliferation. Based on these results, we hypothesized that sebum components affect calcium dynamics in the keratinocyte and consequently induce abnormal keratinization. To test this idea, we first observed the effects of topical application of sebum components, triglycerides (triolein), saturated fatty acids (palmitic acid and stearic acid), and unsaturated fatty acids (oleic acid and palmitoleic acid) on hairless mouse skin. Neither triglyceride nor saturated fatty acids affected the skin surface morphology or epidermal proliferation. On the other hand, application of unsaturated fatty acids, oleic acid, and palmitoleic acid induced scaly skin, abnormal keratinization, and epidermal hyperplasia. Application of triglycerides and saturated fatty acids on cultured human keratinocytes did not affect the intracellular calcium concentration ([Ca(2+)](i)), whereas unsaturated fatty acids increased the [Ca(2+)](i) of the keratinocytes. Moreover, application of oleic acid on hairless mouse skin induced an abnormal calcium distribution in the epidermis. These results suggest that unsaturated fatty acids in sebum alter the calcium dynamics in epidermal keratinocytes and induce abnormal follicular keratinization.

  11. A Glutathione-Nrf2-Thioredoxin Cross-Talk Ensures Keratinocyte Survival and Efficient Wound Repair

    PubMed Central

    Telorack, Michèle; Meyer, Michael; Ingold, Irina; Conrad, Marcus; Bloch, Wilhelm; Werner, Sabine

    2016-01-01

    The tripeptide glutathione is the most abundant cellular antioxidant with high medical relevance, and it is also required as a co-factor for various enzymes involved in the detoxification of reactive oxygen species and toxic compounds. However, its cell-type specific functions and its interaction with other cytoprotective molecules are largely unknown. Using a combination of mouse genetics, functional cell biology and pharmacology, we unraveled the function of glutathione in keratinocytes and its cross-talk with other antioxidant defense systems. Mice with keratinocyte-specific deficiency in glutamate cysteine ligase, which catalyzes the rate-limiting step in glutathione biosynthesis, showed a strong reduction in keratinocyte viability in vitro and in the skin in vivo. The cells died predominantly by apoptosis, but also showed features of ferroptosis and necroptosis. The increased cell death was associated with increased levels of reactive oxygen and nitrogen species, which caused DNA and mitochondrial damage. However, epidermal architecture, and even healing of excisional skin wounds were only mildly affected in the mutant mice. The cytoprotective transcription factor Nrf2 was strongly activated in glutathione-deficient keratinocytes, but additional loss of Nrf2 did not aggravate the phenotype, demonstrating that the cytoprotective effect of Nrf2 is glutathione dependent. However, we show that deficiency in glutathione biosynthesis is efficiently compensated in keratinocytes by the cysteine/cystine and thioredoxin systems. Therefore, our study highlights a remarkable antioxidant capacity of the epidermis that ensures skin integrity and efficient wound healing. PMID:26808544

  12. Melatonin protects skin keratinocyte from hydrogen peroxide-mediated cell death via the SIRT1 pathway

    PubMed Central

    Lee, Ju-Hee; Moon, Ji-Hong; Nazim, Uddin MD.; Lee, You-Jin; Seol, Jae-Won; Eo, Seong-Kug; Lee, John-Hwa; Park, Sang-Youel

    2016-01-01

    Melatonin (N-acetyl-5-methoxytryptamine), which is primarily synthesized in and secreted from the pineal gland, plays a pivotal role in cell proliferation as well as in the regulation of cell metastasis and cell survival in a diverse range of cells. The aim of this study is to investigate protection effect of melatonin on H2O2-induced cell damage and the mechanisms of melatonin in human keratinocytes. Hydrogen peroxide dose-dependently induced cell damages in human keratinocytes and co-treatment of melatonin protected the keratinocytes against H2O2-induced cell damage. Melatonin treatment activated the autophagy flux signals, which were identified by the decreased levels of p62 protein. Inhibition of autophagy flux via an autophagy inhibitor and ATG5 siRNA technique blocked the protective effects of melatonin against H2O2-induced cell death in human keratinocytes. And we found the inhibition of sirt1 using sirtinol and sirt1 siRNA reversed the protective effects of melatonin and induces the autophagy process in H2O2-treated cells. This is the first report demonstrating that autophagy flux activated by melatonin protects human keratinocytes through sirt1 pathway against hydrogen peroxide-induced damages. And this study also suggest that melatonin could potentially be utilized as a therapeutic agent in skin disease. PMID:26918354

  13. Effective inhibition of melanosome transfer to keratinocytes by lectins and niacinamide is reversible.

    PubMed

    Greatens, Amanda; Hakozaki, Tomohiro; Koshoffer, Amy; Epstein, Howard; Schwemberger, Sandy; Babcock, George; Bissett, Donald; Takiwaki, Hirotsugu; Arase, Seiji; Wickett, R Randall; Boissy, Raymond E

    2005-07-01

    Skin pigmentation results in part from the transfer of melanized melanosomes synthesized by melanocytes to neighboring keratinocytes. Plasma membrane lectins and their glycoconjugates expressed by these epidermal cells are critical molecules involved in this transfer process. In addition, the derivative of vitamin B(3), niacinamide, can inhibit melanosome transfer and induce skin lightening. We investigated the effects of these molecules on the viability of melanocytes and keratinocytes and on the reversibility of melanosome-transfer inhibition induced by these agents using an in vitro melanocyte-keratinocyte coculture model system. While lectins and neoglycoproteins could induce apoptosis in a dose-dependent manner to melanocytes or keratinocytes in monoculture, similar dosages of the lectins, as opposed to neoglycoproteins, did not induce apoptosis to either cell type when treated in coculture. The dosages of lectins and niacinamide not affecting cell viability produced an inhibitory effect on melanosome transfer, when used either alone or together in cocultures of melanocytes-keratinocytes. Cocultures treated with lectins or niacinamide resumed normal melanosome transfer in 3 days after removal of the inhibitor, while cocultures treated with a combination of lectins and niacinamide demonstrated a lag in this recovery. Subsequently, we assessed the effect of niacinamide on facial hyperpigmented spots using a vehicle-controlled, split-faced design human clinical trial. Topical application of niacinamide resulted in a dose-dependent and reversible reduction in hyperpigmented lesions. These results suggest that lectins and niacinamide at concentrations that do not affect cell viability are reversible inhibitors of melanosome transfer.

  14. Induction of proteins and mRNAs after uv irradiation of human epidermal keratinocytes

    SciTech Connect

    Kartasova, T.; Ponec, M.; van de Putte, P.

    1988-02-01

    uv sensitivity of cultured human epidermal keratinocytes was analyzed at different growth conditions and compared with the sensitivity of dermal fibroblasts derived from the same skin specimen. No significant differences in survival curves were found between these two cell types, although keratinocytes grown under standard conditions were slightly more resistant to uv irradiation than fibroblasts. The extracellular concentration of calcium appeared to be critical not only in the regulation of keratinocyte proliferation and differentiation, but also in the uv sensitivity of these cells: keratinocytes grown under conditions which favor cell proliferation (low calcium concentration) are more resistant to uv irradiation than those grown under conditions favoring differentiation (high calcium concentration). Two-dimensional protein gel electrophoresis was used to detect a possible effect of uv irradiation on the accumulation of specific mRNAs in the cytoplasm and/or on the synthesis of specific proteins. Proteins were pulse labeled in vivo with (/sup 35/S)methionine or synthesized in vitro in rabbit reticulocyte lysates on mRNA isolated from keratinocytes that were irradiated with different uv doses at different periods of time prior to isolation. Alterations in expression were demonstrated for several proteins in both in vivo and in vitro experiments.

  15. Improvement of human keratinocyte migration by a redox active bioelectric dressing.

    PubMed

    Banerjee, Jaideep; Das Ghatak, Piya; Roy, Sashwati; Khanna, Savita; Sequin, Emily K; Bellman, Karen; Dickinson, Bryan C; Suri, Prerna; Subramaniam, Vish V; Chang, Christopher J; Sen, Chandan K

    2014-01-01

    Exogenous application of an electric field can direct cell migration and improve wound healing; however clinical application of the therapy remains elusive due to lack of a suitable device and hence, limitations in understanding the molecular mechanisms. Here we report on a novel FDA approved redox-active Ag/Zn bioelectric dressing (BED) which generates electric fields. To develop a mechanistic understanding of how the BED may potentially influence wound re-epithelialization, we direct emphasis on understanding the influence of BED on human keratinocyte cell migration. Mapping of the electrical field generated by BED led to the observation that BED increases keratinocyte migration by three mechanisms: (i) generating hydrogen peroxide, known to be a potent driver of redox signaling, (ii) phosphorylation of redox-sensitive IGF1R directly implicated in cell migration, and (iii) reduction of protein thiols and increase in integrinαv expression, both of which are known to be drivers of cell migration. BED also increased keratinocyte mitochondrial membrane potential consistent with its ability to fuel an energy demanding migration process. Electric fields generated by a Ag/Zn BED can cross-talk with keratinocytes via redox-dependent processes improving keratinocyte migration, a critical event in wound re-epithelialization.

  16. Sharpin prevents skin inflammation by inhibiting TNFR1-induced keratinocyte apoptosis

    PubMed Central

    Kumari, Snehlata; Redouane, Younes; Lopez-Mosqueda, Jaime; Shiraishi, Ryoko; Romanowska, Malgorzata; Lutzmayer, Stefan; Kuiper, Jan; Martinez, Conception; Dikic, Ivan; Pasparakis, Manolis; Ikeda, Fumiyo

    2014-01-01

    Linear Ubiquitin chain Assembly Complex (LUBAC) is an E3 ligase complex that generates linear ubiquitin chains and is important for tumour necrosis factor (TNF) signaling activation. Mice lacking Sharpin, a critical subunit of LUBAC, spontaneously develop inflammatory lesions in the skin and other organs. Here we show that TNF receptor 1 (TNFR1)-associated death domain (TRADD)-dependent TNFR1 signaling in epidermal keratinocytes drives skin inflammation in Sharpin-deficient mice. Epidermis-restricted ablation of Fas-associated protein with death domain (FADD) combined with receptor-interacting protein kinase 3 (RIPK3) deficiency fully prevented skin inflammation, while single RIPK3 deficiency only delayed and partly ameliorated lesion development in Sharpin-deficient mice, showing that inflammation is primarily driven by TRADD- and FADD-dependent keratinocyte apoptosis while necroptosis plays a minor role. At the cellular level, Sharpin deficiency sensitized primary murine keratinocytes, human keratinocytes, and mouse embryonic fibroblasts to TNF-induced apoptosis. Depletion of FADD or TRADD in Sharpin-deficient HaCaT cells suppressed TNF-induced apoptosis, indicating the importance of FADD and TRADD in Sharpin-dependent anti-apoptosis signaling in keratinocytes. DOI: http://dx.doi.org/10.7554/eLife.03422.001 PMID:25443631

  17. A Glutathione-Nrf2-Thioredoxin Cross-Talk Ensures Keratinocyte Survival and Efficient Wound Repair.

    PubMed

    Telorack, Michèle; Meyer, Michael; Ingold, Irina; Conrad, Marcus; Bloch, Wilhelm; Werner, Sabine

    2016-01-01

    The tripeptide glutathione is the most abundant cellular antioxidant with high medical relevance, and it is also required as a co-factor for various enzymes involved in the detoxification of reactive oxygen species and toxic compounds. However, its cell-type specific functions and its interaction with other cytoprotective molecules are largely unknown. Using a combination of mouse genetics, functional cell biology and pharmacology, we unraveled the function of glutathione in keratinocytes and its cross-talk with other antioxidant defense systems. Mice with keratinocyte-specific deficiency in glutamate cysteine ligase, which catalyzes the rate-limiting step in glutathione biosynthesis, showed a strong reduction in keratinocyte viability in vitro and in the skin in vivo. The cells died predominantly by apoptosis, but also showed features of ferroptosis and necroptosis. The increased cell death was associated with increased levels of reactive oxygen and nitrogen species, which caused DNA and mitochondrial damage. However, epidermal architecture, and even healing of excisional skin wounds were only mildly affected in the mutant mice. The cytoprotective transcription factor Nrf2 was strongly activated in glutathione-deficient keratinocytes, but additional loss of Nrf2 did not aggravate the phenotype, demonstrating that the cytoprotective effect of Nrf2 is glutathione dependent. However, we show that deficiency in glutathione biosynthesis is efficiently compensated in keratinocytes by the cysteine/cystine and thioredoxin systems. Therefore, our study highlights a remarkable antioxidant capacity of the epidermis that ensures skin integrity and efficient wound healing. PMID:26808544

  18. Exogenous hydrogen sulfide promotes cell proliferation and differentiation by modulating autophagy in human keratinocytes.

    PubMed

    Xie, Xin; Dai, Hui; Zhuang, Binyu; Chai, Li; Xie, Yanguang; Li, Yuzhen

    2016-04-01

    The effects and the underlying mechanisms of hydrogen sulfide (H2S) on keratinocyte proliferation and differentiation are still less known. In the current study, we investigated the effects and the underlying mechanisms of exogenous H2S on keratinocyte proliferation and differentiation. Human keratinocytes (HaCaT cells) were treated with various concentrations (0.05, 0.25, 0.5 and 1 mM) of sodium hydrosulfide (NaHS, a donor of H2S) for 24 h. A CCK-8 assay was used to assess cell viability. Western blot analysis was performed to determine the expression levels of proteins associated with differentiation and autophagy. Transmission electron microscopy was performed to observe autophagic vacuoles, and flow cytometry was applied to evaluate apoptosis. NaHS promoted the viability, induced the differentiation, and enhanced autophagic activity in a dose-dependent manner in HaCaT cells but had no effect on cell apoptosis. Blockage of autophagy by ATG5 siRNA inhibited NaHS-induced cell proliferation and differentiation. The current study demonstrated that autophagy in response to exogenous H2S treatment promoted keratinocyte proliferation and differentiation. Our results provide additional insights into the potential role of autophagy in keratinocyte proliferation and differentiation.

  19. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts

    PubMed Central

    Ramos-Jerz, Maria del R.; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M.

    2013-01-01

    Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes. PMID:24371457

  20. Improvement of Human Keratinocyte Migration by a Redox Active Bioelectric Dressing

    PubMed Central

    Banerjee, Jaideep; Das Ghatak, Piya; Roy, Sashwati; Khanna, Savita; Sequin, Emily K.; Bellman, Karen; Dickinson, Bryan C.; Suri, Prerna; Subramaniam, Vish V.; Chang, Christopher J.; Sen, Chandan K.

    2014-01-01

    Exogenous application of an electric field can direct cell migration and improve wound healing; however clinical application of the therapy remains elusive due to lack of a suitable device and hence, limitations in understanding the molecular mechanisms. Here we report on a novel FDA approved redox-active Ag/Zn bioelectric dressing (BED) which generates electric fields. To develop a mechanistic understanding of how the BED may potentially influence wound re-epithelialization, we direct emphasis on understanding the influence of BED on human keratinocyte cell migration. Mapping of the electrical field generated by BED led to the observation that BED increases keratinocyte migration by three mechanisms: (i) generating hydrogen peroxide, known to be a potent driver of redox signaling, (ii) phosphorylation of redox-sensitive IGF1R directly implicated in cell migration, and (iii) reduction of protein thiols and increase in integrinαv expression, both of which are known to be drivers of cell migration. BED also increased keratinocyte mitochondrial membrane potential consistent with its ability to fuel an energy demanding migration process. Electric fields generated by a Ag/Zn BED can cross-talk with keratinocytes via redox-dependent processes improving keratinocyte migration, a critical event in wound re-epithelialization. PMID:24595050

  1. Human keratinocytes restrict chikungunya virus replication at a post-fusion step

    SciTech Connect

    Bernard, Eric; Simmons, Graham; Chazal, Nathalie; and others

    2015-02-15

    Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses. - Highlights: • Human keratinocytes support endocytosis of CHIKV and fusion of viral membranes. • CHIKV replication is blocked at a post entry step in these cells. • Infection upregulates type-I, –II and –III IFN genes expression. • Keratinocytes behave as immune sentinels against CHIKV.

  2. Production of Superoxide Anions by Keratinocytes Initiates P. acnes-Induced Inflammation of the Skin

    PubMed Central

    Grange, Philippe A.; Chéreau, Christiane; Raingeaud, Joël; Nicco, Carole; Weill, Bernard

    2009-01-01

    Acne vulgaris is a chronic inflammatory disorder of the sebaceous follicles. Propionibacterium acnes (P. acnes), a gram-positive anareobic bacterium, plays a critical role in the development of these inflammatory lesions. This study aimed at determining whether reactive oxygen species (ROS) are produced by keratinocytes upon P. acnes infection, dissecting the mechanism of this production, and investigating how this phenomenon integrates in the general inflammatory response induced by P. acnes. In our hands, ROS, and especially superoxide anions (O2•−), were rapidly produced by keratinocytes upon stimulation by P. acnes surface proteins. In P. acnes-stimulated keratinocytes, O2•− was produced by NAD(P)H oxidase through activation of the scavenger receptor CD36. O2•− was dismuted by superoxide dismutase to form hydrogen peroxide which was further detoxified into water by the GSH/GPx system. In addition, P. acnes-induced O2•− abrogated P. acnes growth and was involved in keratinocyte lysis through the combination of O2•− with nitric oxide to form peroxynitrites. Finally, retinoic acid derivates, the most efficient anti-acneic drugs, prevent O2•− production, IL-8 release and keratinocyte apoptosis, suggesting the relevance of this pathway in humans. PMID:19629174

  3. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts.

    PubMed

    Ramos-Jerz, Maria Del R; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M

    2013-01-01

    Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes. PMID:24371457

  4. Inhibition of apoptosis by expression of antiapoptotic proteins in recombinant human keratinocytes.

    PubMed

    Choi, Claudia Y U; Reimers, Kerstin; Allmeling, Christina; Kall, Susanne; Choi, Yeong-Hoon; Vogt, Peter M

    2007-01-01

    The Fas ligand/Fas interaction plays an important role in the regulation of immune responses. Allografted cells undergo Fas-mediated apoptosis induced by CD8+ T cells. Our objective was to prevent human keratinocytes from immunologically induced apoptosis. We focused on three proteins with inhibitory function on Fas-mediated apoptosis. Human keratinocytes were transfected with either Flip, Faim, or Lifeguard (LFG). The treatment proved to be practicable and efficient. The recombinant keratinocytes with expression of our target proteins were cocultured with CD8+ T cells and the apoptotic activity was then evaluated. Activation of caspase-8 was detectable in control but not in the recombinant cells. Quantitative analysis revealed significant induction of T-cell-induced apoptosis in nontransfected keratinocytes (p = 0.04, n = 12) but not in Flip (p = 0.66), Faim (p = 0.42), or LFG (p = 0.44) expressing cells. Our results suggest that heterotopic expression of antiapoptotic proteins can induce the resistance of keratinocytes to a major mechanism of rejection.

  5. Increased migration of murine keratinocytes under hypoxia is mediated by induction of urokinase plasminogen activator.

    PubMed

    Daniel, Richard J; Groves, Richard W

    2002-12-01

    One of the key consequences of cutaneous wounding is the development of tissue hypoxia. Recent data have suggested that this is a potent stimulus for increased keratinocyte migration and hence re-epithelialization, although the mechanisms responsible for this remain unclear. In this study we have investigated the relationship between hypoxia, plasminogen activation, and in vitro wound healing. Exposure of keratinocyte cultures to hypoxia resulted in upregulation of urokinase plasminogen activator mRNA and a subsequent increase in urokinase plasminogen activator-mediated plasminogen activation, as determined by indirect chromogenic peptide assay and plasminogen-linked zymography. Analysis of keratinocyte wound healing in vitro confirmed enhanced wound closure in hypoxic cultures compared with normoxic cultures after 16 h. Pretreatment of normoxic and hypoxic cultures with mitomycin C and cytochalasin B indicated that in this system wound closure was due to keratinocyte migration rather than proliferation. Addition of the broad-spectrum serine proteinase inhibitor, p-aminobenzamidine, or the specific urokinase plasminogen activator inhibitors, amiloride and WX-293, significantly reduced wound closure in hypoxic cultures and abrogated the hypoxic enhancement of wound closure. These data indicate a central role for urokinase plasminogen activators in hypoxic keratinocyte migration and suggest a potential mechanism for enhanced re-epithelialization of wounds under low oxygen tensions. PMID:12485432

  6. Extracellular vesicles are transferred from melanocytes to keratinocytes after UVA irradiation

    PubMed Central

    Wäster, Petra; Eriksson, Ida; Vainikka, Linda; Rosdahl, Inger; Öllinger, Karin

    2016-01-01

    Ultraviolet (UV) irradiation induces skin pigmentation, which relies on the intercellular crosstalk of melanin between melanocytes to keratinocytes. However, studying the separate effects of UVA and UVB irradiation reveals differences in cellular response. Herein, we show an immediate shedding of extracellular vesicles (EVs) from the plasma membrane when exposing human melanocytes to UVA, but not UVB. The EV-shedding is preceded by UVA-induced plasma membrane damage, which is rapidly repaired by Ca2+-dependent lysosomal exocytosis. Using co-cultures of melanocytes and keratinocytes, we show that EVs are preferably endocytosed by keratinocytes. Importantly, EV-formation is prevented by the inhibition of exocytosis and increased lysosomal pH but is not affected by actin and microtubule inhibitors. Melanosome transfer from melanocytes to keratinocytes is equally stimulated by UVA and UVB and depends on a functional cytoskeleton. In conclusion, we show a novel cell response after UVA irradiation, resulting in transfer of lysosome-derived EVs from melanocytes to keratinocytes. PMID:27293048

  7. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts.

    PubMed

    Ramos-Jerz, Maria Del R; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M

    2013-01-01

    Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes.

  8. Consistency of the Proteome in Primary Human Keratinocytes With Respect to Gender, Age, and Skin Localization*

    PubMed Central

    Sprenger, Adrian; Weber, Sebastian; Zarai, Mostafa; Engelke, Rudolf; Nascimento, Juliana M.; Gretzmeier, Christine; Hilpert, Martin; Boerries, Melanie; Has, Cristina; Busch, Hauke; Bruckner-Tuderman, Leena; Dengjel, Jörn

    2013-01-01

    Keratinocytes account for 95% of all cells of the epidermis, the stratified squamous epithelium forming the outer layer of the skin, in which a significant number of skin diseases takes root. Immortalized keratinocyte cell lines are often used as research model systems providing standardized, reproducible, and homogenous biological material. Apart from that, primary human keratinocytes are frequently used for medical studies because the skin provides an important route for drug administration and is readily accessible for biopsies. However, comparability of these cell systems is not known. Cell lines may undergo phenotypic shifts and may differ from the in vivo situation in important aspects. Primary cells, on the other hand, may vary in biological functions depending on gender and age of the donor and localization of the biopsy specimen. Here we employed metabolic labeling in combination with quantitative mass spectrometry-based proteomics to assess A431 and HaCaT cell lines for their suitability as model systems. Compared with cell lines, comprehensive profiling of the primary human keratinocyte proteome with respect to gender, age, and skin localization identified an unexpected high proteomic consistency. The data were analyzed by an improved ontology enrichment analysis workflow designed for the study of global proteomics experiments. It enables a quick, comprehensive and unbiased overview of altered biological phenomena and links experimental data to literature. We guide through our workflow, point out its advantages compared with other methods and apply it to visualize differences of cell lines compared with primary human keratinocytes. PMID:23722187

  9. Dual mechanisms of green tea extract (EGCG)-induced cell survival in human epidermal keratinocytes.

    PubMed

    Chung, Jin Ho; Han, Ji Hyun; Hwang, Eun Ju; Seo, Jin Young; Cho, Kwang Hyun; Kim, Kyu Han; Youn, Jai Il; Eun, Hee Chul

    2003-10-01

    Beneficial effects attributed to green tea, such as its anticancer and antioxidant properties, may be mediated by (-)-epigallocatechin-3-gallate (EGCG). In this study, the effects of EGCG on cell proliferation and UV-induced apoptosis were investigated in normal epidermal keratinocytes. When topically applied to aged human skin, EGCG stimulated the proliferation of epidermal keratinocytes, which increased the epidermal thickness. In addition, this topical application also inhibited the UV-induced apoptosis of epidermal keratinocytes. EGCG was found to increase the phosphorylation of Bad protein at the Ser112 and Ser136. Moreover, EGCG-induced Erk phosphorylation was found to be critical for the phosphorylation of Ser112 in Bad protein, and the EGCG-induced activation of the Akt pathway was found to be involved in the phosphorylation of Ser136. Furthermore, EGCG increased Bcl-2 expression but decreased Bax expression, causing an increase in the Bcl-2-to-Bax ratio. In addition, we demonstrate the differential growth inhibitory effects of EGCG on cancer cells. In conclusion, this study demonstrates that EGCG promotes keratinocyte survival and inhibits the UV-induced apoptosis via two mechanisms: by phosphorylating Ser112 and Ser136 of Bad protein through Erk and Akt pathways, respectively, and by increasing the Bcl-2-to-Bax ratio. Moreover, these two proposed mechanisms of EGCG-induced cell proliferation may differ kinetically to promote keratinocyte survival.

  10. Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes.

    PubMed

    Hung, Sandy S C; Pébay, Alice; Wong, Raymond C B

    2015-01-01

    Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

  11. Human papillomavirus causes an angiogenic switch in keratinocytes which is sufficient to alter endothelial cell behavior

    SciTech Connect

    Chen, W.; Li, F.; Mead, L.; White, H.; Walker, J.; Ingram, D.A.; Roman, A.

    2007-10-10

    One of the requirements for tumor growth is the ability to recruit a blood supply, a process known as angiogenesis. Angiogenesis begins early in the progression of cervical disease from mild to severe dysplasia and on to invasive cancer. We have previously reported that expression of human papillomavirus type 16 E6 and E7 (HPV16 E6E7) proteins in primary foreskin keratinocytes (HFKs) decreases expression of two inhibitors and increases expression of two angiogenic inducers [Toussaint-Smith, E., Donner, D.B., Roman, A., 2004. Expression of human papillomavirus type 16 E6 and E7 oncoproteins in primary foreskin keratinocytes is sufficient to alter the expression of angiogenic factors. Oncogene 23, 2988-2995]. Here we report that HPV-induced early changes in the keratinocyte phenotype are sufficient to alter endothelial cell behavior both in vitro and in vivo. Conditioned media from HPV16 E6E7 expressing HFKs as well as from human cervical keratinocytes containing the intact HPV16 were able to stimulate proliferation and migration of human microvascular endothelial cells. In addition, introduction of the conditioned media into immunocompetent mice using a Matrigel plug model resulted in a clear angiogenic response. These novel data support the hypothesis that HPV proteins contribute not only to the uncontrolled keratinocyte growth seen following HPV infection but also to the angiogenic response needed for tumor formation.

  12. The Effect of Titanium Dioxide Nanoparticles on Keratinocyte Cell (KC) and Squamous Cell Carcinoma (SCC-13)

    NASA Astrophysics Data System (ADS)

    Lin, Chienhsiu; Simon, Marcia; Jurukovski, Vladimir; Lee, Wilson; Rafailovich, Miriam

    2009-03-01

    We have studied the effects of TiO2 nanoparticles on cell keratinocyte and SCC (Squamous Cell Carcinoma) cells. We found that the concentration of particles required to adversely affect the cells was many times higher for keratinocyte than SCC cells. Confocal microscope shows that the particles in keratinocyte culture are sequestered in membranes between the cell colonies. The particles penetrated into the cells in the case of the SCC cells. TEM images revealed very few particles in the keratinocyte, many more particles were observed sequestered in vacuole of the SCC cells. These results indicate that the keratinocyte layer behaves very different from the fibroblast layers which are much more sensate to TiO2 nanoparticle damage and may suggest a protection mechanism of the dermal tissue. The effect of UV exposure in the presence of DNA was also investigated. We found that adsorbed proteins, as well as grafted polymer provided a measure of protection against free radical formation. The effects of low level UV exposure when the particles are near in-vitro cell culture will be presented.

  13. Integrin-Linked Kinase Is Indispensable for Keratinocyte Differentiation and Epidermal Barrier Function.

    PubMed

    Sayedyahossein, Samar; Rudkouskaya, Alena; Leclerc, Valerie; Dagnino, Lina

    2016-02-01

    A functional permeability barrier is essential to prevent the passage of water and electrolytes, macromolecules, and pathogens through the epidermis. This is accomplished in terminally differentiated keratinocytes through formation of a cornified envelope and the assembly of tight intercellular junctions. Integrin-linked kinase (ILK) is a scaffold protein essential for hair follicle morphogenesis and epidermal attachment to the basement membrane. However, the biological functions of ILK in differentiated keratinocytes remain poorly understood. Furthermore, whether ILK is implicated in keratinocyte differentiation and intercellular junction formation has remained an unresolved issue. Here we describe a pivotal role for ILK in keratinocyte differentiation responses to increased extracellular Ca(2+), regulation of adherens and tight junction assembly, and the formation of an outside-in permeability barrier toward macromolecules. In the absence of ILK, the calcium sensing receptor, E-cadherin, and ZO-1 fail to translocate to the cell membrane, through mechanisms that involve abnormalities in microtubules and in RhoA activation. In situ, ILK-deficient epidermis exhibits reduced tight junction formation and increased outside-in permeability to a dextran tracer, indicating reduced barrier properties toward macromolecules. Therefore, ILK is an essential component of keratinocyte differentiation programs that contribute to epidermal integrity and the establishment of its barrier properties. PMID:26967476

  14. Sodium fluoride influences the expression of keratins in cultured keratinocytes.

    PubMed

    Prado, Euridice; Wurtz, Tilmann; Ferbus, Didier; Shabana, El-Hassan; Forest, Nadine; Berdal, Ariane

    2011-02-01

    Epithelia in lung, skin, and kidney are often exposed to fluoride, and tissue damage in lung and kidney due to fluoride is well documented. Nevertheless, the biological effects of fluoride on epithelia are poorly investigated. In the present study, we report effects of sodium fluoride (NaF) on the differentiation of a human epithelial cell line, HaCaT. These cells may serve as a keratinocyte model, because they express a wide spectrum of keratins (Ks), and they associate into stratified tissue-like arrangements along with changes in their keratin pattern. NaF was added to the culture medium at concentrations of 0.5 and 5 mM. Cell proliferation remained intact, but cell functions were altered at high dose, and HSP70 was induced. Reverse transcription-polymerase chain reaction and Western blotting revealed that keratin (K) 15 mRNA and protein expression, associated with stratification of epithelia, were inhibited. Also, expression of keratins typical for terminal differentiation, K1 and K10, was decreased and so was the expression of the K1/10 regulating enhancer binding protein c/EBP alpha. Stratification of HaCaT cells was abolished at high fluoride dose, as assessed by electron microscopy. The changes in keratin expression were not reversed by withdrawal of fluoride. Taken together, NaF at high dose blocked terminal differentiation of HaCaT cells, visible by keratin expression and failing stratification. This effect may disturb tissue formation due to altered cell interactions.

  15. The transcriptional coactivator DRIP/mediator complex is involved in vitamin D receptor function and regulates keratinocyte proliferation and differentiation.

    PubMed

    Oda, Yuko; Chalkley, Robert J; Burlingame, Alma L; Bikle, Daniel D

    2010-10-01

    Mediator is a multisubunit coactivator complex that facilitates transcription of nuclear receptors. We investigated the role of the mediator complex as a coactivator for vitamin D receptor (VDR) in keratinocytes. Using VDR affinity beads, the vitamin D receptor interacting protein (DRIP)/mediator complex was purified from primary keratinocytes, and its subunit composition was determined by mass spectrometry. The complex included core subunits, such as DRIP205/MED1 (MED1), that directly binds to VDR. Additional subunits were identified that are components of the RNA polymerase II complex. The functions of different mediator components were investigated by silencing its subunits. The core subunit MED1 facilitates VDR activity and regulating keratinocyte proliferation and differentiation. A newly described subunit MED21 also has a role in promoting keratinocyte proliferation and differentiation, whereas MED10 has an inhibitory role. Blocking MED1/MED21 expression caused hyperproliferation of keratinocytes, accompanied by increases in mRNA expression of the cell cycle regulator cyclin D1 and/or glioma-associated oncogene homolog. Blocking MED1 or MED21 expression also resulted in defects in calcium-induced keratinocyte differentiation, as indicated by decreased expression of differentiation markers and decreased translocation of E-cadherin to the membrane. These results show that keratinocytes use the transcriptional coactivator mediator to regulate VDR functions and control keratinocyte proliferation and differentiation.

  16. Platelets Regulate the Migration of Keratinocytes via Podoplanin/CLEC-2 Signaling during Cutaneous Wound Healing in Mice.

    PubMed

    Asai, Jun; Hirakawa, Satoshi; Sakabe, Jun-ichi; Kishida, Tsunao; Wada, Makoto; Nakamura, Naomi; Takenaka, Hideya; Mazda, Osam; Urano, Tetsumei; Suzuki-Inoue, Katsue; Tokura, Yoshiki; Katoh, Norito

    2016-01-01

    Podoplanin is an endogenous ligand for C-type lectin-like receptor 2 (CLEC-2), which is expressed on platelets. Recent evidence indicates that this specific marker of lymphatic endothelial cells is also expressed by keratinocytes at the edge of wounds. However, whether podoplanin or platelets play a role in keratinocyte activity during wound healing remains unknown. We evaluated the effect of podoplanin expression levels on keratinocyte motility using cultured primary normal human epidermal keratinocytes (NHEKs). Down-regulation of podoplanin in NHEKs via transfection with podoplanin siRNA inhibited their migration, indicating that podoplanin plays a mandatory role in this process. In addition, down-regulation of podoplanin was correlated with up-regulation of E-cadherin, suggesting that podoplanin-mediated stimulation of keratinocyte migration is associated with a loss of E-cadherin. Both the addition of platelets and treatment with CLEC-2 inhibited the migration of NHEKs. The down-regulation of RhoA activity and the up-regulation of E-cadherin in keratinocytes were also induced by CLEC-2. In conclusion, these results suggest that podoplanin/CLEC-2 signaling regulates keratinocyte migration via modulating E-cadherin expression through RhoA signaling. Altering the regulation of keratinocyte migration by podoplanin might be a novel therapeutic approach to improve wound healing. PMID:26597882

  17. Peptides from Tetraspanin CD9 Are Potent Inhibitors of Staphylococcus Aureus Adherence to Keratinocytes.

    PubMed

    Ventress, Jennifer K; Partridge, Lynda J; Read, Robert C; Cozens, Daniel; MacNeil, Sheila; Monk, Peter N

    2016-01-01

    Staphylococcus aureus is one of the primary causative agents of skin and wound infections. As bacterial adherence is essential for infection, blocking this step can reduce invasion of host tissues by pathogens. An anti-adhesion therapy, based on a host membrane protein family, the tetraspanins, has been developed that can inhibit the adhesion of S. aureus to human cells. Synthetic peptides derived from a keratinocyte-expressed tetraspanin, CD9, were tested for anti-adhesive properties and at low nanomolar concentrations were shown to inhibit bacterial adhesion to cultured keratinocytes and to be effective in a tissue engineered model of human skin infection. These potential therapeutics had no effect on keratinocyte viability, migration or proliferation, indicating that they could be a valuable addition to current treatments for skin infection. PMID:27467693

  18. Insulin binding properties of normal and transformed human epidermal cultured keratinocytes

    SciTech Connect

    Verrando, P.; Ortonne, J.P.

    1985-10-01

    Insulin binding to its receptors was studied in cultured normal and transformed (A431 line) human epidermal keratinocytes. The specific binding was a temperature-dependent, saturable process. Normal keratinocytes possess a mean value of about 80,000 receptors per cell. Fifteen hours exposure of the cells to insulin lowered their receptor number (about 65% loss in available sites); these reappeared when the hormone was removed from the culture medium. In the A431 epidermoid carcinoma cell line, there is a net decrease in insulin binding (84% of the initial bound/free hormone ratio in comparison with normal cells) essentially related to a loss in receptor affinity for insulin. Thus, cultured human keratinocytes which express insulin receptors may be a useful tool in understanding skin pathology related to insulin disorders.

  19. Isorhamnetin Protects Human Keratinocytes against Ultraviolet B-Induced Cell Damage

    PubMed Central

    Han, Xia; Piao, Mei Jing; Kim, Ki Cheon; Madduma Hewage, Susara Ruwan Kumara; Yoo, Eun Sook; Koh, Young Sang; Kang, Hee Kyoung; Shin, Jennifer H; Park, Yeunsoo; Yoo, Suk Jae; Chae, Sungwook; Hyun, Jin Won

    2015-01-01

    Isorhamnetin (3-methylquercetin) is a flavonoid derived from the fruits of certain medicinal plants. This study investigated the photoprotective properties of isorhamnetin against cell damage and apoptosis resulting from excessive ultraviolet (UV) B exposure in human HaCaT keratinocytes. Isorhamnetin eliminated UVB-induced intracellular reactive oxygen species (ROS) and attenuated the oxidative modification of DNA, lipids, and proteins in response to UVB radiation. Moreover, isorhamnetin repressed UVB-facilitated programmed cell death in the keratinocytes, as evidenced by a reduction in apoptotic body formation, and nuclear fragmentation. Additionally, isorhamnetin suppressed the ability of UVB light to trigger mitochondrial dysfunction. Taken together, these results indicate that isorhamnetin has the potential to protect human keratinocytes against UVB-induced cell damage and death. PMID:26157553

  20. Peptides from Tetraspanin CD9 Are Potent Inhibitors of Staphylococcus Aureus Adherence to Keratinocytes

    PubMed Central

    Ventress, Jennifer K.; Partridge, Lynda J.; Read, Robert C.; Cozens, Daniel; MacNeil, Sheila

    2016-01-01

    Staphylococcus aureus is one of the primary causative agents of skin and wound infections. As bacterial adherence is essential for infection, blocking this step can reduce invasion of host tissues by pathogens. An anti-adhesion therapy, based on a host membrane protein family, the tetraspanins, has been developed that can inhibit the adhesion of S. aureus to human cells. Synthetic peptides derived from a keratinocyte-expressed tetraspanin, CD9, were tested for anti-adhesive properties and at low nanomolar concentrations were shown to inhibit bacterial adhesion to cultured keratinocytes and to be effective in a tissue engineered model of human skin infection. These potential therapeutics had no effect on keratinocyte viability, migration or proliferation, indicating that they could be a valuable addition to current treatments for skin infection. PMID:27467693

  1. Collagen-gel-induced resistance of overlying keratinocyte cultures to photosensitization

    NASA Astrophysics Data System (ADS)

    Katsantonis, John C.; Georgiou, Savas K.; Providaki, Mary G.; Vlahonikolis, John G.; Tosca, Andronicki D.

    1997-12-01

    In the present study it was found that human keratinocytes grown on collagen substrate, exhibited increased resistance to the hematoprophyrin-mediated photodynamic treatment, in comparison to keratinocytes grown on Petri dishes without collagen. Interestingly, no protection was afforded by the collagen gel to the cells in the corresponding control ('hematoprophyrin only' and 'light only') experiments. This observation was found to be independent of light dose and drug concentration, and the relative degree of resistance was the same for both normal and malignant cells. The degree of keratinocyte resistance was found to be closely related to the duration of cell attachment on the collagen substrate. These results are indicative of an active interference of collagen gel with the cellular evolution of the photodynamic phenomenon and they are also suggestive of variation in the photodynamic treatment efficacy according to the cellular environment.

  2. Human keratinocytes restrict chikungunya virus replication at a post-fusion step.

    PubMed

    Bernard, Eric; Hamel, Rodolphe; Neyret, Aymeric; Ekchariyawat, Peeraya; Molès, Jean-Pierre; Simmons, Graham; Chazal, Nathalie; Desprès, Philippe; Missé, Dorothée; Briant, Laurence

    2015-02-01

    Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses.

  3. Constitutive phenolics of Harpephyllum caffrum (Anacardiaceae) and their biological effects on human keratinocytes.

    PubMed

    Nawwar, Mahmoud; Hussein, Sahar; Ayoub, Nahla; Hashim, Amani; El-Sharawy, Reham; Lindequist, Urlike; Harms, Manualle; Wende, Kristian

    2011-12-01

    Assessment of the UV protecting potential of an aqueous methanol leaf extract of Harpephyllum caffrum proved that it possesses a distinct radical scavenging effect and inhibits the production of the proinflammatory cytokine IL-6 by human keratinocytes (HaCaT cells) following UV radiation. Phytochemical investigation of this extract led to isolation and structural determination of the hitherto unknown phenolics, kaempferol 3-O-(2″-sulphatogalactopyranoside), its quercetin analogue and 3-methoxyellagic acid 4-O-galactopyranoside in addition to 18 known phenolic compounds. The structures were determined by spectroscopic and conventional methods of analysis. Flavonoid sulphatoglycosides which have been rarely found in nature were major phenolic constituents of this plant, and this is the first report of the isolation of any of them from Anacardiaceae. The extract was found to diminish UV phototoxic reaction of keratinocytes. However, the isolated kaempferol sulphatogalactopyranoside did not interact with UVB triggered IL-6 production of HaCaT keratinocytes.

  4. Testosterone Stimulates Duox1 Activity through GPRC6A in Skin Keratinocytes*

    PubMed Central

    Ko, Eunbi; Choi, Hyun; Kim, Borim; Kim, Minsun; Park, Kkot-Nara; Bae, Il-Hong; Sung, Young Kwan; Lee, Tae Ryong; Shin, Dong Wook; Bae, Yun Soo

    2014-01-01

    Testosterone is an endocrine hormone with functions in reproductive organs, anabolic events, and skin homeostasis. We report here that GPRC6A serves as a sensor and mediator of the rapid action of testosterone in epidermal keratinocytes. The silencing of GPRC6A inhibited testosterone-induced intracellular calcium ([Ca2+]i) mobilization and H2O2 generation. These results indicated that a testosterone-GPRC6A complex is required for activation of Gq protein, IP3 generation, and [Ca2+]i mobilization, leading to Duox1 activation. H2O2 generation by testosterone stimulated the apoptosis of keratinocytes through the activation of caspase-3. The application of testosterone into three-dimensional skin equivalents increased the apoptosis of keratinocytes between the granular and stratified corneum layers. These results support an understanding of the molecular mechanism of testosterone-dependent apoptosis in which testosterone stimulates H2O2 generation through the activation of Duox1. PMID:25164816

  5. Isorhamnetin Protects Human Keratinocytes against Ultraviolet B-Induced Cell Damage.

    PubMed

    Han, Xia; Piao, Mei Jing; Kim, Ki Cheon; Madduma Hewage, Susara Ruwan Kumara; Yoo, Eun Sook; Koh, Young Sang; Kang, Hee Kyoung; Shin, Jennifer H; Park, Yeunsoo; Yoo, Suk Jae; Chae, Sungwook; Hyun, Jin Won

    2015-07-01

    Isorhamnetin (3-methylquercetin) is a flavonoid derived from the fruits of certain medicinal plants. This study investigated the photoprotective properties of isorhamnetin against cell damage and apoptosis resulting from excessive ultraviolet (UV) B exposure in human HaCaT keratinocytes. Isorhamnetin eliminated UVB-induced intracellular reactive oxygen species (ROS) and attenuated the oxidative modification of DNA, lipids, and proteins in response to UVB radiation. Moreover, isorhamnetin repressed UVB-facilitated programmed cell death in the keratinocytes, as evidenced by a reduction in apoptotic body formation, and nuclear fragmentation. Additionally, isorhamnetin suppressed the ability of UVB light to trigger mitochondrial dysfunction. Taken together, these results indicate that isorhamnetin has the potential to protect human keratinocytes against UVB-induced cell damage and death. PMID:26157553

  6. Experience gained during the long term cultivation of keratinocytes for treatment of burns patients.

    PubMed

    Dragúňová, Jana; Kabát, Peter; Koller, Ján; Jarabinská, Valéria

    2012-08-01

    Both allogenic and autologous cultured skin cells have been used clinically on burn patients. In vitro cultivation of human keratinocytes has been routinely provided by the Central Tissue Bank in Bratislava since 1996, with an average annual production of around 7,000 cm(2). Keratinocytes have been cultivated using a version of the original by Rheinwald and Green (Cell 6:317-330, 1975) methodology which has been modified over time in our laboratory as we gained more experience with this serial passage system. We have observed that the growth of cultured keratinocytes depends on several important factors, including the timing of skin sample procurement, the method of skin sample procurement, the general condition of the patient, the quality and composition of the culture media and, to a lesser extent, the age of the patient. We aim to share our experience with other cell cultivation facilities. PMID:21847560

  7. RIP2: A novel player in the regulation of keratinocyte proliferation and cutaneous wound repair?

    SciTech Connect

    Adams, Stephanie; Valchanova, Ralitsa S.; Munz, Barbara

    2010-03-10

    We could recently demonstrate an important role of receptor interacting protein 4 (RIP4) in the regulation of keratinocyte differentiation. Now, we analyzed a potential role of the RIP4 homolog RIP2 in keratinocytes. Specifically, we demonstrate here that rip2 expression is induced by scratch-wounding and after the induction of differentiation in these cells. Furthermore, serum growth factors and cytokines can induce rip2, with TNF-{alpha}-dependent induction being dependent on p38 MAPK. In addition, we demonstrate that scratch-induced upregulation of rip2 expression is completely blocked by the steroid dexamethasone. Since we also show that RIP2 is an important player in the regulation of keratinocyte proliferation, these data suggest that inhibition of rip2 upregulation after wounding might contribute to the reduced and delayed wound re-epithelialization phenotype seen in glucocorticoid-treated patients.

  8. Regulation of gene expression by glucose in pancreatic beta -cells (MIN6) via insulin secretion and activation of phosphatidylinositol 3'-kinase.

    PubMed

    da Silva Xavier, G; Varadi, A; Ainscow, E K; Rutter, G A

    2000-11-17

    Increases in glucose concentration control the transcription of the preproinsulin (PPI) gene and several other genes in the pancreatic islet beta-cell. Although recent data have demonstrated that secreted insulin may regulate the PPI gene (Leibiger, I. B., Leibiger, B., Moede, T., and Berggren, P. O. (1998) Mol. Cell 1, 933-938), the role of insulin in the control of other beta-cell genes is unexplored. To study the importance of insulin secretion in the regulation of the PPI and liver-type pyruvate kinase (L-PK) genes by glucose, we have used intranuclear microinjection of promoter-luciferase constructs into MIN6 beta-cells and photon-counting imaging. The activity of each promoter was increased either by 30 (versus 3) mm glucose or by 1-20 nm insulin. These effects of insulin were not due to enhanced glucose metabolism since culture with the hormone had no impact on the stimulation of increases in intracellular ATP concentration caused by 30 mm glucose. Furthermore, the islet-specific glucokinase promoter and cellular glucokinase immunoreactivity were unaffected by 30 mm glucose or 20 nm insulin. Inhibition of insulin secretion with the Ca(2+) channel blocker verapamil, the ATP-sensitive K(+) channel opener diazoxide, or the alpha(2)-adrenergic agonist clonidine blocked the effects of glucose on L-PK gene transcription. Similarly, 30 mm glucose failed to induce the promoter after inhibition of phosphatidylinositol 3'-kinase activity with LY294002 and the expression of dominant negative-acting phosphatidylinositol 3'-kinase (Deltap85) or the phosphoinositide 3'-phosphatase PTEN (phosphatase and tensin homologue). LY294002 also diminished the activation of the L-PK gene caused by inhibition of 5'-AMP-activated protein kinase with anti-5'-AMP-activated protein kinase alpha2 antibodies. Conversely, stimulation of insulin secretion with 13 mm KCl or 10 microm tolbutamide strongly activated the PPI and L-PK promoters. These data indicate that, in MIN6 beta

  9. MicroRNA-191 triggers keratinocytes senescence by SATB1 and CDK6 downregulation

    SciTech Connect

    Lena, A.M.; Mancini, M.; Rivetti di Val Cervo, P. [University of 'Tor Vergata', Department of Experimental Medicine and Biochemical Sciences, Via Montpellier 1, Rome 00133; Istituto Dermopatico dell'Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico , Laboratory of Biochemistry c Saintigny, G.; Mahe, C. [CHANEL Parfums Beaute, 135 av. Charles de Gaulle, F 92521, Neuilly Melino, G. [University of 'Tor Vergata', Department of Experimental Medicine and Biochemical Sciences, Via Montpellier 1, Rome 00133; Istituto Dermopatico dell'Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico , Laboratory of Biochemistry c Association Cell Death and Differentiation c and others

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer miR-191 expression is upregulated in senescencent human epidermal keratinocytes. Black-Right-Pointing-Pointer miR-191 overexpression is sufficient per se to induce senescence in keratinocytes. Black-Right-Pointing-Pointer SATB1 and CDK6 are downregulated in senescence and are direct miR-191 targets. Black-Right-Pointing-Pointer SATB1 and CDK6 silencing by siRNA triggers senescence in HEKn cells. -- Abstract: Keratinocyte replicative senescence has an important role in time-dependent changes of the epidermis, a tissue with high turnover. Senescence encompasses growth arrest during which cells remain metabolically active but acquire a typical enlarged, vacuolar and flattened morphology. It is also accompanied by the expression of endogenous senescence-associated-{beta}-galactosidase and specific gene expression profiles. MicroRNAs levels have been shown to be modulated during keratinocytes senescence, playing key roles in inhibiting proliferation and in the acquisition of senescent markers. Here, we identify miR-191 as an anti-proliferative and replicative senescence-associated miRNA in primary human keratinocytes. Its overexpression is sufficient per se to induce senescence, as evaluated by induction of several senescence-associated markers. We show that SATB1 and CDK6 3 Prime UTRs are two miR-191 direct targets involved in this pathway. Cdk6 and Satb1 protein levels decrease during keratinocytes replicative senescence and their silencing by siRNA is able to induce a G1 block in cell cycle, accompanied by an increase in senescence-associated markers.

  10. Antimycotics suppress the Malassezia extract-induced production of CXC chemokine ligand 10 in human keratinocytes.

    PubMed

    Hau, Carren S; Kanda, Naoko; Makimura, Koichi; Watanabe, Shinichi

    2014-02-01

    Malassezia, a lipophilic yeast, exacerbates atopic dermatitis. Malassezia products can penetrate the disintegrated stratum corneum and encounter subcorneal keratinocytes in the skin of atopic dermatitis patients. Type 1 helper T (Th1) cells infiltrate chronic lesions with atopic dermatitis, and antimycotic agents improve its symptoms. We aimed to identify Malassezia-induced chemokines in keratinocytes and examine whether antimycotics suppressed this induction. Normal human keratinocytes were incubated with a Malassezia restricta extract and antimycotics. Chemokine expression was analyzed by enzyme-linked immunosorbent assays and real-time polymerase chain reaction. Signal transducer and activator of transcription (STAT)1 activity was examined by luciferase assays. The tyrosine-phosphorylation of STAT1 was analyzed by western blotting. The M. restricta extract increased the mRNA and protein expression of Th1-attracting CXC chemokine ligand (CXCL)10 and STAT1 activity and phosphorylation in keratinocytes, which was suppressed by a Janus kinase inhibitor. The antimycotics itraconazole, ketoconazole, luliconazole, terbinafine, butenafine and amorolfine suppressed M. restricta extract-induced CXCL10 mRNA and protein expression and STAT1 activity and phosphorylation. These effects were similarly induced by 15-deoxy-Δ-(12