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Sample records for pkc inhibitor calphostin

  1. Design, synthesis, and investigation of protein kinase C inhibitors: total syntheses of (+)-calphostin D, (+)-phleichrome, cercosporin, and new photoactive perylenequinones.

    PubMed

    Morgan, Barbara J; Dey, Sangeeta; Johnson, Steven W; Kozlowski, Marisa C

    2009-07-08

    The total syntheses of the PKC inhibitors (+)-calphostin D, (+)-phleichrome, cercosporin, and 10 novel perylenequinones are detailed. The highly convergent and flexible strategy developed employed an enantioselective oxidative biaryl coupling and a double cuprate epoxide opening, allowing the selective syntheses of all the possible stereoisomers in pure form. In addition, this strategy permitted rapid access to a broad range of analogues, including those not accessible from the natural products. These compounds provided a powerful means for evaluation of the perylenequinone structural features necessary to PKC activity. Simpler analogues were discovered with superior PKC inhibitory properties and superior photopotentiation in cancer cell lines relative to the more complex natural products.

  2. PKC-β as a therapeutic target in CLL: PKC inhibitor AEB071 demonstrates preclinical activity in CLL.

    PubMed

    El-Gamal, Dalia; Williams, Katie; LaFollette, Taylor D; Cannon, Matthew; Blachly, James S; Zhong, Yiming; Woyach, Jennifer A; Williams, Erich; Awan, Farrukh T; Jones, Jeffrey; Andritsos, Leslie; Maddocks, Kami; Wu, Chia-Hsien; Chen, Ching-Shih; Lehman, Amy; Zhang, Xiaoli; Lapalombella, Rosa; Byrd, John C

    2014-08-28

    Targeting B-cell receptor (BCR) signaling in chronic lymphocytic leukemia (CLL) has been successful with durable remissions observed with several targeted therapeutics. Protein kinase C-β (PKC-β) is immediately downstream of BCR and has been shown to be essential to CLL cell survival and proliferation in vivo. We therefore evaluated sotrastaurin (AEB071), an orally administered potent PKC inhibitor, on CLL cell survival both in vitro and in vivo. AEB071 shows selective cytotoxicity against B-CLL cells in a dose-dependent manner. Additionally, AEB071 attenuates BCR-mediated survival pathways, inhibits CpG-induced survival and proliferation of CLL cells in vitro, and effectively blocks microenvironment-mediated survival signaling pathways in primary CLL cells. Furthermore, AEB071 alters β-catenin expression, resulting in decreased downstream transcriptional genes as c-Myc, Cyclin D1, and CD44. Lastly, our preliminary in vivo studies indicate beneficial antitumor properties of AEB071 in CLL. Taken together, our results indicate that targeting PKC-β has the potential to disrupt signaling from the microenvironment contributing to CLL cell survival and potentially drug resistance. Future efforts targeting PKC with the PKC inhibitor AEB071 as monotherapy in clinical trials of relapsed and refractory CLL patients are warranted.

  3. Pre-clinical characterization of PKC412, a multi-kinase inhibitor, against colorectal cancer cells

    PubMed Central

    Zhou, Yi-Chan; Shao, Yun; He, Xiao-Pu; Chen, Su-Rong; Wang, Dong-Dong; Qin, Li-Sen; Sun, Wei-Hao

    2016-01-01

    The potential effect of PKC412, a small molecular multi-kinase inhibitor, in colorectal cancer (CRC) cells was evaluated here. We showed that PKC412 was cytotoxic and anti-proliferative against CRC cell lines (HT-29, HCT-116, HT-15 and DLD-1) and primary CRC cells. PKC412 provoked caspase-dependent apoptotic death, and induced G2-M arrest in the CRC cells. AKT activation was inhibited by PKC412 in CRC cells. Reversely, expression of constitutively-active AKT1 (CA-AKT1) decreased the PKC412's cytotoxicity against HT-29 cells. We propose that Bcl-2 could be a primary resistance factor of PKC412. ABT-737, a Bcl-2 inhibitor, or Bcl-2 siRNA knockdown, dramatically potentiated PKC412's lethality against CRC cells. Forced Bcl-2 over-expression, on the other hand, attenuated PKC412's cytotoxicity. Significantly, PKC412 oral administration suppressed AKT activation and inhibited HT-29 tumor growth in nude mice. Mice survival was also improved with PKC412 administration. These results indicate that PKC412 may have potential value for CRC treatment. PMID:27780925

  4. Novel protein kinase C inhibitors: synthesis and PKC inhibition of beta-substituted polythiophene derivatives.

    PubMed

    Xu, W C; Zhou, Q; Ashendel, C L; Chang, C T; Chang, C J

    1999-08-02

    A series of beta-substituted polythiophene derivatives was synthesized through palladium-catalyzed coupling reaction. Their structure-protein kinase C (PKC) inhibitory activity relationship was studied. The carboxaldehyde and hydroxymethyl derivatives of alpha-terthiophene were potent PKC inhibitors (IC50 = 10(-7) M).

  5. PKC/MEK inhibitors suppress oxaliplatin-induced neuropathy and potentiate the antitumor effects.

    PubMed

    Tsubaki, Masanobu; Takeda, Tomoya; Tani, Tadahumi; Shimaoka, Hirotaka; Suzuyama, Naohiro; Sakamoto, Kotaro; Fujita, Arisa; Ogawa, Naoki; Itoh, Tatsuki; Imano, Motohiro; Funakami, Yoshinori; Ichida, Seiji; Satou, Takao; Nishida, Shozo

    2015-07-01

    Oxaliplatin is a key drug commonly used in colorectal cancer treatment. Despite high clinical efficacy, its therapeutic application is limited by common, dose-limiting occurrence of neuropathy. As usual symptomatic neuropathy treatments fail to improve the patients' condition, there is an urgent need to advance our understanding of the pathogenesis of neuropathy to propose effective therapy and ensure adequate pain management. Oxaliplatin-induced neuropathy was recently reported to be associated with protein kinase C (PKC) activation. It is unclear, however, whether PKC inhibition can prevent neuropathy. In our current studies, we found that a PKC inhibitor, tamoxifen, inhibited oxaliplatin-induced neuropathy via the PKC/extracellular signal-regulated kinase (ERK)/c-Fos pathway in lumbar spinal cords (lumbar segments 4-6). Additionally, tamoxifen was shown to act in synergy with oxaliplatin to inhibit growth in tumor cells-implanted mice. Moreover, mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor, PD0325901, suppressed oxaliplatin-induced neuropathy and enhanced oxaliplatin efficacy. Our results indicate that oxaliplatin-induced neuropathy is associated with PKC/ERK/c-Fos pathway in lumbar spinal cord. Additionally, we demonstrate that disruption of this pathway by PKC and MEK inhibitors suppresses oxaliplatin-induced neuropathy, thereby suggesting that PKC and MEK inhibitors may be therapeutically useful in preventing oxaliplatin-induced neuropathy and could aid in combination antitumor pharmacotherapy.

  6. Calphostin-C induction of vascular smooth muscle cell apoptosis proceeds through phospholipase D and microtubule inhibition.

    PubMed

    Zheng, Xi-Long; Gui, Yu; Du, Guangwei; Frohman, Michael A; Peng, Dao-Quan

    2004-02-20

    Calphostin-C, a protein kinase C inhibitor, induces apoptosis of cultured vascular smooth muscle cells. However, the mechanisms are not completely defined. Because apoptosis of vascular smooth muscle cells is critical in several proliferating vascular diseases such as atherosclerosis and restenosis after angioplasty, we decided to investigate the mechanisms underlying the calphostin-C-induced apoptotic pathway. We show here that apoptosis is inhibited by the addition of exogenous phosphatidic acid, a metabolite of phospholipase D (PLD), and that calphostin-C inhibits completely the activities of both isoforms of PLD, PLD1 and PLD2. Overexpression of either PLD1 or PLD2 prevented the vascular smooth muscle cell apoptosis induced by serum withdrawal but not the calphostin-C-elicited apoptosis. These data suggest that PLDs have anti-apoptotic effects and that complete inhibition of PLD activity by calphostin-C induces smooth muscle cell apoptosis. We also report that calphostin-C induced microtubule disruption and that the addition of exogenous phosphatidic acid inhibits calphostin-C effects on microtubules, suggesting a role for PLD in stabilizing the microtubule network. Overexpressing PLD2 in Chinese hamster ovary cells phenocopies this result, providing strong support for the hypothesis. Finally, taxol, a microtubule stabilizer, not only inhibited the calphostin-C-induced microtubule disruption but also inhibited apoptosis. We therefore conclude that calphostin-C induces apoptosis of cultured vascular smooth muscle cells through inhibiting PLD activity and subsequent microtubule polymerization.

  7. Structural investigation of protein kinase C inhibitors.

    PubMed

    Barak, D; Shibata, M; Rein, R

    1991-01-01

    The phospholipid and Ca2+ dependent protein kinase (PKC) plays an essential role in a variety of cellular events. Inhibition of PKC was shown to arrest growth in tumor cell cultures making it a target for possible antitumor therapy. Calphostins are potent inhibitors of PKC with high affinity for the enzyme regulatory site. Structural characteristics of calphostins, which confer the inhibitory activity, are investigated by comparing their optimized structures with the existing models for PKC activation. The resulting model of inhibitory activity assumes interaction with two out of the three electrostatic interaction sites postulated for activators. The model shows two sites of hydrophobic interaction and enables the inhibitory activity of gossypol to be accounted for.

  8. Structural investigation of protein kinase C inhibitors

    NASA Technical Reports Server (NTRS)

    Barak, D.; Shibata, M.; Rein, R.

    1991-01-01

    The phospholipid and Ca2+ dependent protein kinase (PKC) plays an essential role in a variety of cellular events. Inhibition of PKC was shown to arrest growth in tumor cell cultures making it a target for possible antitumor therapy. Calphostins are potent inhibitors of PKC with high affinity for the enzyme regulatory site. Structural characteristics of calphostins, which confer the inhibitory activity, are investigated by comparing their optimized structures with the existing models for PKC activation. The resulting model of inhibitory activity assumes interaction with two out of the three electrostatic interaction sites postulated for activators. The model shows two sites of hydrophobic interaction and enables the inhibitory activity of gossypol to be accounted for.

  9. ETV6-NTRK3 as a therapeutic target of small molecule inhibitor PKC412

    SciTech Connect

    Chi, Hoang Thanh; Ly, Bui Thi Kim; Kano, Yasuhiko; Tojo, Arinobu; Sato, Yuko

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer ETV6-NTRK3 is an oncogene with transformation activity in multiple cell lineages. Black-Right-Pointing-Pointer PKC412 could block ETV6-NTRK3 activation. Black-Right-Pointing-Pointer Loss of ETV6-NTRK3 phosphorylation leads to inactivation of its downstream signaling pathway. Black-Right-Pointing-Pointer Inhibition of ETV6-NTRK3 activation by PKC412 could be a novel strategy for the treatment. -- Abstract: The ETV6-NTRK3 (EN) fusion gene which encodes a chimeric tyrosine kinase was first identified by cloning of the t(12;15)(p13;q25) translocation in congenital fibrosarcoma (CFS). Since then, EN has been also found in congenital mesoblastic nephroma (CMN), secretory breast carcinoma (SBC) and acute myelogenous leukemia (AML). Using IMS-M2 and M0-91 cell lines harboring the EN fusion gene, and Ba/F3 cells stably transfected with EN, we demonstrated that PKC412, also known as midostaurin, is an inhibitor of EN. Inhibition of EN activity by PKC412 suppressed the activity of it downstream molecules leading to inhibition of cell proliferation and induction of apoptosis. Our data for the first time suggested that PKC412 could serve as therapeutic drug for treatment of patients with this fusion.

  10. CGX1037 is a novel PKC isoform delta selective inhibitor in platelets.

    PubMed

    Bhavanasi, Dheeraj; Kostyak, John C; Swindle, John; Kilpatrick, Laurie E; Kunapuli, Satya P

    2015-01-01

    Platelets upon activation change their shape, aggregate and secrete alpha and dense granule contents among which ADP acts as a feedback activator. Different Protein Kinase C (PKC) isoforms have specific non-redundant roles in mediating platelet responses including secretion and thrombus formation. Murine platelets lacking specific PKC isoforms have been used to evaluate the isoform specific functions. Novel PKC isoform δ has been shown to play an important role in some pathological processes. Lack of specific inhibitors for PKCδ has restricted analysis of its role in various cells. The current study was carried out to evaluate a novel small molecule PKCδ inhibitor, CGX1037 in platelets. Platelet aggregation, dense granule secretion and western blotting experiments were performed to evaluate CGX1037. In human platelets, CGX1037 inhibited PAR4-mediated phosphorylation on PKD2, a PKCδ-specific substrate. Pre-treatment of human or murine platelets with CGX1037 inhibited PAR4-mediated dense granule secretion whereas it potentiated GPVI-mediated dense granule secretion similar to the responses observed in murine platelets lacking PKCδ· Furthermore, pre-treatment of platelets from PKCδ(-/-) mice with CGX1037 had no significant additive effect on platelet responses suggesting the specificity of CGX1037. Hence, we show that CGX1037 is a selective small molecule inhibitor of PKCδ in platelets.

  11. Effects of the PKC inhibitors chelerythrine and bisindolylmaleimide I (GF 109203X) on delayed rectifier K+ currents.

    PubMed

    Harmati, Gábor; Papp, Ferenc; Szentandrássy, Norbert; Bárándi, László; Ruzsnavszky, Ferenc; Horváth, Balázs; Bányász, Tamás; Magyar, János; Panyi, György; Krasznai, Zoltán; Nánási, Péter P

    2011-02-01

    Protein kinase C (PKC) inhibitors are useful tools for studying PKC-dependent regulation of ion channels. For this purpose, high PKC specificity is a basic requirement excluding any direct interaction between the PKC inhibitor and the ion channel. In the present study, the effects of two frequently applied PKC inhibitors, chelerythine and bisindolylmaleimide I, were studied on the rapid and slow components of the delayed rectifier K(+) current (I(Kr) and I(Ks)) in canine ventricular cardiomyocytes and on the human ether-à-go-go-related gene (hERG) channels expressed in human embryonic kidney (HEK) cells. The whole cell version of the patch clamp technique was used in all experiments. Chelerythrine and bisindolylmaleimide I (both 1 μM) suppressed I(Kr) in canine ventricular cells. This inhibition developed rapidly, suggesting a direct drug-channel interaction. In HEK cells heterologously expressing hERG channels, chelerythrine and bisindolylmaleimide I blocked hERG current in a concentration-dependent manner, having EC(50) values of 0.11 ± 0.01 and 0.76 ± 0.04 μM, respectively. Both chelerythrine and bisindolylmaleimide I strongly modified gating kinetics of hERG--voltage dependence of activation was shifted towards more negative voltages and activation was accelerated. Deactivation was slowed by bisindolylmaleimide I but not by chelerythrine. I(Ks) was not significantly altered by bisindolylmaleimide I and chelerythrine. No significant effect of 0.1 μM bisindolylmaleimide I or 0.1 μM PMA (PKC activator) was observed on I(Kr) arguing against significant contribution of PKC to regulation of I(Kr). It is concluded that neither chelerythrine nor bisindolylmaleimide I is suitable for selective PKC blockade due to their direct blocking actions on the hERG channel.

  12. Effect of rottlerin, a PKC-{delta} inhibitor, on TLR-4-dependent activation of murine microglia

    SciTech Connect

    Kim, Dong-Chan; Kim, Sun-Hee; Jeong, Min-Woo; Baek, Nam-in; Kim, Kyong-Tai . E-mail: ktk@postech.ac.kr

    2005-11-11

    In microglia, Toll-like receptors have been shown to recognize pathogen-associated molecular patterns and initiate innate immune responses upon interaction with infectious agents. The effect of rottlerin, a PKC-{delta} specific inhibitor, on TLR-4-mediated signaling was investigated in murine microglia stimulated with lipopolysaccharide and taxol. Pretreatment of microglia cells with rottlerin decreased LPS- and taxol-induced nitric oxide production in a concentration-dependent manner (IC{sub 50} = 99.1 {+-} 1.5 nM). Through MTT and FACS analysis, we found that the inhibition effect of rottlerin was not due to microglial cell death. Rottlerin pretreatment also attenuated LPS-induced phosphorylation of I{kappa}B-{alpha}, nuclear translocation of NF-{kappa}B, and expression of type II nitric oxide synthase. In addition, microglial phagocytosis in response to TLR-4 activation was diminished in which rottlerin was pretreated. Together, these data raise the possibility that certain PKC-{delta} specific inhibitors can modulate TLR-4-derived signaling and inflammatory target gene expression, and can alter susceptibility to microbial infection and chronic inflammatory diseases in central nervous system.

  13. A novel and selective inhibitor of PKC ζ potently inhibits human breast cancer metastasis in vitro and in mice.

    PubMed

    Wu, Jing; Liu, Shuye; Fan, Zhijuan; Zhang, Lei; Tian, Yaqiong; Yang, Rui

    2016-06-01

    Cell motility and chemotaxis play pivotal roles in the process of tumor development and metastasis. Protein kinase C ζ (PKC ζ) mediates epidermal growth factor (EGF)-stimulated chemotactic signaling pathway through regulating cytoskeleton rearrangement and cell adhesion. The purpose of this study was to develop anti-PKC ζ therapeutics for breast cancer metastasis. In this study, a novel and high-efficient PKC ζ inhibitor named PKCZI195.17 was screened out through a substrate-specific strategy. MTT assay was used to determine the cell viability of human breast cancer MDA-MB-231, MDA-MB-435, and MCF-7 cells while under PKCZI195.17 treatment. Wound-healing, chemotaxis, and Matrigel invasion assays were performed to detect the effects of PKCZI195.17 on breast cancer cells migration and invasion. Adhesion, actin polymerization, and Western blotting were performed to detect the effects of PKCZI195.17 on cells adhesion and actin polymerization, and explore the downsteam signaling mechanisms involved in PKC ζ inhibition. MDA-MB-231 xenograft was used to measure the in vivo anti-metastasis efficacy of PKCZI195.17. The compound PKCZI195.17 selectively inhibited PKC ζ kinase activity since it failed to inhibit PKC α, PKC β, PKC δ, PKC η, AKT2, as well as FGFR2 activity. PKCZI195.17 significantly impaired spontaneous migration, chemotaxis, and invasion of human breast cancer MDA-MB-231, MDA-MB-435, and MCF-7 cells, while PKCZI195.17 did not obviously inhibited cells viability. PKCZI195.17 also inhibited cells adhesion and actin polymerization through attenuating the phosphorylations of integrin β1, LIMK, and cofilin, which might be the downstream effectors of PKC ζ-mediated chemotaxis in MDA-MB-231 cells. Furthermore, PKCZI195.17 suppressed the breast cancer metastasis and increased the survival time of breast tumor-bearing mice. In summary, PKCZI195.17 was a PKC ζ-specific inhibitor which dampened cancer cell migration and metastasis and may serve as a novel

  14. Structure-based modelling, scoring, screening, and in vitro kinase assay of anesthetic pkc inhibitors against a natural medicine library.

    PubMed

    Shi, B X; Chen, F R; Sun, X

    2017-02-01

    Protein kinase C (PKC) is an intracellular effector of the inositol phosphate-mediated signal transduction pathway. Evidence is emerging that certain general anaesthetics can influence the activity of PKC by interacting with the regulatory domain of the enzyme, and targeting PKC kinase domain is considered as a strategy to modulate the anaesthetic effects. Here, an integrated method was used to perform virtual screening against a large library of natural compounds for the discovery of new and potent PKC modulators. A number of hits were identified and their inhibitory activity against PKC kinase domain was measured by using a standard kinase assay protocol. Three and five compounds were determined to have high and moderate activities with IC50 values at nanomolar and micromolar levels, respectively. These compounds can be considered as promising lead molecular entities to develop efficacious anaesthetic modulators. Structural examination revealed a variety of nonbonded interactions such as hydrogen bonds, cation-π contacts, and hydrophobic forces across the complex interface of PKC with the identified compounds. This study helps to establish an integrative approach to rational kinase inhibitor discovery by efficiently exploiting various existing natural products.

  15. PKC delta-isoform translocation and enhancement of tonic contractions of gastrointestinal smooth muscle.

    PubMed

    Poole, Daniel P; Furness, John B

    2007-03-01

    PKC is involved in mediating the tonic component of gastrointestinal smooth muscle contraction in response to stimulation by agonists for G protein-coupled receptors. Here, we present pharmacological and immunohistochemical evidence indicating that a member of the novel PKC isoforms, PKC-delta, is involved in maintaining muscarinic receptor-coupled tonic contractions of the guinea pig ileum. The tonic component of carbachol-evoked contractions was enhanced by an activator of conventional and novel PKCs, phorbol 12,13-dibutyrate (PDBu; 200 nM or 1 microM), and by an activator of novel PKCs, ingenol 3,20-dibenzoate (IDB; 100 or 500 nM). Enhancement was unaffected by concentrations of bisindolylmaleimide I (BIM-I; 22 nM) that block conventional PKCs or by a PKC-epsilon-specific inhibitor peptide but was attenuated by higher doses of BIM-I (2.2 microM). Relevant proteins were localized at a cellular and subcellular level using confocal analysis. Immunohistochemical staining of the ileum showed that PKC-delta was exclusively expressed in smooth muscles distributed throughout the layers of the gut wall. PKC-epsilon immunoreactivity was prominent in enteric neurons but was largely absent from smooth muscle of the muscularis externa. Treatment with PDBu, IDB, or carbachol resulted in a time- and concentration-dependent translocation of PKC-delta from the cytoplasm to filamentous structures within smooth muscle cells. These were parallel to, but distinct from, actin filaments. The translocation of PKC-delta in response to carbachol was significantly reduced by scopolamine or calphostin C. The present study indicates that the tonic carbachol-induced contraction of the guinea pig ileum is mediated through a novel PKC, probably PKC-delta.

  16. Stress Signals, Mediated by Membranous Glucocorticoid Receptor, Activate PLC/PKC/GSK-3β/β-catenin Pathway to Inhibit Wound Closure.

    PubMed

    Jozic, Ivan; Vukelic, Sasa; Stojadinovic, Olivera; Liang, Liang; Ramirez, Horacio A; Pastar, Irena; Tomic Canic, Marjana

    2016-12-23

    Glucocorticoids (GCs), key mediators of stress signals, are also potent wound healing inhibitors. To understand how stress signals inhibit wound healing, we investigated the role of membranous glucocorticoid receptor (mbGR) by using cell-impermeable BSA-conjugated dexamethasone. We found that mbGR inhibits keratinocyte migration and wound closure by activating a Wnt-like phospholipase (PLC)/ protein kinase C (PKC) signaling cascade. Rapid activation of mbGR/PLC/PKC further leads to activation of known biomarkers of nonhealing found in patients, β-catenin and c-myc. Conversely, a selective inhibitor of PKC, calphostin C, blocks mbGR/PKC pathway, and rescues GC-mediated inhibition of keratinocyte migration in vitro and accelerates wound epithelialization of human wounds ex vivo. This novel signaling mechanism may have a major impact on understanding how stress response via GC signaling regulates homeostasis and its role in development and treatments of skin diseases, including wound healing. To test tissue specificity of this nongenomic signaling mechanism, we tested retinal and bronchial human epithelial cells and fibroblasts. We found that mbGR/PLC/PKC signaling cascade exists in all cell types tested, suggesting a more general role. The discovery of this nongenomic signaling pathway, in which glucocorticoids activate Wnt pathway via mbGR, provides new insights into how stress-mediated signals may activate growth signals in various epithelial and mesenchymal tissues.

  17. PKC delta and tissue transglutaminase are novel inhibitors of autophagy in pancreatic cancer cells.

    PubMed

    Ozpolat, Bulent; Akar, Ugur; Mehta, Kapil; Lopez-Berestein, Gabriel

    2007-01-01

    Apoptosis (type I) and autophagy (type II) are both highly regulated forms of programmed cell death and play crucial roles in physiological processes such as the development, homeostasis and selective, moderate to massive elimination of cells, if needed. Accumulating evidence suggests that cancer cells, including pancreatic cancer cells, in general tend to have reduced autophagy relative to their normal counterparts and premalignant lesions, supporting the contention that defective autophagy provides resistance to metabolic stress such as hypoxia, acidity and chemotherapeutics, promotes tumor cell survival and plays a role in the process of tumorigenesis. However, the mechanisms underlying the reduced capability of undergoing autophagy in pancreatic cancer remain elusive. In a recent study, we demonstrated a novel mechanism for regulation of autophagy in pancreatic ductal carcinoma cells. We found that protein kinase C-delta (PKC delta) constitutively suppresses autophagy through induction of tissue transglutaminase (TG2). Inhibition of PKC delta/TG2 signaling resulted in significant autophagic cell death that was mediated by Beclin 1. Elevated expression of TG2 in pancreatic cancer cells has been implicated in the development of drug resistance, metastatic phenotype and poor patient prognosis. In conclusion, our data suggest a novel role of PKC delta/TG2 in regulation of autophagy, and that TG2 may serve as an excellent therapeutic target in pancreatic cancer cells.

  18. Impairment of neurovascular coupling in Type 1 Diabetes Mellitus in rats is prevented by pancreatic islet transplantation and reversed by a semi-selective PKC inhibitor.

    PubMed

    Vetri, Francesco; Qi, Meirigeng; Xu, Haoliang; Oberholzer, Jose; Paisansathan, Chanannait

    2017-01-15

    Streptozotocin (STZ)-induced chronic hyperglycemia has a detrimental effect on neurovascular coupling, linked to increased PKC-mediated phosphorylation and PKC isoform expression changes. Here, we sought to determine whether: 1) selective PKC-α/β/γ inhibitor, GF109203X, could reverse the effects of chronic hyperglycemia on cerebrovascular reactivity; 2) pancreatic islet transplantation could prevent the development of cerebrovascular impairment seen in a rat model of Type 1 Diabetes. We studied the effect of GF109203X in diabetic (DM), non-diabetic (ND), and transplanted (TR) Lewis rats during either sciatic nerve stimulation (SNS) or the topical applications of the large-conductance Ca(2+)-operated K(+)(BKCa) channel opener, NS1619, or the K(+) inward rectifier (Kir) channel agonist, KCl. Pial arteriole diameter changes were monitored using a closed cranial window in vivo microscopy technique. The pial arteriole dilatory response associated with SNS was decreased by ~45%, when comparing DM vs either ND or TR rats. Also, pial arteriolar dilations to topical KCl and NS1619 were largely attenuated in DM rats, but not in ND or TR animals. These responses were completely restored by the acute application of GF109203X to the brain surface. The PKC inhibitor had no effect on vascular responses in normoglycemic and TR animals. In conclusion, DM-associated chronic impairment of neurovascular coupling may be readily reversed by a PKC-α/β/γ inhibitor or prevented via pancreatic islet transplantation. We believe that specific PCK isoforms (α/β/γ) are mechanistically linked to the neurovascular uncoupling seen with hyperglycemia.

  19. Brassica juncea nitric oxide synthase like activity is stimulated by PKC activators and calcium suggesting modulation by PKC-like kinase.

    PubMed

    Talwar, Pooja Saigal; Gupta, Ravi; Maurya, Arun Kumar; Deswal, Renu

    2012-11-01

    Nitric oxide (NO) is an important signaling molecule having varied physiological and regulatory roles in biological systems. The fact that nitric oxide synthase (NOS) is responsible for NO generation in animals, prompted major search for a similar enzyme in plants. Arginine dependent NOS like activity (BjNOSla) was detected in Brassica juncea seedlings using oxyhemoglobin and citrulline assays. BjNOSla showed 25% activation by NADPH (0.4 mM) and 40% by calcium (0.4 mM) but the activity was flavin mononucleotide (FMN), flavin dinucleotide (FAD) and calmodulin (CaM) independent. Pharmacological approach using mammalian NOS inhibitors, NBT (300 μM) and l-NAME (5 mM), showed significant inhibition (100% and 67% respectively) supporting that the BjNOSla operates via the oxidative pathway. Most of the BjNOSla activity (80%) was confined to shoot while root showed only 20% activity. Localization studies by NADPH-diaphorase and DAF-2DA staining showed the presence of BjNOSla in guard cells. Kinetic analysis showed positive cooperativity with calcium as reflected by a decreased K(m) (∼13%) and almost two fold increase in V(max). PMA (438 nM), a kinase activator, activated BjNOSla ∼1.9 fold while its inactive analog 4αPDD was ineffective. Calcium and PMA activated the enzyme to ∼3 folds. Interestingly, 1,2-DG6 (2.5 μM) and PS (1 μM) with calcium activated the enzyme activity to ∼7 fold. A significant inhibition of BjNOSla by PKC inhibitors-staurosporine (∼90%) and calphostin-C (∼40%), further supports involvement of PKC-like kinase. The activity was also enhanced by abiotic stress conditions (7-46%). All these findings suggest that BjNOSla generates NO via oxidative pathway and is probably regulated by phosphorylation.

  20. A novel mouse PKC{delta} splice variant, PKC{delta}IX, inhibits etoposide-induced apoptosis

    SciTech Connect

    Kim, Jung D.; Seo, Kwang W.; Lee, Eun A.; Quang, Nguyen N.; Cho, Hong R.; Kwon, Byungsuk

    2011-07-01

    Highlights: {yields} A novel PKC{delta} isoform, named PKC{delta}IX, that lacks the C1 domain and the ATP-binding site is ubiquitously expressed. {yields} PKC{delta}IX inhibits etoposide-induced apoptosis. {yields} PKC{delta}IX may function as an endogenous dominant negative isoform for PKC{delta}. -- Abstract: Protein kinase C (PKC) {delta} plays an important role in cellular proliferation and apoptosis. The catalytic fragment of PKC{delta} generated by caspase-dependent cleavage is essential for the initiation of etoposide-induced apoptosis. In this study, we identified a novel mouse PKC{delta} isoform named PKC{delta}IX (Genebank Accession No. (HQ840432)). PKC{delta}IX is generated by alternative splicing and is ubiquitously expressed, as seen in its full-length PKC{delta}. PKC{delta}IX lacks the C1 domain, the caspase 3 cleavage site, and the ATP binding site but preserves an almost intact c-terminal catalytic domain and a nuclear localization signal (NLS). The structural characteristics of PKC{delta}IX provided a possibility that this PKC{delta} isozyme functions as a novel dominant-negative form for PKC{delta} due to its lack of the ATP-binding domain that is required for the kinase activity of PKC{delta}. Indeed, overexpression of PKC{delta}IX significantly inhibited etoposide-induced apoptosis in NIH3T3 cells. In addition, an in vitro kinase assay showed that recombinant PKC{delta}IX protein could competitively inhibit the kinase activity of PKC{delta}. We conclude that PKC{delta}IX can function as a natural dominant-negative inhibitor of PKC{delta}in vivo.

  1. Protein Kinase C (PKC) Activity Regulates Functional Effects of Kvβ1.3 Subunit on KV1.5 Channels

    PubMed Central

    David, Miren; Macías, Álvaro; Moreno, Cristina; Prieto, Ángela; Martínez-Mármol, Ramón; Vicente, Rubén; González, Teresa; Felipe, Antonio; Tamkun, Michael M.; Valenzuela, Carmen

    2012-01-01

    Kv1.5 channels are the primary channels contributing to the ultrarapid outward potassium current (IKur). The regulatory Kvβ1.3 subunit converts Kv1.5 channels from delayed rectifiers with a modest degree of slow inactivation to channels with both fast and slow inactivation components. Previous studies have shown that inhibition of PKC with calphostin C abolishes the fast inactivation induced by Kvβ1.3. In this study, we investigated the mechanisms underlying this phenomenon using electrophysiological, biochemical, and confocal microscopy approaches. To achieve this, we used HEK293 cells (which lack Kvβ subunits) transiently cotransfected with Kv1.5+Kvβ1.3 and also rat ventricular and atrial tissue to study native α-β subunit interactions. Immunocytochemistry assays demonstrated that these channel subunits colocalize in control conditions and after calphostin C treatment. Moreover, coimmunoprecipitation studies showed that Kv1.5 and Kvβ1.3 remain associated after PKC inhibition. After knocking down all PKC isoforms by siRNA or inhibiting PKC with calphostin C, Kvβ1.3-induced fast inactivation at +60 mV was abolished. However, depolarization to +100 mV revealed Kvβ1.3-induced inactivation, indicating that PKC inhibition causes a dramatic positive shift of the inactivation curve. Our results demonstrate that calphostin C-mediated abolishment of fast inactivation is not due to the dissociation of Kv1.5 and Kvβ1.3. Finally, immunoprecipitation and immunocytochemistry experiments revealed an association between Kv1.5, Kvβ1.3, the receptor for activated C kinase (RACK1), PKCβI, PKCβII, and PKCθ in HEK293 cells. A very similar Kv1.5 channelosome was found in rat ventricular tissue but not in atrial tissue. PMID:22547057

  2. An inhibitor of the δPKC interaction with the d subunit of F1Fo ATP synthase reduces cardiac troponin I release from ischemic rat hearts: utility of a novel ammonium sulfate precipitation technique.

    PubMed

    Ogbi, Mourad; Obi, Ijeoma; Johnson, John A

    2013-01-01

    We have previously reported protection against hypoxic injury by a cell-permeable, mitochondrially-targeted δPKC-d subunit of F1Fo ATPase (dF1Fo) interaction inhibitor [NH2-YGRKKRRQRRRMLA TRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK-COOH] in neonatal cardiac myo-cytes. In the present work we demonstrate the partitioning of this peptide to the inner membrane and matrix of mitochondria when it is perfused into isolated rat hearts. We also used ammonium sulfate ((NH4)2SO4) and chloroform/methanol precipitation of heart effluents to demonstrate reduced card-iac troponin I (cTnI) release from ischemic rat hearts perfused with this inhibitor. 50% (NH4)2SO4 saturation of perfusates collected from Langendorff rat heart preparations optimally precipitated cTnI, allowing its detection in Western blots. In hearts receiving 20 min of ischemia followed by 30, or 60 min of reperfusion, the Mean±S.E. (n=5) percentage of maximal cTnI release was 30 ± 7 and 60 ± 17, respectively, with additional cTnI release occurring after 150 min of reperfusion. Perfusion of hearts with the δPKC-dF1Fo interaction inhibitor, prior to 20 min of ischemia and 60-150 min of reperfusion, reduced cTnI release by 80%. Additionally, we found that when soybean trypsin inhibitor (SBTI), was added to rat heart effluents, it could also be precipitated using (NH4)2SO4 and detected in western blots. This provided a convenient method for normalizing protein recoveries between groups. Our results support the further development of the δPKC-dF1Fo inhibitor as a potential therapeutic for combating cardiac ischemic injury. In addition, we have developed an improved method for the detection of cTnI release from perfused rat hearts.

  3. An Inhibitor of the δPKC Interaction with the d Subunit of F1Fo ATP Synthase Reduces Cardiac Troponin I Release from Ischemic Rat Hearts: Utility of a Novel Ammonium Sulfate Precipitation Technique

    PubMed Central

    Ogbi, Mourad; Obi, Ijeoma; Johnson, John A.

    2013-01-01

    We have previously reported protection against hypoxic injury by a cell-permeable, mitochondrially-targeted δPKC-d subunit of F1Fo ATPase (dF1Fo) interaction inhibitor [NH2-YGRKKRRQRRRMLA TRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK-COOH] in neonatal cardiac myo-cytes. In the present work we demonstrate the partitioning of this peptide to the inner membrane and matrix of mitochondria when it is perfused into isolated rat hearts. We also used ammonium sulfate ((NH4)2SO4) and chloroform/methanol precipitation of heart effluents to demonstrate reduced card-iac troponin I (cTnI) release from ischemic rat hearts perfused with this inhibitor. 50% (NH4)2SO4 saturation of perfusates collected from Langendorff rat heart preparations optimally precipitated cTnI, allowing its detection in Western blots. In hearts receiving 20 min of ischemia followed by 30, or 60 min of reperfusion, the Mean±S.E. (n = 5) percentage of maximal cTnI release was 30±7 and 60±17, respectively, with additional cTnI release occurring after 150 min of reperfusion. Perfusion of hearts with the δPKC-dF1Fo interaction inhibitor, prior to 20 min of ischemia and 60–150 min of reperfusion, reduced cTnI release by 80%. Additionally, we found that when soybean trypsin inhibitor (SBTI), was added to rat heart effluents, it could also be precipitated using (NH4)2SO4 and detected in western blots. This provided a convenient method for normalizing protein recoveries between groups. Our results support the further development of the δPKC-dF1Fo inhibitor as a potential therapeutic for combating cardiac ischemic injury. In addition, we have developed an improved method for the detection of cTnI release from perfused rat hearts. PMID:23936451

  4. Perylenequinone natural products: total syntheses of the diastereomers (+)-phleichrome and (+)-calphostin D by assembly of centrochiral and axial chiral fragments.

    PubMed

    Morgan, Barbara J; Mulrooney, Carol A; O'Brien, Erin M; Kozlowski, Marisa C

    2010-01-01

    The first total synthesis of (+)-calphostin D and the total synthesis of (+)-phleichrome are outlined. The convergent syntheses utilize an enantiopure biaryl common intermediate, which is formed via an enantioselective catalytic biaryl coupling. The established axial chirality is transferred to the perylenequinone helical stereochemistry with good fidelity. Additionally, efforts focused on the installation of the stereogenic C7,C7'-2-hydroxypropyl groups. Three routes were evaluated to establish the C7,C7'-stereochemistry, in which the successful route involved a double epoxide alkylation with a complex axial chiral biscuprate. This strategy not only allowed the synthesis of the unnatural isomers of calphostin D and phleichrome for assessment in biological systems but also provided valuable information for the syntheses of the more complex cercosporin and hypocrellin A.

  5. Dexmedetomidine-Induced Contraction in the Isolated Endothelium-Denuded Rat Aorta Involves PKC-δ-mediated JNK Phosphorylation.

    PubMed

    Yu, Jongsun; Ok, Seong-Ho; Kim, Won Ho; Cho, Hyunhoo; Park, Jungchul; Shin, Il-Woo; Lee, Heon Keun; Chung, Young-Kyun; Choi, Mun-Jeoung; Kwon, Seong-Chun; Sohn, Ju-Tae

    2015-01-01

    Vasoconstriction mediated by the highly selective alpha-2 adrenoceptor agonist dexmedetomidine leads to transiently increased blood pressure and severe hypertension. The dexmedetomidine-induced contraction involves the protein kinase C (PKC)-mediated pathway. However, the main PKC isoform involved in the dexmedetomidine-induced contraction remains unknown. The goal of this in vitro study was to examine the specific PKC isoform that contributes to the dexmedetomidine-induced contraction in the isolated rat aorta. The endothelium-denuded rat aorta was suspended for isometric tension recording. Dexmedetomidine dose-response curves were generated in the presence or absence of the following inhibitors: the pan-PKC inhibitor, chelerythrine; the PKC-α and -β inhibitor, Go6976; the PKCinhibitor, safingol; the PKCinhibitor, ruboxistaurin; the PKCinhibitor, rottlerin; the c-Jun NH2-terminal kinase (JNK) inhibitor, SP600125; and the myosin light chain kinase inhibitor, ML-7 hydrochloride. Western blot analysis was used to examine the effect of rottlerin on dexmedetomidine-induced PKC-δ expression and JNK phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) and to investigate the effect of dexmedetomidine on PKC-δ expression in VSMCs transfected with PKC-δ small interfering RNA (siRNA) or control siRNA. Chelerythrine as well as SP600125 and ML-7 hydrochloride attenuated the dexmedetomidine-induced contraction. Go6976, safingol, and ruboxistaurin had no effect on the dexmedetomidine-induced contraction, whereas rottlerin inhibited the dexmedetomidine-induced contraction. Dexmedetomidine induced PKC-δ expression, whereas rottlerin and PKC-δ siRNA transfection inhibited dexmedetomidine-induced PKC-δ expression. Dexmedetomidine also induced JNK phosphorylation, which was inhibited by rottlerin. Taken together, these results suggest that the dexmedetomidine-induced contraction involves PKC-δ-dependent JNK phosphorylation in the isolated rat aorta.

  6. Effect of Tamoxifen and Brain-Penetrant Protein Kinase C and c-Jun N-Terminal Kinase Inhibitors on Tolerance to Opioid-Induced Respiratory Depression in Mice.

    PubMed

    Withey, Sarah L; Hill, Rob; Lyndon, Abigail; Dewey, William L; Kelly, Eamonn; Henderson, Graeme

    2017-04-01

    Respiratory depression is the major cause of death in opioid overdose. We have previously shown that prolonged treatment of mice with morphine induces profound tolerance to the respiratory-depressant effects of the drug (Hill et al., 2016). The aim of the present study was to investigate whether tolerance to opioid-induced respiratory depression is mediated by protein kinase C (PKC) and/or c-Jun N-terminal kinase (JNK). We found that although mice treated for up to 6 days with morphine developed tolerance, as measured by the reduced responsiveness to an acute challenge dose of morphine, administration of the brain-penetrant PKC inhibitors tamoxifen and calphostin C restored the ability of acute morphine to produce respiratory depression in morphine-treated mice. Importantly, reversal of opioid tolerance was dependent on the nature of the opioid ligand used to induce tolerance, as these PKC inhibitors did not reverse tolerance induced by prolonged treatment of mice with methadone nor did they reverse the protection to acute morphine-induced respiratory depression afforded by prolonged treatment with buprenorphine. We found no evidence for the involvement of JNK in morphine-induced tolerance to respiratory depression. These results indicate that PKC represents a major mechanism underlying morphine tolerance, that the mechanism of opioid tolerance to respiratory depression is ligand-dependent, and that coadministration of drugs with PKC-inhibitory activity and morphine (as well as heroin, largely metabolized to morphine in the body) may render individuals more susceptible to overdose death by reversing tolerance to the effects of morphine.

  7. Inhibition of PKC activity blocks the increase of ETB receptor expression in cerebral arteries

    PubMed Central

    Henriksson, Marie; Vikman, Petter; Stenman, Emelie; Beg, Saema; Edvinsson, Lars

    2006-01-01

    Background Previous studies have shown that there is a time-dependent upregulation of contractile endothelin B (ETB) receptors in middle cerebral arteries (MCA) after organ culture. This upregulation is dependent on mitogen-activated protein kinases and possibly protein kinase C (PKC). The aim of this study was to examine the effect of PKC inhibitors with different profiles on the upregulation of contractile ETB receptors in rat MCA. Artery segments were incubated for 24 hours at 37°C. To investigate involvement of PKC, inhibitors were added to the medium before incubation. The contractile endothelin-mediated responses were measured and real-time PCR was used to detect endothelin receptor mRNA levels. Furthermore, immunohistochemistry was used to demonstrate the ETB receptor protein distribution in the MCA and Western blot to measure which of the PKC subtypes that were affected by the inhibitors. Results The PKC inhibitors bisindolylmaleimide I, Ro-32-0432 and PKC inhibitor 20–28 attenuated the ETB receptor mediated contractions. Furthermore, Ro-32-0432 and bisindolylmaleimide I decreased ETB receptor mRNA levels while PKC inhibitor 20–28 reduced the amount of receptor protein on smooth muscle cells. PKC inhibitor 20–28 also decreased the protein levels of the five PKC subtypes studied (α, βI, γ, δ and ε). Conclusion The results show that PKC inhibitors are able to decrease the ETB receptor contraction and expression in MCA smooth muscle cells following organ culture. The PKC inhibitor 20–28 affects the protein levels, while Ro-32-0432 and bisindolylmaleimide I affect the mRNA levels, suggesting differences in activity profile. Since ETB receptor upregulation is seen in cerebral ischemia, the results of the present study provide a way to interfere with the vascular involvement in cerebral ischemia. PMID:17129394

  8. PKA and PKC Are Required for Long-Term but Not Short-Term in Vivo Operant Memory in "Aplysia"

    ERIC Educational Resources Information Center

    Michel, Maximilian; Green, Charity L.; Lyons, Lisa C.

    2011-01-01

    We investigated the involvement of PKA and PKC signaling in a negatively reinforced operant learning paradigm in "Aplysia", learning that food is inedible (LFI). In vivo injection of PKA or PKC inhibitors blocked long-term LFI memory formation. Moreover, a persistent phase of PKA activity, although not PKC activity, was necessary for long-term…

  9. Role of protein kinase C (PKC) in short- and long-term cellular responses: inhibition of agonist-mediated calcium transients and down-regulation of PKC

    SciTech Connect

    Fabbro, D.; Mazurek, N.; Borner, C.; Conscience, J.F.; Erne, P.

    1988-01-01

    Active tumor promoters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or membrane-diffusible synthetic diacylglycerols such as 1,2-dioctanoyl-sn-glycerol (DiC8), which specifically activate protein kinase C (PKC), inhibited the agonist-mediated rise in cytosolic calcium ((Ca2+)i) in a mast cell line (PB-3c) and human platelets. TPA inhibition of agonist-mediated calcium transient in platelets was readily reversed by the PKC inhibitor staurosporine. In contrast to DiCs, only active tumor promoters induced a time- and dose-dependent translocation of cytosolic PKC to membranes as determined both enzymatically or by immunoblotting. However, the concentration of TPA required to induce a half-maximal subcellular redistribution of immunodetectable PKC activity was an order of magnitude greater than the half-maximal dose required to inhibit the intracellular rise in (Ca2+)i. Thus, activation of PKC seems not to be exclusively coupled to its translocation to membranes, suggesting that translocation of PKC is mainly involved in the down-regulation of PKC. Down-regulation of immunoprecipitable PKC was studied in various human breast cancer cell lines that display differential growth inhibitory responses toward the tumor promoter. TPA induced translocation of (35S)methionine-prelabeled cytosolic 80 kDa PKC to membranes followed by complete degradation of the enzyme (t1/2 = 2 h) without affecting PKC synthesis. During prolonged TPA exposure, 20-80% of total 80 kDa PKC of control cells was still synthetized as a membrane-bound 74/80 kDa PKC doublet. Although both proteins lacked PKC activity and phorbol ester binding, they revealed structural similarity with the active 80 kDa PKC form of untreated cells.

  10. CCK causes PKD1 activation in pancreatic acini by signaling through PKC-δ and PKC-independent pathways

    PubMed Central

    Berna, Marc J.; Hoffmann, K. Martin; Tapia, Jose A.; Thill, Michelle; Pace, Andrea; Mantey, Samuel A.; Jensen, Robert T.

    2007-01-01

    Summary Protein kinase D1 (PKD1) is involved in cellular processes including protein secretion, proliferation and apoptosis. Studies suggest PKD1 is activated by various stimulants including gastrointestinal (GI) hormones/neurotransmitters and growth factors in a protein kinase C (PKC)-dependent pathway. However, little is known about the mechanisms of PKD1 activation in physiologic GI tissues. We explored PKD1 activation by GI hormones/neurotransmitters and growth factors and the mediators involved in rat pancreatic acini. Only hormones/neurotransmitters activating phospholipase C caused PKD1 phosphorylation (S916, S744/748). CCK activated PKD1 and caused a time- and dose-dependant increase in serine phosphorylation by activation of high- and low-affinity CCKA receptor states. Inhibition of CCK-stimulated increases in phospholipase C, PKC activity or intracellular calcium decreased PKD1 S916 phosphorylation by 56%, 62% and 96%, respectively. PKC inhibitors GF109203X/Go6976/Go6983/PKC-ζ pseudosubstrate caused a 62/43/49/0% inhibition of PKD1 S916 phosphorylation and an 87/13/82/0% inhibition of PKD1 S744/748 phosphorylation. Expression of dominant negative PKC-δ, but not PKC-ε, or treatment with PKC-δ translocation inhibitor caused marked inhibition of PKD phosphorylation. Inhibition of Src/PI3K/MAPK/tyrosine phosphorylation had no effect. In unstimulated cells, PKD1 was mostly located in the cytoplasm. CCK stimulated translocation of total and phosphorylated PKD1 to the membrane. These results demonstrate that CCKA receptor activation leads to PKD activation by signaling through PKC-dependent and PKC-independent pathways. PMID:17306383

  11. SP6616 as a new Kv2.1 channel inhibitor efficiently promotes β-cell survival involving both PKC/Erk1/2 and CaM/PI3K/Akt signaling pathways

    PubMed Central

    Zhou, T T; Quan, L L; Chen, L P; Du, T; Sun, K X; Zhang, J C; Yu, L; Li, Y; Wan, P; Chen, L L; Jiang, B H; Hu, L H; Chen, J; Shen, X

    2016-01-01

    Kv2.1 as a voltage-gated potassium (Kv) channel subunit has a pivotal role in the regulation of glucose-stimulated insulin secretion (GSIS) and pancreatic β-cell apoptosis, and is believed to be a promising target for anti-diabetic drug discovery, although the mechanism underlying the Kv2.1-mediated β-cell apoptosis is obscure. Here, the small molecular compound, ethyl 5-(3-ethoxy-4-methoxyphenyl)-2-(4-hydroxy-3-methoxybenzylidene)-7-methyl-3-oxo-2,3-dihydro-5H-[1,3]thiazolo[3,2–a]pyrimidine-6-carboxylate (SP6616) was discovered to be a new Kv2.1 inhibitor. It was effective in both promoting GSIS and protecting β cells from apoptosis. Evaluation of SP6616 on either high-fat diet combined with streptozocin-induced type 2 diabetic mice or db/db mice further verified its efficacy in the amelioration of β-cell dysfunction and glucose homeostasis. SP6616 treatment efficiently increased serum insulin level, restored β-cell mass, decreased fasting blood glucose and glycated hemoglobin levels, and improved oral glucose tolerance. Mechanism study indicated that the promotion of SP6616 on β-cell survival was tightly linked to its regulation against both protein kinases C (PKC)/extracellular-regulated protein kinases 1/2 (Erk1/2) and calmodulin(CaM)/phosphatidylinositol 3-kinase(PI3K)/serine/threonine-specific protein kinase (Akt) signaling pathways. To our knowledge, this may be the first report on the underlying pathway responsible for the Kv2.1-mediated β-cell protection. In addition, our study has also highlighted the potential of SP6616 in the treatment of type 2 diabetes. PMID:27148689

  12. A pseudosubstrate of PKC inhibits the phorbol dibutyrate (PDBu) effect on permeabilized smooth muscle

    SciTech Connect

    Sullivan, T.S.; Wells, J.N. )

    1991-03-11

    Phorbol esters can induce contraction of vascular smooth muscle and potentiate calcium-induced contractions of permeabilized smooth muscle strips. The authors have used a synthetic peptide inhibitor based on residues 19-31 of PKC (PKC-I) to determine the importance of PKC in the PDBu potentiation of calcium-induced contractions in permeabilized coronary artery smooth muscle. Although peptides similar to PKC-I have been shown to also inhibit MLCK in vitro, MLCK was presumably not inhibited in our system since 30 {mu}M PKC-I alone did not alter the calcium-induced contractions. However, the potentiation of these contractions by 1 {mu}M PDBu was reduced by about 50% in the presence of 10 {mu}M PKC-I, and the potentiation was completely abolished by 30 {mu}M PKC-I. These data indicate that, in this system, PKC is not involved in calcium-induced contractions but that activation of PKC may be the mechanism by which PDBu potentiates calcium-induced contractions in permeabilized coronary artery smooth muscle.

  13. Impairment of neurovascular coupling in type 1 diabetes mellitus in rats is linked to PKC modulation of BK(Ca) and Kir channels.

    PubMed

    Vetri, Francesco; Xu, Haoliang; Paisansathan, Chanannait; Pelligrino, Dale A

    2012-03-15

    We hypothesized that chronic hyperglycemia has a detrimental effect on neurovascular coupling in the brain and that this may be linked to protein kinase C (PKC)-mediated phosphorylation. Therefore, in a rat model of streptozotocin-induced chronic type 1 diabetes mellitus (T1DM), and in nondiabetic (ND) controls, we monitored pial arteriole diameter changes during sciatic nerve stimulation and topical applications of the large-conductance Ca(2+)-operated K(+) channel (BK(Ca)) opener, NS-1619, or the K(+) inward rectifier (Kir) channel agonist, K(+). In the T1DM vs. ND rats, the dilatory response associated with sciatic nerve stimulation was decreased by ∼30%, whereas pial arteriolar dilations to NS-1619 and K(+) were largely suppressed. These responses were completely restored by the acute topical application of a PKC antagonist, calphostin C. Moreover, the suffusion of a PKC activator, phorbol 12,13-dibutyrate, in ND rats was able to reproduce the vascular reactivity impairments found in T1DM rats. Assay of PKC activity in brain samples from T1DM vs. ND rats revealed a significant gain in activity only in specimens harvested from the pial and superficial glia limitans tissue, but not in bulk cortical gray matter. Altogether, these findings suggest that the T1DM-associated impairment of neurovascular coupling may be mechanistically linked to a readily reversible PKC-mediated depression of BK(Ca) and Kir channel activity.

  14. Regulation of the viability of Nf1 deficient cells by PKC isoforms.

    PubMed

    Zhou, Xiaodong; Shen, Ling; Parris, Toshima; Huang, Junchi; Yi, Bo; Helou, Khalil; Chen, Changyan

    2014-11-15

    Suppression of protein kinase C (PKC) is known to be synthetically lethal with ras mutations in various types of cancer cells. The studies also showed that blockade of PKC affected the viability of Nf1 deficient cells. Since PKC family consists of more than 10 isoforms, our study aimed at identifying which isoform(s) played the crucial role in sensitizing Nf1 deficient cells to apoptosis. Using genetic and chemical PKC inhibitors, we demonstrated that the concurrent inhibition of PKC α and β induced Nf1 deficient ST or 96.2 cells, but not SNF02.2 cells with a normal Nf1 or ST cells ectopically expressing Nf1 effective domain gene, to apoptosis. In this process, PKC δ in Nf1 deficient cells, but not in ST/Nf1 cells, was upregulated and translocated to the nucleus. Furthermore, caspase 3 was cleaved and cytochrome c was released to the cytosol. Thus, it appeared that PKC δ and α/β are the crucial components for sustaining the aberrant Ras signaling and further viability of Nf1 deficient cells. The abrogation of these two isoforms activated their opponent PKC δ for switching on the caspase 3-governed apoptotic machinery.

  15. Absence of catalytic domain in a putative protein kinase C (PkcA) suppresses tip dominance in Dictyostelium discoideum.

    PubMed

    Mohamed, Wasima; Ray, Sibnath; Brazill, Derrick; Baskar, Ramamurthy

    2015-09-01

    A number of organisms possess several isoforms of protein kinase C but little is known about the significance of any specific isoform during embryogenesis and development. To address this we characterized a PKC ortholog (PkcA; DDB_G0288147) in Dictyostelium discoideum. pkcA expression switches from prestalk in mound to prespore in slug, indicating a dynamic expression pattern. Mutants lacking the catalytic domain of PkcA (pkcA(-)) did not exhibit tip dominance. A striking phenotype of pkcA- was the formation of an aggregate with a central hollow, and aggregates later fragmented to form small mounds, each becoming a fruiting body. Optical density wave patterns of cAMP in the late aggregates showed several cAMP wave generation centers. We attribute these defects in pkcA(-) to impaired cAMP signaling, altered cell motility and decreased expression of the cell adhesion molecules - CadA and CsaA. pkcA(-) slugs showed ectopic expression of ecmA in the prespore region. Further, the use of a PKC-specific inhibitor, GF109203X that inhibits the activity of catalytic domain phenocopied pkcA(-).

  16. Absence of catalytic domain in a putative protein kinase C (PkcA) suppresses tip dominance in Dictyostelium discoideum

    PubMed Central

    Mohamed, Wasima; Ray, Sibnath; Brazill, Derrick; Baskar, Ramamurthy

    2017-01-01

    A number of organisms possess several isoforms of protein kinase C but little is known about the significance of any specific isoform during embryogenesis and development. To address this we characterized a PKC ortholog (PkcA; DDB_G0288147) in Dictyostelium discoideum. pkcA expression switches from prestalk in mound to prespore in slug, indicating a dynamic expression pattern. Mutants lacking the catalytic domain of PkcA (pkcA−) did not exhibit tip dominance. A striking phenotype of pkcA− was the formation of an aggregate with a central hollow, and aggregates later fragmented to form small mounds, each becoming a fruiting body. Optical density wave patterns of cAMP in the late aggregates showed several cAMP wave generation centers. We attribute these defects in pkcA− to impaired cAMP signaling, altered cell motility and decreased expression of the cell adhesion molecules – CadA and CsaA. pkcA− slugs showed ectopic expression of ecmA in the prespore region. Further, the use of a PKC-specific inhibitor, GF109203X that inhibits the activity of catalytic domain phenocopied pkcA−. PMID:26183108

  17. Investigating the Potential Signaling Pathways That Regulate Activation of the Novel PKC Downstream of Serotonin in Aplysia

    PubMed Central

    Farah, Carole A.; Rourke, Bryan; Shin, Unkyung; Ferguson, Larissa; Luna, María José

    2016-01-01

    Activation of the novel PKC Apl II in sensory neurons by serotonin (5HT) underlies the ability of 5HT to reverse synaptic depression, but the pathway from 5HT to PKC Apl II activation remains unclear. Here we find no evidence for the Aplysia-specific B receptors, or for adenylate cyclase activation, to translocate fluorescently-tagged PKC Apl II. Using an anti-PKC Apl II antibody, we monitor translocation of endogenous PKC Apl II and determine the dose response for PKC Apl II translocation, both in isolated sensory neurons and sensory neurons coupled with motor neurons. Using this assay, we confirm an important role for tyrosine kinase activation in 5HT mediated PKC Apl II translocation, but rule out roles for intracellular tyrosine kinases, epidermal growth factor (EGF) receptors and Trk kinases in this response. A partial inhibition of translocation by a fibroblast growth factor (FGF)-receptor inhibitor led us to clone the Aplysia FGF receptor. Since a number of related receptors have been recently characterized, we use bioinformatics to define the relationship between these receptors and find a single FGF receptor orthologue in Aplysia. However, expression of the FGF receptor did not affect translocation or allow it in motor neurons where 5HT does not normally cause PKC Apl II translocation. These results suggest that additional receptor tyrosine kinases (RTKs) or other molecules must also be involved in translocation of PKC Apl II. PMID:28002451

  18. PKC{eta} confers protection against apoptosis by inhibiting the pro-apoptotic JNK activity in MCF-7 cells

    SciTech Connect

    Rotem-Dai, Noa; Oberkovitz, Galia; Abu-Ghanem, Sara; Livneh, Etta

    2009-09-10

    Apoptosis is frequently regulated by different protein kinases including protein kinase C family enzymes. Both inhibitory and stimulatory effects were demonstrated for several of the different PKC isoforms. Here we show that the novel PKC isoform, PKC{eta}, confers protection against apoptosis induced by the DNA damaging agents, UVC irradiation and the anti-cancer drug - Camptothecin, of the breast epithelial adenocarcinoma MCF-7 cells. The induced expression of PKC{eta} in MCF-7 cells, under the control of the tetracycline-responsive promoter, resulted in increased cell survival and inhibition of cleavage of the apoptotic marker PARP-1. Activation of caspase-7 and 9 and the release of cytochrome c were also inhibited by the inducible expression of PKC{eta}. Furthermore, JNK activity, required for apoptosis in MCF-7, as indicated by the inhibition of both caspase-7 cleavage and cytochrome c release from the mitochondria in the presence of the JNK inhibitor SP600125, was also suppressed by PKC{eta} expression. Hence, in contrast to most PKC isoforms enhancing JNK activation, our studies show that PKC{eta} is an anti-apoptotic protein, acting as a negative regulator of JNK activity. Thus, PKC{eta} could represent a target for intervention aimed to reduce resistance to anti-cancer treatments.

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

    PubMed

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

    2011-06-01

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

  20. Novel PKC-ζ to p47phox interaction is necessary for transformation from blebbishields

    PubMed Central

    Jinesh, Goodwin G.; Taoka, Rikiya; Zhang, Qiang; Gorantla, Siddharth; Kamat, Ashish M.

    2016-01-01

    Cancer stem cells are capable of transformation after apoptosis through the blebbishield emergency program. Reactive oxygen species (ROS) play an essential role in transformation. Understanding how ROS are linked to blebbishield-mediated transformation is necessary to develop efficient therapeutics that target the resurrection of cancer stem cells. Here we demonstrate that a novel PKC-ζ to p47phox interaction is required for ROS production in cancer cells. The combined use of the S6K inhibitor BI-D1870 with TNF-α inhibited the PKC-ζ to p47phox interaction, inhibited ROS production, degraded PKC-ζ, and activated caspases-3 and -8 to block transformation from blebbishields. BI-D1870 also inhibited transformation from cycloheximide-generated blebbishields. Thus ROS and the PKC-ζ to p47phox interaction are valid therapeutic targets to block transformation from blebbishields. PMID:27040869

  1. Lacrimal gland PKC isoforms are differentially involved in agonist-induced protein secretion.

    PubMed

    Zoukhri, D; Hodges, R R; Sergheraert, C; Toker, A; Dartt, D A

    1997-01-01

    In the present study, we have synthesized and N-myristoylated peptides derived from the pseudosubstrate sequences of protein kinase C (PKC)-alpha, -delta, and -epsilon [Myr-PKC-alpha-(15-28), Myr-PKC-delta-(142-153), and Myr-PKC-epsilon-(149-164)], three isoforms present in rat lacrimal gland, and a peptide derived from the sequence of the endogenous inhibitor of protein kinase A [Myr-PKI-(17-25)]. Lacrimal gland acini were preincubated for 60 min with the myristoylated peptides (10(-10) to 3 x 10(-7) M), then protein secretion was stimulated with a phorbol ester, phorbol 12,13-dibutyrate (10(-6) M); vasoactive intestinal peptide (10(-8) M); a cholinergic agonist, carbachol (10(-5) M); or an alpha 1-adrenergic agonist, phenylephrine (10(-4) M), for 20 min. In intact lacrimal gland acini, Myr-PKC-alpha-(15-28) inhibited phorbol 12,13-dibutyrate-induced protein secretion. This effect was not reproduced by the acetylated peptide or by the myristoylated PKI, which inhibited vasoactive intestinal peptide-induced protein secretion, a response mediated by protein kinase A. Carbachol-induced protein secretion was inhibited by all three peptides. In contrast, phenylephrine-induced protein secretion was inhibited only by Myr-PKC-epsilon-(149-164), whereas Myr-PKC-alpha-(15-28) and Myr-PKC-delta-(142-153) had a stimulatory effect. None of these myristoylated peptides affected the calcium increase evoked by cholinergic or alpha 1-adrenergic agonists. We concluded that phorbol ester- and receptor-induced protein secretion involve different PKC isoforms in lacrimal gland.

  2. McEliece PKC Calculator

    NASA Astrophysics Data System (ADS)

    Marek, Repka

    2015-01-01

    The original McEliece PKC proposal is interesting thanks to its resistance against all known attacks, even using quantum cryptanalysis, in an IND-CCA2 secure conversion. Here we present a generic implementation of the original McEliece PKC proposal, which provides test vectors (for all important intermediate results), and also in which a measurement tool for side-channel analysis is employed. To our best knowledge, this is the first such an implementation. This Calculator is valuable in implementation optimization, in further McEliece/Niederreiter like PKCs properties investigations, and also in teaching. Thanks to that, one can, for example, examine side-channel vulnerability of a certain implementation, or one can find out and test particular parameters of the cryptosystem in order to make them appropriate for an efficient hardware implementation. This implementation is available [1] in executable binary format, and as a static C++ library, as well as in form of source codes, for Linux and Windows operating systems.

  3. PKC412 (CGP41251) modulates the proliferation and lipopolysaccharide-induced inflammatory responses of RAW 264.7 macrophages

    SciTech Connect

    Miyatake, Katsutoshi; Inoue, Hiroshi . E-mail: hinoue@genome.tokushima-u.ac.jp; Hashimoto, Kahoko; Takaku, Hiroshi; Takata, Yoichiro; Nakano, Shunji; Yasui, Natsuo; Itakura, Mitsuo

    2007-08-17

    PKC412 (CGP41251) is a multitarget protein kinase inhibitor with anti-tumor activities. Here, we investigated the effects of PKC412 on macrophages. PKC412 inhibited the proliferation of murine RAW 264.7 macrophages through induction of G2/M cell cycle arrest and apoptosis. At non-toxic drug concentrations, PKC412 significantly suppressed the lipopolysaccharide (LPS)-induced release of TNF-{alpha} and nitric oxide, while instead enhancing IL-6 secretion. PKC412 attenuated LPS-induced phosphorylations of MKK4 and JNK, as well as AP-1 DNA binding activities. Furthermore, PKC412 suppressed LPS-induced Akt and GSK-3{beta} phosphorylations. These results suggest that the anti-proliferative and immunomodulatory effects of PKC412 are, at least in part, mediated through its interference with the MKK4/JNK/AP-1 and/or Akt/GSK-3{beta} pathways. Since macrophages contribute significantly to the development of both acute and chronic inflammation, PKC412 may have therapeutic potential and applications in treating inflammatory and/or autoimmune diseases.

  4. The interrelation between aPKC and glucose uptake in the skeletal muscle during contraction and insulin stimulation.

    PubMed

    Santos, J M; Benite-Ribeiro, S A; Queiroz, G; Duarte, J A

    2014-12-01

    Contraction and insulin increase glucose uptake in skeletal muscle. While the insulin pathway, better characterized, requires activation of phosphoinositide 3-kinase (PI3K) and atypical protein kinase (aPKC), muscle contraction seems to share insulin-activated components to increase glucose uptake. This study aimed to investigate the interrelation between the pathway involved in glucose uptake evoked by insulin and muscle contraction. Isolated muscle of rats was treated with solvent (control), insulin, wortmannin (PI3K inhibitor) and the combination of insulin plus wortmannin. After treatment, muscles were electrically stimulated (contracted) or remained at rest. Glucose transporter 4 (GLUT4) localization, glucose uptake and phospho-aPKC (aPKC activated form) were assessed. Muscle contraction and insulin increased glucose uptake in all conditions when compared with controls not stimulating an effect that was accompanied by an increase in GLUT4 and of phospho-aPKC at the muscle membrane. Contracted muscles treated with insulin did not show additive effects on glucose uptake or aPKC activity compared with the response when these stimuli were applied alone. Inhibition of PI3K blocked insulin effect on glucose uptake and aPKC but not in the contractile response. Thus, muscle contraction seems to stimulate aPKC and glucose uptake independently of PI3K. Therefore, aPKC may be a convergence point and a rate limit step in the pathway by which, insulin and contraction, increase glucose uptake in skeletal muscle.

  5. Induction of mitotic catastrophe by PKC inhibition in Nf1-deficient cells.

    PubMed

    Zhou, Xiaodong; Kim, Sung-Hoon; Shen, Ling; Lee, Hyo-Jung; Chen, Changyan

    2014-01-01

    Mutations of tumor suppressor Nf1 gene deregulate Ras-mediated signaling, which confers the predisposition for developing benign or malignant tumors. Inhibition of protein kinase C (PKC) was shown to be in synergy with aberrant Ras for the induction of apoptosis in various types of cancer cells. However, it has not been investigated whether loss of PKC is lethal for Nf1-deficient cells. In this study, using HMG (3-hydroxy-3-methylgutaryl, a PKC inhibitor), we demonstrate that the inhibition of PKC by HMG treatment triggered a persistently mitotic arrest, resulting in the occurrence of mitotic catastrophe in Nf1-deficient ST8814 cells. However, the introduction of the Nf1 effective domain gene into ST8814 cells abolished this mitotic crisis. In addition, HMG injection significantly attenuated the growth of the xenografted ST8814 tumors. Moreover, Chk1 was phosphorylated, accompanied with the persistent increase of cyclin B1 expression in HMG-treated ST8814 cells. The knockdown of Chk1 by the siRNA prevented the Nf1-deficient cells from undergoing HMG-mediated mitotic arrest as well as mitotic catastrophe. Thus, our data suggested that the suppression of PKC activates the Chk1-mediated mitotic exit checkpoint in Nf1-deficient cells, leading to the induction of apoptosis via mitotic catastrophe. Collectively, the study indicates that targeting PKC may be a potential option for developing new strategies to treat Nf1-deficiency-related diseases.

  6. A cell-death-defying factor, anamorsin mediates cell growth through inactivation of PKC and p38MAPK

    SciTech Connect

    Saito, Yuri; Shibayama, Hirohiko; Tanaka, Hirokazu; Tanimura, Akira; Kanakura, Yuzuru

    2011-02-11

    Research highlights: {yields} Anamorsin (AM) (also called CIAPIN-1) is a cell-death-defying factor. {yields} Biological mechanisms of AM functions have not been elucidated yet. {yields} PKC{theta} , PKC{delta} and p38MAPK were more phosphorylated in AM deficient MEF cells. {yields} AM may negatively regulates PKCs and p38MAPK in MEF cells. -- Abstract: Anamorsin (AM) plays crucial roles in hematopoiesis and embryogenesis. AM deficient (AM KO) mice die during late gestation; AM KO embryos are anemic and very small compared to wild type (WT) embryos. To determine which signaling pathways AM utilizes for these functions, we used murine embryonic fibroblast (MEF) cells generated from E-14.5 AM KO or WT embryos. Proliferation of AM KO MEF cells was markedly retarded, and PKC{theta}, PKC{delta}, and p38MAPK were more highly phosphorylated in AM KO MEF cells. Expression of cyclinD1, the target molecule of p38MAPK, was down-regulated in AM KO MEF cells. p38MAPK inhibitor as well as PKC inhibitor restored expression of cyclinD1 and cell growth in AM KO MEF cells. These data suggest that PKC{theta}, PKC{delta}, and p38MAPK activation lead to cell cycle retardation in AM KO MEF cells, and that AM may negatively regulate novel PKCs and p38MAPK in MEF cells.

  7. PKC-dependent activation of human K2P18.1 K+ channels

    PubMed Central

    Rahm, Ann-Kathrin; Gierten, Jakob; Kisselbach, Jana; Staudacher, Ingo; Staudacher, Kathrin; Schweizer, Patrick A; Becker, Rüdiger; Katus, Hugo A; Thomas, Dierk

    2012-01-01

    BACKGROUND AND PURPOSE Two-pore-domain K+ channels (K2P) mediate K+ background currents that modulate the membrane potential of excitable cells. K2P18.1 (TWIK-related spinal cord K+ channel) provides hyperpolarizing background currents in neurons. Recently, a dominant-negative loss-of-function mutation in K2P18.1 has been implicated in migraine, and activation of K2P18.1 channels was proposed as a therapeutic strategy. Here we elucidated the molecular mechanisms underlying PKC-dependent activation of K2P18.1 currents. EXPERIMENTAL APPROACH Human K2P18.1 channels were heterologously expressed in Xenopus laevis oocytes, and currents were recorded with the two-electrode voltage clamp technique. KEY RESULTS Stimulation of PKC using phorbol 12-myristate-13-acetate (PMA) activated the hK2P18.1 current by 3.1-fold in a concentration-dependent fashion. The inactive analogue 4α-PMA had no effect on channel activity. The specific PKC inhibitors bisindolylmaleimide I, Ro-32-0432 and chelerythrine reduced PMA-induced channel activation indicating that PKC is involved in this effect of PMA. Selective activation of conventional PKC isoforms with thymeleatoxin (100 nM) did not reproduce K2P18.1 channel activation. Current activation by PMA was not affected by pretreatment with CsA (calcineurin inhibitor) or KT 5720 (PKA inhibitor), ruling out a significant contribution of calcineurin or cross-talk with PKA to the PKC-dependent hK2P18.1 activation. Finally, mutation of putative PKC phosphorylation sites did not prevent PMA-induced K2P18.1 channel activation. CONCLUSIONS AND IMPLICATIONS We demonstrated that activation of hK2P18.1 (TRESK) by PMA is mediated by PKC stimulation. Hence, PKC-mediated activation of K2P18.1 background currents may serve as a novel molecular target for migraine treatment. PMID:22168364

  8. PKC Isoform Expression in Modeled Microgravity

    NASA Technical Reports Server (NTRS)

    Risin, Diana; Sundaresan, Alamelu; Pellis, Neal R.; Dawson, David L. (Technical Monitor)

    1999-01-01

    Our previous studies showed that modeled (MMG) and true (USA Space Shuttle Missions STS-54 and STS-56) microgravity (MG) inhibit human lymphocyte locomotion, Modeled MG also suppressed polyclonal and antigen-specific lymphocyte activation. Activation of PKC by phorbol myristate acetate (PMA) restored the microgravity-inhibited lymphocyte locomotion as well as activation by phytohaemagglutinin (PHA), whereas calcium ionophore (ionomycin) was unable to restore these functions. Based on these results we hypothesized that MG-induced changes in lymphocyte functions are caused by a fundamental defect in signal transduction mechanism. This defect may be localized either at the PKC level or upstream of PKC, most likely, at the cell membrane level. In this study we examined the expression of PKC isoforms alpha, epsilon and delta in PBMC cultured in rotating wall vessel bioreactor, developed at NASA JSC, which models microgravity by sustaining cells in continuous free fall. The assessment of the isoforms was performed by FACS analysis following cell permeabilization. A decrease in the expression of isoforms epsilon and delta, but not isoform a, was observed in PBMC cultured in microgravity conditions. These data suggest that MMG might selectively affect the expression of Ca2+ independent isoforms of PKC Molecular analysis confirm selective suppression of Ca2+ independent isoforms of PKC.

  9. PKC δ mediates pro-inflammatory responses in a mouse model of caerulein-induced acute pancreatitis.

    PubMed

    Ramnath, Raina Devi; Sun, Jia; Bhatia, Madhav

    2010-10-01

    Acute pancreatitis is an inflammatory disorder of the pancreas. Protein kinase C (PKC) δ plays an important role in mediating chemokine production in mouse pancreatic acinar cells. This study aims to investigate the role of PKC δ in the pathogenesis of acute pancreatitis and to explore the mechanisms through which PKC δ mediates pro-inflammatory signaling. Acute pancreatitis was induced in mice by ten hourly intraperitoneal injections of caerulein. PKC δ translocation inhibitor peptide (δV1-1) at a dose of 1.0 mg/kg or Tat (carrier peptide) at a dose of 1.0 mg/kg was administered to mice either 1 h before or 1 h after the first caerulein injection. One hour after the last caerulein injection, the mice were killed and pancreas, lungs, and blood were collected. Prophylactic and therapeutic treatment with δV1-1 attenuated caerulein-induced plasma amylase levels and pancreatic edema. Treatment with δV1-1 decreased myeloperoxidase activity and monocyte chemotactic protein-1 levels in both pancreas and plasma. PKC δ mediated acute pancreatitis by activating pancreatic nuclear factor κB, activator protein-1, and mitogen-activated protein kinases. Moreover, blockade of PKC δ attenuated lung myeloperoxidase activity and edema. Histological examination of pancreatic and lung sections confirmed protection against acute pancreatitis. Treatment with Tat had no protective effect on acute pancreatitis. Blockade of PKC δ represents a promising prophylactic and/or therapeutic tool for the treatment of acute pancreatitis.

  10. Coordinated activation of mitochondrial respiration and exocytosis mediated by PKC signaling in pancreatic β cells.

    PubMed

    Santo-Domingo, Jaime; Chareyron, Isabelle; Dayon, Loïc; Núñez Galindo, Antonio; Cominetti, Ornella; Pilar Giner Giménez, María; De Marchi, Umberto; Canto, Carles; Kussmann, Martin; Wiederkehr, Andreas

    2017-03-01

    Mitochondria play a central role in pancreatic β-cell nutrient sensing by coupling their metabolism to plasma membrane excitability and insulin granule exocytosis. Whether non-nutrient secretagogues stimulate mitochondria as part of the molecular mechanism to promote insulin secretion is not known. Here, we show that PKC signaling, which is employed by many non-nutrient secretagogues, augments mitochondrial respiration in INS-1E (rat insulinoma cell line clone 1E) and human pancreatic β cells. The phorbol ester, phorbol 12-myristate 13-acetate, accelerates mitochondrial respiration at both resting and stimulatory glucose concentrations. A range of inhibitors of novel PKC isoforms prevent phorbol ester-induced respiration. Respiratory response was blocked by oligomycin that demonstrated PKC-dependent acceleration of mitochondrial ATP synthesis. Enhanced respiration was observed even when glycolysis was bypassed or fatty acid transport was blocked, which suggested that PKC regulates mitochondrial processes rather than upstream catabolic fluxes. A phosphoproteome study of phorbol ester-stimulated INS-1E cells maintained under resting (2.5 mM) glucose revealed a large number of phosphorylation sites that were altered during short-term activation of PKC signaling. The data set was enriched for proteins that are involved in gene expression, cytoskeleton remodeling, secretory vesicle transport, and exocytosis. Interactome analysis identified PKC, C-Raf, and ERK1/2 as the central phosphointeraction cluster. Prevention of ERK1/2 signaling by using a MEK1 inhibitor caused a marked decreased in phorbol 12-myristate 13-acetate-induced mitochondrial respiration. ERK1/2 signaling module therefore links PKC activation to downstream mitochondrial activation. We conclude that non-nutrient secretagogues act, in part, via PKC and downstream ERK1/2 signaling to stimulate mitochondrial energy production to compensate for energy expenditure that is linked to β-cell activation

  11. Contraction stimulates muscle glucose uptake independent of atypical PKC.

    PubMed

    Yu, Haiyan; Fujii, Nobuharu L; Toyoda, Taro; An, Ding; Farese, Robert V; Leitges, Michael; Hirshman, Michael F; Mul, Joram D; Goodyear, Laurie J

    2015-11-01

    Exercise increases skeletal muscle glucose uptake, but the underlying mechanisms are only partially understood. The atypical protein kinase C (PKC) isoforms λ and ζ (PKC-λ/ζ) have been shown to be necessary for insulin-, AICAR-, and metformin-stimulated glucose uptake in skeletal muscle, but not for treadmill exercise-stimulated muscle glucose uptake. To investigate if PKC-λ/ζ activity is required for contraction-stimulated muscle glucose uptake, we used mice with tibialis anterior muscle-specific overexpression of an empty vector (WT), wild-type PKC-ζ (PKC-ζ(WT)), or an enzymatically inactive T410A-PKC-ζ mutant (PKC-ζ(T410A)). We also studied skeletal muscle-specific PKC-λ knockout (MλKO) mice. Basal glucose uptake was similar between WT, PKC-ζ(WT), and PKC-ζ(T410A) tibialis anterior muscles. In contrast, in situ contraction-stimulated glucose uptake was increased in PKC-ζ(T410A) tibialis anterior muscles compared to WT or PKC-ζ(WT) tibialis anterior muscles. Furthermore, in vitro contraction-stimulated glucose uptake was greater in soleus muscles of MλKO mice than WT controls. Thus, loss of PKC-λ/ζ activity increases contraction-stimulated muscle glucose uptake. These data clearly demonstrate that PKC-λζ activity is not necessary for contraction-stimulated glucose uptake.

  12. Pilocarpine-induced status epilepticus alters hippocampal PKC expression in mice.

    PubMed

    Liu, Jian Xin; Liu, Yong; Tang, Feng Ru

    2011-01-01

    We investigated the protein expression of different protein kinase C (PKC) isoforms (PKC-alpha, PKC-beta1, PKC-beta2, PKC-gamma, PKC-delta, PKC-epsilon, PKC-eta and PKC-zeta) in the hippocampus of normal control mice and progressive changes in PKC isoforms expression during and after pilocarpine induced status epilepticus (PISE). We showed the reduced expression of PKC-delta, PKC-eta and PKC-zeta in interneurons in the CA1 area and in the hilus of the dentate gyrus during or after PISE. Increased expression of PKC-alpha and PKC-beta1 was demonstrated in the stratum pyramidale of CA3 area, and PKC-epsilon was up-regulated in the stratum lucidum of the CA3 area during or after PISE. Our results suggest that hippocampal PKC isoforms may play different roles in seizure generation, and be targets for development of anti-convulsive drugs.

  13. PKC and PTPα participate in Src activation by 1α,25OH2 vitamin D3 in C2C12 skeletal muscle cells.

    PubMed

    Buitrago, Claudia; Costabel, Marcelo; Boland, Ricardo

    2011-06-06

    We previously demonstrated that 1α,25(OH)(2)-vitamin D(3) [1α,25(OH)(2)D(3)] induces Src activation, which mediates the hormone-dependent ERK1/2 and p38 MAPK phosphorylation in skeletal muscle cells. In the present study, we have investigated upstream steps whereby 1α,25(OH)(2)D(3) may act to transmit its signal to Src. Preincubation with the PKC inhibitor Ro318220 demonstrated the participation of PKC in 1α,25(OH)(2)D(3)-dependent Src activation. Of interest, the hormone promoted the activation of δ the isoform of PKC. We also explored the role of PTPα in PKC-mediated Src stimulation. Silencing of PTPα with a specific siRNA suppressed Src activation induced by 1α,25(OH)(2)D(3). Hormone treatment increased PTPα (Tyr789) phosphorylation and PKC-dependent phosphatase activity. Accordingly, 1α,25(OH)(2)D(3) promoted serine phosphorylation of PTPα in a PKC-dependent manner. Confocal immunocytochemistry and co-immunoprecipitation assays revealed that the hormone induces the co-localization of Src and PTPα with PKC participation. Computational analysis revealed that the electrostatic interaction between Src and PTPα is favored when PTPα is phosphorylated in Tyr789. These data suggest that 1α,25(OH)(2)D(3) acts in skeletal muscle upstream on MAPK cascades sequentially activating PKC, PTPα and Src.

  14. Posttetanic potentiation critically depends on an enhanced Ca2+ sensitivity of vesicle fusion mediated by presynaptic PKC

    PubMed Central

    Korogod, Natalya; Lou, Xuelin; Schneggenburger, Ralf

    2007-01-01

    Activity-dependent enhancement of transmitter release is a common form of presynaptic plasticity, but the underlying signaling mechanisms have remained largely unknown, perhaps because of the inaccessibility of most CNS nerve terminals. Here we investigated the signaling steps that underlie posttetanic potentiation (PTP), a form of presynaptic plasticity found at many CNS synapses. Direct whole-cell recordings from the large calyx of Held nerve terminals with the perforated patch-clamp technique showed that PTP was not mediated by changes in the presynaptic action potential waveform. Ca2+ imaging revealed a slight increase of the presynaptic Ca2+ transient during PTP (≈15%), which, however, was too small to explain a large part of PTP. The presynaptic PKC pathway was critically involved in PTP because (i) PTP was occluded by activation of PKC with phorbol esters, and (ii) PTP was largely (by approximately two-thirds) blocked by the PKC inhibitors, Ro31-8220 or bisindolylmaleimide. Activation of PKC during PTP most likely acts directly on the presynaptic release machinery, because in presynaptic Ca2+ uncaging experiments, activation of PKC by phorbol ester greatly increased the Ca2+ sensitivity of vesicle fusion in a Ro31-8220-sensitive manner (≈300% with small Ca2+ uncaging stimuli), but only slightly increased presynaptic voltage-gated Ca2+ currents (≈15%). We conclude that a PKC-dependent increase in the Ca2+ sensitivity of vesicle fusion is a key step in the enhancement of transmitter release during PTP. PMID:17884983

  15. CHBPR-Angiotensin II stimulates renin in inner medullary collecting duct cells via PKC and independent of ENaC and mineralocorticoid receptor activity

    PubMed Central

    Gonzalez, Alexis A.; Liu, Liu; Lara, Lucienne S.; Seth, Dale M; Navar, L. Gabriel; Prieto, Minolfa C

    2011-01-01

    Collecting duct (CD) renin is stimulated by angiotensin (Ang) II providing a pathway for Ang I generation and further conversion to Ang II. Ang II stimulates epithelial sodium channel (ENaC) via Ang II type 1 receptor (AT1R) and increases mineralocorticoid receptor (MR) activity due to increased aldosterone release. Our objective was to determine if CD renin augmentation is mediated directly by AT1R or via ENaC and MR. In vivo studies examined the effects of ENaC blockade (amiloride; 5 mg/kg/day) on CD renin expression and urinary renin content (URC) in Ang II-infused rats (80 ng/min, 2 weeks). Ang II infusion increased systolic blood pressure (SBP), medullary renin mRNA, URC and intrarenal Ang II levels. Amiloride co-treatment did not alter these responses despite reduction in the rate of progression of SBP. In primary cultures of inner medullary CD (IMCD) cells, renin mRNA and (pro)renin protein levels increased with Ang II (100 nmol/L), and candesartan (AT1R antagonist) prevented this effect. Aldosterone (10−10 to 10−7 mol/L) with or without amiloride did not modify the upregulation of renin mRNA in Ang II treated cells. However, inhibition of protein kinase C (PKC) with calphostin C prevented the Ang II-mediated increases in renin mRNA and (pro)renin protein levels. Furthermore, PKC activation with phorbol 12-myristate 13-acetate (PMA) increased renin expression to the same extent as Ang II. These data indicate that AT1R-mediated increase in CD renin is induced directly by Ang II via PKC pathway and that this regulation is independent of MR activation or ENaC activity. PMID:21282553

  16. IKKε inhibits PKC to promote Fascin-dependent actin bundling

    PubMed Central

    Ogura, Yosuke; Misaki, Kazuyo; Maeda, Takuya; Kimpara, Akiyo; Yonemura, Shigenobu; Hayashi, Shigeo

    2016-01-01

    Signaling molecules have pleiotropic functions and are activated by various extracellular stimuli. Protein kinase C (PKC) is activated by diverse receptors, and its dysregulation is associated with diseases including cancer. However, how the undesired activation of PKC is prevented during development remains poorly understood. We have previously shown that a protein kinase, IKKε, is active at the growing bristle tip and regulates actin bundle organization during Drosophila bristle morphogenesis. Here, we demonstrate that IKKε regulates the actin bundle localization of a dynamic actin cross-linker, Fascin. IKKε inhibits PKC, thereby protecting Fascin from inhibitory phosphorylation. Excess PKC activation is responsible for the actin bundle defects in IKKε-deficient bristles, whereas PKC is dispensable for bristle morphogenesis in wild-type bristles, indicating that PKC is repressed by IKKε in wild-type bristle cells. These results suggest that IKKε prevents excess activation of PKC during bristle morphogenesis. PMID:27578797

  17. Amoebic PI3K and PKC is required for Jurkat T cell death induced by Entamoeba histolytica.

    PubMed

    Lee, Young Ah; Kim, Kyeong Ah; Min, Arim; Shin, Myeong Heon

    2014-08-01

    The enteric protozoan parasite Entamoeba histolytica is the causative agent of human amebiasis. During infection, adherence of E. histolytica through Gal/GalNAc lectin on the surface of the amoeba can induce caspase-3-dependent or -independent host cell death. Phosphorylinositol 3-kinase (PI3K) and protein kinase C (PKC) in E. histolytica play an important function in the adhesion, killing, or phagocytosis of target cells. In this study, we examined the role of amoebic PI3K and PKC in amoeba-induced apoptotic cell death in Jurkat T cells. When Jurkat T cells were incubated with E. histolytica trophozoites, phosphatidylserine (PS) externalization and DNA fragmentation in Jurkat cells were markedly increased compared to those of cells incubated with medium alone. However, when amoebae were pretreated with a PI3K inhibitor, wortmannin before being incubated with E. histolytica, E. histolytica-induced PS externalization and DNA fragmentation in Jurkat cells were significantly reduced compared to results for amoebae pretreated with DMSO. In addition, pretreatment of amoebae with a PKC inhibitor, staurosporine strongly inhibited Jurkat T cell death. However, E. histolytica-induced cleavage of caspase-3, -6, and -7 were not inhibited by pretreatment of amoebae with wortmannin or staurosporin. In addition, we found that amoebic PI3K and PKC have an important role on amoeba adhesion to host compartment. These results suggest that amebic PI3K and PKC activation may play an important role in caspase-independent cell death in Entamoeba-induced apoptosis.

  18. Polycystin-1 promotes PKC{alpha}-mediated NF-{kappa}B activation in kidney cells

    SciTech Connect

    Banzi, Manuela; Aguiari, Gianluca; Trimi, Viky; Mangolini, Alessandra; Pinton, Paolo; Witzgall, Ralph; Rizzuto, Rosario; Senno, Laura del . E-mail: sen@unife.it

    2006-11-17

    Polycystin-1 (PC1), the PKD1 gene product, is a membrane receptor which regulates many cell functions, including cell proliferation and apoptosis, both typically increased in cyst lining cells in autosomal dominant polycystic kidney disease. Here we show that PC1 upregulates the NF-{kappa}B signalling pathway in kidney cells to prevent cell death. Human embryonic kidney cell lines (HEK293{sup CTT}), stably expressing a PC1 cytoplasmic terminal tail (CTT), presented increased NF-{kappa}B nuclear levels and NF-{kappa}B-mediated luciferase promoter activity. This, consistently, was reduced in HEK293 cells in which the endogenous PC1 was depleted by RNA interference. CTT-dependent NF-{kappa}B promoter activation was mediated by PKC{alpha} because it was blocked by its specific inhibitor Ro-320432. Furthermore, it was observed that apoptosis, which was increased in PC1-depleted cells, was reduced in HEK293{sup CTT} cells and in porcine kidney LtTA cells expressing a doxycycline-regulated CTT. Staurosporine, a PKC inhibitor, and parthenolide, a NF-{kappa}B inhibitor, significantly reduced the CTT-dependent antiapoptotic effect. These data reveal, therefore, a novel pathway by which polycystin-1 activates a PKC{alpha}-mediated NF-{kappa}B signalling and cell survival.

  19. Sevoflurane Stimulates MAP Kinase Signal transduction through the Activation of PKC α and βII in Fetal Rat Cerebral Cortex Cultured Neuron

    PubMed Central

    Hasegawa, Jun; Takekoshi, Susumu; Nagata, Hidetaka; Osamura, R. Yoshiyuki; Suzuki, Toshiyasu

    2006-01-01

    Protein kinase C (PKC) is a key enzyme that participates in various neuronal functions. PKC has also been identified as a target molecule for general anesthetic actions. Raf, mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK1/2) have been thought to be target effectors of PKC. In the present study, we attempted to evaluate the effect of sevoflurane on PKC/MAPK cascade signaling in cultured fetal rat cerebral ­cortex neurons, prepared from embryonic day 18 fetuses. The effects of sevoflurane on the translocation of 7 PKC isoforms (α, βI, βII, γ, δ, ɛ and ζ) were observed by immunoblotting using isoform-selective antibodies to PKCs. The treatment of neurons with sevoflurane induced the translocation of PKC α and PKC βII species from the cytosol to the membrane fraction, which indicated the activation of these PKC isoforms. In contrast, there was no clear change in the distribution of other PKC isoforms. We next examined whether the specific activation of PKC α and βII by sevoflurane could stimulate the MAP kinase signaling pathway in cultured neurons. Raf phosphorylation was increased by the administration of 0.25 mM sevoflurane. The phosphorylation of Raf proteins reached a maximum at 5–10 min. Subsequently, the phosphorylation of MEK proteins was increased at 10–15 min after sevoflurane treatments. That of ERK proteins was induced at 15–60 min. Moreover, the phosphorylation of ERK induced by sevoflurane was significantly decreased by the treatment of PKC inhibitor (staurosporine) and MEK inhibitor (PD98059). On the other hand, the contents of total Raf, MEK and ERK proteins were relatively constant at all times examined. To examine the ­localization of phosphorylated-ERK protein, immunohistochemical staining of sevoflurane-treated cultured neurons was performed. The phosphorylated-ERK proteins were markedly accumulated in both the cytosol of the cell body and the neurites in the neuronal cells with time after 0

  20. miR-940 Upregulation Suppresses Cell Proliferation and Induces Apoptosis by Targeting PKC-δ in Ovarian Cancer OVCAR3 Cells.

    PubMed

    Wang, Fang; Wang, Zhihong; Gu, Xiaoli; Cui, Jinquan

    2017-01-02

    Ovarian cancer remains as one of the most threatening malignancies for females in the world. This study investigated the pivotal role of miR-940 in the progression of ovarian cancer and to reveal the possible molecular mechanism of its action. Ovarian cancer OVCAR3 cells were transfected with the miR-940 vector, miR-940 inhibitor, and/or small interfering RNA (siRNA) targeting PKC-δ (si-PKC-δ), respectively. After transfection, cell viability and cell apoptosis were analyzed, as well as cell proliferation and apoptosis-related protein expression. Compared to the control, miR-940 upregulation suppressed cell viability but induced cell apoptosis. miR-940 upregulation increased the expression of p27, Hes1, survivin, and caspase 3, but decreased the expression of PKC-δ. In addition, elevated cell viability induced by the miR-940 inhibitor was significantly decreased by knockdown of PKC-δ, and reduced cell apoptosis induced by the miR-940 inhibitor was increased by knockdown of PKC-δ. Taken together, the results of our study suggest that upregulation of miR-940 may function as a suppressor in the progression of ovarian cancer by inhibiting cell proliferation and inducing apoptosis by targeting PKC-δ. This study may provide a basis for the possible application of miR-940 in illustrating the molecular pathogenic mechanism of ovarian cancer.

  1. PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90.

    PubMed

    LaFayette, Shantelle L; Collins, Cathy; Zaas, Aimee K; Schell, Wiley A; Betancourt-Quiroz, Marisol; Gunatilaka, A A Leslie; Perfect, John R; Cowen, Leah E

    2010-08-26

    Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug

  2. A PKM Generated by Calpain Cleavage of a Classical PKC Is Required for Activity-Dependent Intermediate-Term Facilitation in the Presynaptic Sensory Neuron of "Aplysia"

    ERIC Educational Resources Information Center

    Farah, Carole A.; Hastings, Margaret H.; Dunn, Tyler W.; Gong, Katrina; Baker-Andresen, Danay; Sossin, Wayne S.

    2017-01-01

    Atypical PKM, a persistently active form of atypical PKC, is proposed to be a molecular memory trace, but there have been few examinations of the role of PKMs generated from other PKCs. We demonstrate that inhibitors used to inhibit PKMs generated from atypical PKCs are also effective inhibitors of other PKMs. In contrast, we demonstrate that…

  3. GBPI, a novel gastrointestinal- and brain-specific PP1-inhibitory protein, is activated by PKC and inactivated by PKA.

    PubMed Central

    Liu, Qing-Rong; Zhang, Ping-Wu; Lin, Zhicheng; Li, Qi-Fu; Woods, Amina S; Troncoso, Juan; Uhl, George R

    2004-01-01

    The activities of PP1 (protein phosphatase 1), a principal cellular phosphatase that reverses serine/threonine protein phosphorylation, can be altered by inhibitors whose activities are themselves regulated by phosphorylation. We now describe a novel PKC (protein kinase C)-dependent PP1 inhibitor, namely GBPI (gut and brain phosphatase inhibitor). The shorter mRNA that encodes this protein, GBPI-1, is expressed in brain, stomach, small intestine, colon and kidney, whereas a longer GBPI-2 splice variant mRNA is found in testis. Human GBPI-1 mRNA encodes a 145-amino-acid, 16.5 kDa protein with pI 7.92. GBPI contains a consensus PP1-binding motif at residues 21-25 and consensus sites for phosphorylation by enzymes, including PKC, PKA (protein kinase A or cAMP-dependent protein kinase) and casein kinase II. Recombinant GBPI-1-fusion protein inhibits PP1 activity with IC50=3 nM after phosphorylation by PKC. Phospho-GBPI can even enhance PP2A activity by >50% at submicromolar concentrations. Non-phosphorylated GBPI-1 is inactive in both assays. Each of the mutations in amino acids located in potential PP1-binding sequences, K21E+K22E and W25A, decrease the ability of GBPI-1 to inhibit PP1. Mutations in the potential PKC phosphoacceptor site T58E also dramatically decrease the ability of GBPI-1 to inhibit PP1. Interestingly, when PKC-phosphorylated GBPI-1 is further phosphorylated by PKA, it no longer inhibits PP1. Thus, GBPI-1 is well positioned to integrate PKC and PKA modulation of PP1 to regulate differentially protein phosphorylation patterns in brain and gut. GBPI, its closest family member CPI (PKC-potentiated PP1 inhibitor) and two other family members, kinase-enhanced phosphatase inhibitor and phosphatase holoenzyme inhibitor, probably modulate integrated control of protein phosphorylation states in these and other tissues. PMID:12974676

  4. Translocation of PKC-betaII is mediated via RACK-1 in the neuronal cells following dioxin exposure.

    PubMed

    Lee, Hyun-Gyo; Kim, Sun-Young; Choi, Eun-Jung; Park, Ki-Yeon; Yang, Jae-Ho

    2007-03-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to induce neurotoxic effects. However, the mechanism of TCDD-mediated signaling pathways and its possible molecular targets in neurons remains unknown. In this study, we analyzed effects of TCDD on neurofilament subunits, receptor for activated C kinase-1 (RACK-1), and PKC-betaII activity in developing neuronal cells. TCDD induced a significant increase of RACK-1, an adaptor protein for protein kinase C (PKC), in cerebellar granule cells in both dose- and time-dependent manner, indicating that RACK-1 is a sensitive molecular target in neuronal cells for TCDD exposure. TCDD induced a dose-dependent translocation of PKC-betaII from cytosol to membrane fractions. However, when RACK-1 induction was blocked by antisense oligonucleotide or alpha-naphthoflavone, Ah receptor (AhR) inhibitor, the translocation of PKC-betaII was inhibited. Our data suggests that TCDD activates PKC-betaII via RACK-1 in an AhR-dependent manner. This is the first report identifying RACK-1 as a target molecule involved in TCDD-mediated signaling pathways. TCDD exposure also increased the level of neurofilament-H mRNA. These results suggest that identification of target molecules may contribute to improve our understanding of TCDD-mediated signaling pathway and the risk assessment of TCDD-induced neurotoxicities.

  5. In vitro neutrophil migration requires protein kinase c-delta (δ-PKC) mediated MARCKS (Myristoylated Alanine Rich C-Kinase Substrate) phosphorylation

    PubMed Central

    Sung, Eui Jae; Adler, Kenneth B.; Jones, Samuel L.

    2015-01-01

    Dysregulated release of neutrophil reactive oxygen species and proteolytic enzymes contributes to both acute and chronic inflammatory diseases. Therefore, molecular regulators of these processes are potential targets for new anti-inflammatory therapies. We have shown previously that MARCKS (Myristoylated Alanine Rich C-Kinase Substrate), a well-known PKC substrate protein, is a key regulator of neutrophil functions. In the current study we investigate the role of PKC-mediated MARCKS phosphorylation in neutrophil migration and adhesion in vitro. We report that treatment of human neutrophils with the δ-PKC inhibitor rottlerin significantly attenuates fMLF induced MARCKS phosphorylation (IC50 = 5.709 μM), adhesion (IC50 = 8.4 uM) and migration (IC50 = 6.7 uM); while α-, β- and ζ-PKC inhibitors had no significant effect. We conclude that δ-PKC mediated MARCKS phosphorylation is essential for human neutrophil migration and adhesion in vitro. These results implicate δ-PKC mediated MARCKS phosphorylation as a key step in the inflammatory response of neutrophils. PMID:25515270

  6. Melatonin potentiates glycine currents through a PLC/PKC signalling pathway in rat retinal ganglion cells.

    PubMed

    Zhao, Wen-Jie; Zhang, Min; Miao, Yanying; Yang, Xiong-Li; Wang, Zhongfeng

    2010-07-15

    In vertebrate retina, melatonin regulates various physiological functions. In this work we investigated the mechanisms underlying melatonin-induced potentiation of glycine currents in rat retinal ganglion cells (RGCs). Immunofluorescence double labelling showed that rat RGCs were solely immunoreactive to melatonin MT(2) receptors. Melatonin potentiated glycine currents of RGCs, which was reversed by the MT(2) receptor antagonist 4-P-PDOT. The melatonin effect was blocked by intracellular dialysis of GDP-beta-S. Either preincubation with pertussis toxin or application of the phosphatidylcholine (PC)-specific phospholipase C (PLC) inhibitor D609, but not the phosphatidylinositol (PI)-PLC inhibitor U73122, blocked the melatonin effect. The protein kinase C (PKC) activator PMA potentiated the glycine currents and in the presence of PMA melatonin failed to cause further potentiation of the currents, whereas application of the PKC inhibitor bisindolylmaleimide IV abolished the melatonin-induced potentiation. The melatonin effect persisted when [Ca(2+)](i) was chelated by BAPTA, and melatonin induced no increase in [Ca(2+)](i). Neither cAMP-PKA nor cGMP-PKG signalling pathways seemed to be involved because 8-Br-cAMP or 8-Br-cGMP failed to cause potentiation of the glycine currents and both the PKA inhibitor H-89 and the PKG inhibitor KT5823 did not block the melatonin-induced potentiation. In consequence, a distinct PC-PLC/PKC signalling pathway, following the activation of G(i/o)-coupled MT(2) receptors, is most likely responsible for the melatonin-induced potentiation of glycine currents of rat RGCs. Furthermore, in rat retinal slices melatonin potentiated light-evoked glycine receptor-mediated inhibitory postsynaptic currents in RGCs. These results suggest that melatonin, being at higher levels at night, may help animals to detect positive or negative contrast in night vision by modulating inhibitory signals largely mediated by glycinergic amacrine cells in the inner

  7. The participation of NMDA receptors, PKC, and MAPK in Lymnaea memory extinction.

    PubMed

    Rosenegger, David; Lukowiak, Ken

    2013-02-01

    The aerial respiratory behavior of Lymnaea can be operantly conditioned to form a long-term memory (LTM) that will persist for >24h. LTM formation is dependent on altered gene activity and new protein synthesis, with the N-methyl-D-aspartate (NMDA) receptors, mitogen activated protein kinase (MAPK), and protein kinase C (PKC) pathways playing a critical role. LTM can also undergo extinction, whereby the original memory is temporarily masked by a new memory. Here we investigate if the formation of an extinction memory uses similar molecular pathways to those required for LTM formation. We find that the formation of the extinction memory can be blocked by inhibitors of NMDA receptors, PKC, and MAPK suggesting that extinction memory formation uses similar mechanisms to that of 'normal' memory formation.

  8. C-Fos Regulation by the MAPK and PKC Pathways in Intervertebral Disc Cells

    PubMed Central

    Yokoyama, Katsuya; Hiyama, Akihiko; Arai, Fumiyuki; Nukaga, Tadashi; Sakai, Daisuke; Mochida, Joji

    2013-01-01

    Background The gene encoding c-fos is an important factor in the pathogenesis of joint disease in patients with osteoarthritis. However, it is unknown whether the signal mechanism of c-fos acts in intervertebral disc (IVD) cells. We investigated whether c-fos is activated in relation to mitogen-activated protein kinases (MAPKs) and the protein kinase C (PKC) pathway in nucleus pulposus (NP) cells. Methodology/Results Reverse transcription-polymerase chain reaction and western blotting analyses were used to measure the expression of c-fos in rat IVD cells. Transfections were performed to determine the effects of c-fos on target gene activity. The effect of c-fos protein expression was examined in transfection experiments and in a 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide cell viability assay. Phorbol 12-myristate 13-acetate (PMA), the most commonly used phorbol ester, binds to and activates protein kinase C (PKC), causing a wide range of effects in cells and tissues. PMA induced the expression of c-fos gene transcription and protein expression, and led to activation of the MAPK pathways in NP cells. The c-fos promoter was suppressed completely in the presence of the MAPK inhibitor PD98059, an inhibitor of the MEK/ERK kinase cascade, but not in the presence of SKF86002, SB202190, or SP600125. The effects of the PKC pathway on the transcriptional activity of the c-fos were evaluated. PKCγ and PKCδ suppressed the promoter activity of c-fos. Treatment with c-fos inhibited aggrecan and Col2 promoter activities and the expression of these genes in NP cells. Conclusions This study demonstrated, for the first time, that the MAPK and PKC pathways had opposing effects on the regulation of c-fos in NP cells. Thus, the expression of c-fos can be suppressed in the extracellular matrix of NP cells. PMID:24023832

  9. Lead acetate induces EGFR activation upstream of SFK and PKC{alpha} linkage to the Ras/Raf-1/ERK signaling

    SciTech Connect

    Wang, C.-Y.; Wang, Y.-T.; Tzeng, D.-W.; Yang, J.-L.

    2009-03-01

    Lead acetate (Pb), a probable human carcinogen, can activate protein kinase C (PKC) upstream of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Yet, it remains unclear whether Pb activation of PKC {yields} ERK1/2 involves receptor/non-receptor tyrosine kinases and the Ras signaling transducer. Here we demonstrate a novel mechanism elicited by Pb for transmitting ERK1/2 signaling in CL3 human non-small-cell lung adenocarcinoma cells. Pb induction of higher steady-state levels of Ras-GTP was essential for increasing phospho-Raf-1{sup S338} and phospho-ERK1/2. Pre-treatment of the cells with a conventional PKC inhibitor Goe6976 or depleting PKC{alpha} using specific small interfering RNA blocked Pb induction of Ras-GTP. Pb also activated cellular tyrosine kinases. Specific pharmacological inhibitors, PD153035 for epidermal growth factor receptor (EGFR) and SU6656 for Src family tyrosine kinases (SFK), but not AG1296 for platelet-derived growth factor receptor, could suppress the Pb-induced tyrosine kinases, PKC{alpha}, Ras-GTP, phospho-Raf-1{sup S338} and phospho-ERK1/2. Furthermore, phosphorylation of tyrosines on the EGFR multiple autophosphorylation sites and the conserved SFK autophosphorylation site occurred during exposure of cells to Pb for 1-5 min and 5-30 min, respectively. Intriguingly, Pb activation of EGFR required the intrinsic kinase activity but not dimerization of the receptor. Inhibition of SFK or PKC{alpha} activities did not affect EGFR phosphorylation, while knockdown of EGFR blocked SFK phosphorylation and PKC{alpha} activation following Pb. Together, these results indicate that immediate activation of EGFR in response to Pb is obligatory for activation of SFK and PKC{alpha} and subsequent the Ras-Raf-1-MKK1/2-ERK1/2 signaling cascade.

  10. Discovery of a novel class of targeted kinase inhibitors that blocks protein kinase C signaling and ameliorates retinal vascular leakage in a diabetic rat model.

    PubMed

    Grant, Stephan; Tran, Phong; Zhang, Qin; Zou, Aihua; Dinh, Dac; Jensen, Jordan; Zhou, Sue; Kang, Xiaolin; Zachwieja, Joseph; Lippincott, John; Liu, Kevin; Johnson, Sarah Ludlum; Scales, Stephanie; Yin, Chunfeng; Nukui, Seiji; Stoner, Chad; Prasanna, Ganesh; Lafontaine, Jennifer; Wells, Peter; Li, Hui

    2010-02-10

    Protein kinase C (PKC) family members such as PKCbetaII may become activated in the hyperglycemic state associated with diabetes. Preclinical and clinical data implicate aberrant PKC activity in the development of diabetic microvasculature abnormalities. Based on this potential etiological role for PKC in diabetic complications, several therapeutic PKC inhibitors have been investigated in clinical trials for the treatment of diabetic patients. In this report, we present the discovery and preclinical evaluation of a novel class of 3-amino-pyrrolo[3,4-c]pyrazole derivatives as inhibitors of PKC that are structurally distinct from the prototypical indolocarbazole and bisindolylmaleimide PKC inhibitors. From this pyrrolo-pyrazole series, several compounds were identified from biochemical assays as potent, ATP-competitive inhibitors of PKC activity with high specificity for PKC over other protein kinases. These compounds were also found to block PKC signaling activity in multiple cellular functional assays. PF-04577806, a representative from this series, inhibited PKC activity in retinal lysates from diabetic rats stimulated with phorbol myristate acetate. When orally administered, PF-04577806 showed good exposure in the retina of diabetic Long-Evans rats and ameliorated retinal vascular leakage in a streptozotocin-induced diabetic rat model. These novel PKC inhibitors represent a promising new class of targeted protein kinase inhibitors with potential as therapeutic agents for the treatment of patients with diabetic microvascular complications.

  11. Learning-induced reduction in post-burst after-hyperpolarization (AHP) is mediated by activation of PKC.

    PubMed

    Seroussi, Yaron; Brosh, Inbar; Barkai, Edi

    2002-09-01

    We studied the role of protein kinase C (PKC) and protein kinase A (PKA) in mediating learning-related long lasting reduction of the post-burst after-hyperpolarization (AHP) in cortical pyramidal neurons. We have shown previously that pyramidal neurons in the rat piriform (olfactory) cortex from trained (TR) rats have reduced post-burst AHP for 3 days after odour-discrimination learning, and that this reduction is due to decreased conductance of calcium-dependent potassium current. In the present study, we examined whether this long-lasting reduction in AHP is mediated by second messenger systems. The broad-spectrum kinase inhibitor, H7, increased the AHP in neurons from TR rats, but not in neurons from pseudo-trained (pseudo-TR) and naive rats. Consequently, the difference in AHP amplitude between neurons from TR and control animals was diminished. This effect was also obtained by application of the specific PKC inhibitor, GF-109203x. The PKC activator, 1-Oleoyl-2-acetyl-sn-glycerol (OAG), significantly reduced the AHP in neurons from naive and pseudo-TR rats, but not in neurons from TR rats, so that the difference between the groups was abolished. The PKA-specific inhibitor, H-89, increased the AHP in neurons from all groups to a similar extent, and the difference in AHP amplitude between neurons from TR rats and neurons from controls was maintained. We suggest that while the post-burst AHP in piriform cortex pyramidal neurons is modulated by both PKC and PKA, a PKC-dependent process maintains the learning-related reduction of the AHP in these cells.

  12. Effects of PKC412, nilotinib and imatinib against GIST-associated PDGFRA mutants with differential imatinib sensitivity

    PubMed Central

    Weisberg, Ellen; Wright, Renee D.; Jiang, Jingrui; Ray, Arghya; Moreno, Daisy; Manley, Paul W.; Fabbro, Doriano; Hall-Meyers, Elizabeth; Catley, Laurie; Podar, Klaus; Kung, Andrew L.; Griffin, James D.

    2007-01-01

    Background & Aims Activating mutations in platelet-derived growth factor receptor alpha (PDGFRA) have been reported in a subset of gastrointestinal stromal tumor (GIST) patients who do not express mutant stem cell factor receptor, c-KIT. The responsiveness of mutant PDGFRA-positive GIST to imatinib depends on the location of the PDGFRA mutation; for example, the V561D juxtamembrane domain mutation is more sensitive to imatinib than the D842V kinase domain mutation. In this study, we compare the effects of three tyrosine kinase inhibitors, PKC412 and nilotinib, and imatinib, on two GIST-related PDGFRA mutants, V561D and D842V, which possess differential sensitivity to imatinib. Methods The effects of PKC412, nilotinib, and imatinib, alone and in combination, were evaluated via in vitro proliferation studies performed with V561D- or D842V-PDGFRA mutants. The effects of nilotinib and PKC412, alone and combined, were investigated in vivo. Results PKC412 potently inhibited the V561D-PDGFRA mutant in vitro and the D842V-PDGFRA mutant in vitro and in vivo. Both imatinib and nilotinib displayed potent activity in vitro against the V561D-PDGFRA mutant, but were significantly less efficacious against D842V-PDGFRA. However, when combined with either imatinib or PKC412, nilotinib showed no evidence for antagonism and acted in a cooperative fashion against D842V-PDGFRA. Conclusions Our findings support the clinical testing of PKC412 for treatment of mutant PDGFRA-GIST. The data also support the use of nilotinib as a treatment option for V561D-PDGFRA-associated GIST, although the reduced sensitivity of D842V-PDGFRA probably limits the potential of nilotinib monotherapy for D842V-PDGFRA-associated GIST. PMID:17087936

  13. PKC-DEPENDENT REGULATION OF Kv7.5 CHANNELS BY THE BRONCHOCONSTRICTOR HISTAMINE IN HUMAN AIRWAY SMOOTH MUSCLE CELLS.

    PubMed

    Haick, Jennifer M; Brueggemann, Lioubov I; Cribbs, Leanne L; Denning, Mitchell F; Schwartz, Jeffrey; Byron, Kenneth L

    2017-03-10

    Kv7 potassium channels have recently been found to be expressed and functionally important for relaxation of airway smooth muscle. Previous research suggests that native Kv7 currents are inhibited following treatment of freshly isolated airway smooth muscle cells with bronchoconstrictor agonists, and in intact airways inhibition of Kv7 channels is sufficient to induce bronchiolar constriction. However, the mechanism by which Kv7 currents are inhibited by bronchoconstrictor agonists has yet to be elucidated. In the present study, native Kv7 currents in cultured human trachealis smooth muscle cells (HTSMCs) were observed to be inhibited upon treatment with histamine; inhibition of Kv7 currents was associated with membrane depolarization and an increase in cytosolic Ca2+ ([Ca2+]cyt). The latter response was inhibited by verapamil, a blocker of L-type voltage sensitive Ca2+ channels (VSCCs). Protein kinase C (PKC) has been implicated as a mediator of bronchoconstrictor actions, though the targets of PKC are not clearly established. We found that histamine treatment significantly and dose-dependently suppressed currents through overexpressed wild-type human Kv7.5 (hKv7.5) channels in cultured HTSMCs, and this effect was inhibited by the PKC inhibitor Ro-31-8220 (3 µM). The PKC-dependent suppression of hKv7.5 currents corresponded with a PKC-dependent increase in hKv7.5 channel phosphorylation. Knocking down or inhibiting PKCα, or mutating hKv7.5 serine 441 to alanine, abolished the inhibitory effects of histamine on hKv7.5 currents. These findings provide the first evidence linking PKC activation to suppression of Kv7 currents, membrane depolarization, and Ca2+ influx via L-type VSCCs as a mechanism for histamine-induced bronchoconstriction.

  14. PKC-mediated potentiation of morphine analgesia by St. John's Wort in rodents and humans.

    PubMed

    Galeotti, Nicoletta; Farzad, Mersedeh; Bianchi, Enrica; Ghelardini, Carla

    2014-01-01

    Our purpose was to combine the use of morphine with clinically available inhibitors of protein kinase C (PKC), finally potentiating morphine analgesia in humans. Thermal tests were performed in rodents and humans previously administered with acute or chronic morphine combined or not with increasing doses of the PKC-blocker St. John's Wort (SJW) or its main component hypericin. Phosphorylation of the γ subunit of PKC enzyme was assayed by western blotting in the periaqueductal grey matter (PAG) from rodents co-administered with morphine and hypericin and was prevented in rodent PAG by SJW or hypericin co-administration with morphine, inducing a potentiation of morphine analgesia in thermal pain. The score of pain assessment in healthy volunteers were decreased by 40% when morphine was co-administered with SJW at a dose largely below those used to obtain an antidepressant or analgesic effect in both rodents and humans. The SJW/hypericin potentiating effect lasted in time and preserved morphine analgesia in tolerant mice. Our findings indicate that, in clinical practice, SJW could reduce the dose of morphine obtaining the same analgesic effect. Therefore, SJW and one of its main components, hypericin, appear ideal to potentiate morphine-induced analgesia.

  15. Spike timing-dependent long-term potentiation in ventral tegmental area dopamine cells requires PKC.

    PubMed

    Luu, Percy; Malenka, Robert C

    2008-07-01

    Long-term potentiation (LTP) of excitatory synapses on ventral tegmental area (VTA) dopamine (DA) cells is thought to play an important role in mediating some of the behavioral effects of drugs of abuse yet little is known about its underlying mechanisms. We find that spike timing-dependent LTP (STD LTP) in VTA DA cells is absent in slices prepared from mice previously administered cocaine, suggesting that cocaine-induced LTP and STD LTP share underlying mechanisms. This form of STD LTP is dependent on NMDA receptor (NMDAR) activation and a rise in postsynaptic calcium but surprisingly was not affected by an inhibitor of calcium/calmodulin-dependent protein kinase II (CaMKII). It was blocked by antagonists of conventional isoforms of PKC, whereas activation of protein kinase C (PKC) using a phorbol ester enhanced synaptic strength. These results suggest that NMDAR-mediated activation of PKC, but not CaMKII, is a critical trigger for LTP in VTA DA cells.

  16. GLP-1 stimulates insulin secretion by PKC-dependent TRPM4 and TRPM5 activation

    PubMed Central

    Shigeto, Makoto; Ramracheya, Reshma; Tarasov, Andrei I.; Cha, Chae Young; Chibalina, Margarita V.; Hastoy, Benoit; Philippaert, Koenraad; Reinbothe, Thomas; Rorsman, Nils; Salehi, Albert; Sones, William R.; Vergari, Elisa; Weston, Cathryn; Gorelik, Julia; Katsura, Masashi; Nikolaev, Viacheslav O.; Vennekens, Rudi; Zaccolo, Manuela; Galione, Antony; Johnson, Paul R.V.; Kaku, Kohei; Ladds, Graham; Rorsman, Patrik

    2015-01-01

    Strategies aimed at mimicking or enhancing the action of the incretin hormone glucagon-like peptide 1 (GLP-1) therapeutically improve glucose-stimulated insulin secretion (GSIS); however, it is not clear whether GLP-1 directly drives insulin secretion in pancreatic islets. Here, we examined the mechanisms by which GLP-1 stimulates insulin secretion in mouse and human islets. We found that GLP-1 enhances GSIS at a half-maximal effective concentration of 0.4 pM. Moreover, we determined that GLP-1 activates PLC, which increases submembrane diacylglycerol and thereby activates PKC, resulting in membrane depolarization and increased action potential firing and subsequent stimulation of insulin secretion. The depolarizing effect of GLP-1 on electrical activity was mimicked by the PKC activator PMA, occurred without activation of PKA, and persisted in the presence of PKA inhibitors, the KATP channel blocker tolbutamide, and the L-type Ca2+ channel blocker isradipine; however, depolarization was abolished by lowering extracellular Na+. The PKC-dependent effect of GLP-1 on membrane potential and electrical activity was mediated by activation of Na+-permeable TRPM4 and TRPM5 channels by mobilization of intracellular Ca2+ from thapsigargin-sensitive Ca2+ stores. Concordantly, GLP-1 effects were negligible in Trpm4 or Trpm5 KO islets. These data provide important insight into the therapeutic action of GLP-1 and suggest that circulating levels of this hormone directly stimulate insulin secretion by β cells. PMID:26571400

  17. Induction of ANGPTL4 expression in human airway smooth muscle cells by PMA through activation of PKC and MAPK pathways.

    PubMed

    Stapleton, Cliona M; Joo, Joung Hyuck; Kim, Yong-Sik; Liao, Grace; Panettieri, Reynold A; Jetten, Anton M

    2010-02-15

    In this study, we demonstrate that protein kinase C (PKC) activators, including phorbol-12-myristate-13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol (DOG), and platelet-derived growth factor alpha are potent inducers of angiopoietin-like protein 4 (ANGPTL4) expression in several normal lung cell types and carcinoma cell lines. In human airway smooth muscle (HASM) cells induction of ANGPTL4 expression is observed as early as 2 h after the addition of PMA. PMA also increases the level of ANGPTL4 protein released in the medium. PKC inhibitors Ro31-8820 and Gö6983 greatly inhibit the induction of ANGPTL4 mRNA by PMA suggesting that this up-regulation involves activation of PKC. Knockdown of several PKCs by corresponding siRNAs suggest a role for PKCalpha. PMA does not activate MAPK p38 and p38 inhibitors have little effect on the induction of ANGPTL4 indicating that p38 is not involved in the regulation of ANGPTL4 by PMA. In contrast, treatment of HASM by PMA induces phosphorylation and activation of Ra, MEK1/2, ERK1/2, JNK, Elk-1, and c-Jun. The Ras inhibitor manumycin A, the MEK1/2 inhibitor U0126, and the JNK inhibitor SP600125, greatly reduce the increase in ANGPTL4 expression by PMA. Knockdown of MEK1/2 and JNK1/2 expression by corresponding siRNAs inhibits the induction of ANGPTL4. Our observations suggest that the induction of ANGPTL4 by PMA in HASM involves the activation of PKC, ERK, and JNK pathways. This induction may play a role in tissue remodeling during lung injury and be implicated in several lung pathologies.

  18. Activation of amygdaloid PKC pathway is necessary for conditioned cues-provoked cocaine memory performance.

    PubMed

    Lai, Yu-Ting; Fan, Hsin-Yi; Cherng, Chianfang G; Chiang, Chih-Yuan; Kao, Gour-Shenq; Yu, Lung

    2008-07-01

    Drug-associated cues are critical in reinstating the drug taking behavior even during prolonged abstinence and thus are thought to be a key factor to induce drug craving and to cause relapse. Amygdaloid complex has been known for its physiological function in mediating emotional experience storage and emotional cues-regulated memory retrieval. This study was undertaken to examine the role of basolateral nuclei of amygdala and the intracellular signaling molecule in drug cues-elicited cocaine memory retrieval. Systemic anisomycin treatment prior to the retrieval test abolished the cues-provoked cocaine conditioned place preference (CPP) memory. Likewise, a similar blockade of cues-provoked cocaine CPP performance was achieved by infusion of anisomycin and cycloheximide into the basolateral nuclei of amygdala before the test. Intra-amygdaloid infusion of H89, a protein kinase A inhibitor, or U0126, a MEK inhibitor, did not affect retrieval of the cues-elicited cocaine CPP memory. In contrast, intra-amygdaloid infusion of NPC 15437, a PKC inhibitor, abolished the cues-elicited cocaine CPP expression, while left the memory per se intact. Intra-amygdaloid infusion of NPC 15437 did not seem to affect locomotor activity or exert observable aversive effect. Taken together, our results suggest that activation of PKC signaling pathway and probably downstream de novo protein synthesis in the basolateral nuclei of amygdala is required for the cues-elicited cocaine memory performance. However, temporary inhibition of this signaling pathway does not seem to affect cocaine CPP memory per se.

  19. Regulation of human monocarboxylate transporter 4 in skeletal muscle cells: the role of protein kinase C (PKC).

    PubMed

    Narumi, Katsuya; Kobayashi, Masaki; Otake, Sho; Furugen, Ayako; Takahashi, Natsuko; Ogura, Jiro; Itagaki, Shirou; Hirano, Takeshi; Yamaguchi, Hiroaki; Iseki, Ken

    2012-05-30

    In the present study, to clarify the role of protein kinase C (PKC) in the regulation of monocarboxylate transporter 4 (MCT4) expression, we examined the regulation mechanism of MCT4 expression in human rhabdomyosarcoma (RD) cells, an in vitro skeletal muscle model. Exposure of RD cells to PMA, a PKC activator, for 24 h resulted in a two-fold increase in the amount of lactic acid in the growth medium. In parallel to an increase in lactic acid release from RD cells, the level of MCT4 mRNA and protein were also significantly increased in RD cells. A PKC inhibitory study indicated that PMA-induced stimulation of MCT4 expression can be mediated through a novel PKC isoform, especially PKCδ. Moreover, rottlerin, a selective PKCδ inhibitor, decreased PMA-induced MCT4 promoter activity. Deletion and mutational analysis suggested that the potential hypoxia-response elements (HREs) played a major role in the observed modulation of MCT4 expression by PMA. Furthermore, we found that small interfering RNA (siRNA)-mediated knockdown of hypoxia-inducible factor 1α (HIF-1α) significantly inhibited PMA-induced MCT4 promoter activity. Our results show that the effects of PMA on MCT4 expression are mediated through an indirect pathway partially involving PKCδ and HIF-1α transcription factor.

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

    PubMed

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

    2016-04-01

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

  1. Heat induces interleukin-6 in skeletal muscle cells via TRPV1/PKC/CREB pathways.

    PubMed

    Obi, Syotaro; Nakajima, Toshiaki; Hasegawa, Takaaki; Kikuchi, Hironobu; Oguri, Gaku; Takahashi, Masao; Nakamura, Fumitaka; Yamasoba, Tatsuya; Sakuma, Masashi; Toyoda, Shigeru; Tei, Chuwa; Inoue, Teruo

    2017-03-01

    Interleukin-6 (IL-6) is released from skeletal muscle cells and induced by exercise, heat, catecholamine, glucose, lipopolysaccharide, reactive oxygen species, and inflammation. However, the mechanism that induces release of IL-6 from skeletal muscle cells remains unknown. Thermosensitive transient receptor potential (TRP) proteins such as TRPV1-4 play vital roles in cellular functions. In this study we hypothesized that TRPV1 senses heat, transmits a signal into the nucleus, and produces IL-6. The purpose of the present study is to investigate the underlying mechanisms whereby skeletal muscle cells sense and respond to heat. When mouse myoblast cells were exposed to 37-42°C for 2 h, mRNA expression of IL-6 increased in a temperature-dependent manner. Heat also increased IL-6 secretion in myoblast cells. A fura 2 fluorescence dual-wavelength excitation method showed that heat increased intracellular calcium flux in a temperature-dependent manner. Intracellular calcium flux and IL-6 mRNA expression were increased by the TRPV1 agonists capsaicin and N-arachidonoyldopamine and decreased by the TRPV1 antagonists AMG9810 and SB366791 and siRNA-mediated knockdown of TRPV1. TRPV2, 3, and 4 agonists did not change intracellular calcium flux. Western blotting with inhibitors demonstrated that heat increased phosphorylation levels of TRPV1, followed by PKC and cAMP response element-binding protein (CREB). PKC inhibitors, Gö6983 and staurosporine, CREB inhibitors, curcumin and naphthol AS-E, and knockdown of CREB suppressed the heat-induced increases in IL-6. These results indicate that heat increases IL-6 in skeletal muscle cells through the TRPV1, PKC, and CREB signal transduction pathway.NEW & NOTEWORTHY Heat increases the release of interleukin-6 (IL-6) from skeletal muscle cells. IL-6 has been shown to serve immune responses and metabolic functions in muscle. It can be anti-inflammatory as well as proinflammatory. However, the mechanism that induces release of IL-6

  2. Dopaminergic neurotoxicant 6-OHDA induces oxidative damage through proteolytic activation of PKC{delta} in cell culture and animal models of Parkinson's disease

    SciTech Connect

    Latchoumycandane, Calivarathan; Anantharam, Vellareddy; Jin, Huajun; Kanthasamy, Anumantha; Kanthasamy, Arthi

    2011-11-15

    The neurotoxicant 6-hydroxydopamine (6-OHDA) is used to investigate the cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in Parkinson's disease (PD). Oxidative stress and caspase activation contribute to the 6-OHDA-induced apoptotic cell death of dopaminergic neurons. In the present study, we sought to systematically characterize the key downstream signaling molecule involved in 6-OHDA-induced dopaminergic degeneration in cell culture and animal models of PD. Treatment of mesencephalic dopaminergic neuronal N27 cells with 6-OHDA (100 {mu}M) for 24 h significantly reduced mitochondrial activity and increased cytosolic cytochrome c, followed by sequential activation of caspase-9 and caspase-3. Co-treatment with the free radical scavenger MnTBAP (10 {mu}M) significantly attenuated 6-OHDA-induced caspase activities. Interestingly, 6-OHDA induced proteolytic cleavage and activation of protein kinase C delta (PKC{delta}) was completely suppressed by treatment with a caspase-3-specific inhibitor, Z-DEVD-FMK (50 {mu}M). Furthermore, expression of caspase-3 cleavage site-resistant mutant PKC{delta}{sup D327A} and kinase dead PKC{delta}{sup K376R} or siRNA-mediated knockdown of PKC{delta} protected against 6-OHDA-induced neuronal cell death, suggesting that caspase-3-dependent PKC{delta} promotes oxidative stress-induced dopaminergic degeneration. Suppression of PKC{delta} expression by siRNA also effectively protected N27 cells from 6-OHDA-induced apoptotic cell death. PKC{delta} cleavage was also observed in the substantia nigra of 6-OHDA-injected C57 black mice but not in control animals. Viral-mediated delivery of PKC{delta}{sup D327A} protein protected against 6-OHDA-induced PKC{delta} activation in mouse substantia nigra. Collectively, these results strongly suggest that proteolytic activation of PKC{delta} is a key downstream event in dopaminergic degeneration, and these results may have important translational value for

  3. Dystrophin/α1-syntrophin scaffold regulated PLC/PKC-dependent store-operated calcium entry in myotubes.

    PubMed

    Sabourin, Jessica; Harisseh, Rania; Harnois, Thomas; Magaud, Christophe; Bourmeyster, Nicolas; Déliot, Nadine; Constantin, Bruno

    2012-12-01

    In skeletal muscles from patient suffering of Duchenne Muscular Dystrophy and from mdx mice, the absence of the cytoskeleton protein dystrophin has been shown to be essential for maintaining a normal calcium influx. We showed that a TRPC store-dependent cation influx is increased by loss of dystrophin or a scaffolding protein α1-syntrophin, however the mechanisms of this calcium mishandling are incompletely understood. First of all, we confirmed that TRPC1 but also STIM1 and Orai1 are supporting the store-operated cation entry which is enhanced in dystrophin-deficient myotubes. Next, we demonstrated that inhibition of PLC or PKC in dystrophin-deficient myotubes restores elevated cation entry to normal levels similarly to enforced minidystrophin expression. In addition, silencing α1-syntrophin also increased cation influx in a PLC/PKC dependent pathway. We also showed that α1-syntrophin and PLCβ are part of a same protein complex reinforcing the idea of their inter-relation in calcium influx regulation. This elevated cation entry was decreased to normal levels by chelating intracellular free calcium with BAPTA-AM. Double treatments with BAPTA-AM and PLC or PKC inhibitors suggested that the elevation of cation influx by PLC/PKC pathway is dependent on cytosolic calcium. All these results demonstrate an involvement in dystrophin-deficient myotubes of a specific calcium/PKC/PLC pathway in elevation of store-operated cation influx supported by the STIM1/Orai1/TRPC1 proteins, which is normally regulated by the α1-syntrophin/dystrophin scaffold.

  4. Extracellular nucleotides inhibit oxalate transport by human intestinal Caco-2-BBe cells through PKC-δ activation.

    PubMed

    Amin, Ruhul; Sharma, Sapna; Ratakonda, Sireesha; Hassan, Hatim A

    2013-07-01

    Nephrolithiasis remains a major health problem in Western countries. Seventy to 80% of kidney stones are composed of calcium oxalate, and small changes in urinary oxalate affect risk of kidney stone formation. Intestinal oxalate secretion mediated by the anion exchanger SLC26A6 plays an essential role in preventing hyperoxaluria and calcium oxalate nephrolithiasis, indicating that understanding the mechanisms regulating intestinal oxalate transport is critical for management of hyperoxaluria. Purinergic signaling modulates several intestinal processes through pathways including PKC activation, which we previously found to inhibit Slc26a6 activity in mouse duodenal tissue. We therefore examined whether purinergic stimulation with ATP and UTP affects oxalate transport by human intestinal Caco-2-BBe (C2) cells. We measured [¹⁴C]oxalate uptake in the presence of an outward Cl⁻ gradient as an assay of Cl⁻/oxalate exchange activity, ≥50% of which is mediated by SLC26A6. We found that ATP and UTP significantly inhibited oxalate transport by C2 cells, an effect blocked by the PKC inhibitor Gö-6983. Utilizing pharmacological agonists and antagonists, as well as PKC-δ knockdown studies, we observed that ATP inhibits oxalate transport through the P2Y₂ receptor, PLC, and PKC-δ. Biotinylation studies showed that ATP inhibits oxalate transport by lowering SLC26A6 surface expression. These findings are of potential relevance to pathophysiology of inflammatory bowel disease-associated hyperoxaluria, where supraphysiological levels of ATP/UTP are expected and overexpression of the P2Y₂ receptor has been reported. We conclude that ATP and UTP inhibit oxalate transport by lowering SLC26A6 surface expression in C2 cells through signaling pathways including the P2Y₂ purinergic receptor, PLC, and PKC-δ.

  5. Effect of PKC-β Signaling Pathway on Expression of MCP-1 and VCAM-1 in Different Cell Models in Response to Advanced Glycation End Products (AGEs).

    PubMed

    Rempel, Lisienny C T; Finco, Alessandra B; Maciel, Rayana A P; Bosquetti, Bruna; Alvarenga, Larissa M; Souza, Wesley M; Pecoits-Filho, Roberto; Stinghen, Andréa E M

    2015-05-14

    Advanced glycation end products (AGEs) are compounds classified as uremic toxins in patients with chronic kidney disease that have several pro-inflammatory effects and are implicated in the development of cardiovascular diseases. To explore the mechanisms of AGEs-endothelium interactions through the receptor for AGEs (RAGE) in the PKC-β pathway, we evaluated the production of MCP-1 and VCAM-1 in human endothelial cells (HUVECs), monocytes, and a coculture of both. AGEs were prepared by albumin glycation and characterized by absorbance and electrophoresis. The effect of AGEs on cell viability was assessed with an MTT assay. The cells were also treated with AGEs with and without a PKCinhibitor. MCP-1 and VCAM-1 in the cell supernatants were estimated by ELISA, and RAGE was evaluated by immunocytochemistry. AGEs exposure did not affect cell viability, but AGEs induced RAGE, MCP-1, and VCAM-1 expression in HUVECs. When HUVECs or monocytes were incubated with AGEs and a PKCinhibitor, MCP-1 and VCAM-1 expression significantly decreased. However, in the coculture, exposure to AGEs and a PKCinhibitor produced no significant effect. This study demonstrates, in vitro, the regulatory mechanisms involved in MCP-1 production in three cellular models and VCAM-1 production in HUVECs, and thus mimics the endothelial dysfunction caused by AGEs in early atherosclerosis. Such mechanisms could serve as therapeutic targets to reduce the harmful effects of AGEs in patients with chronic kidney disease.

  6. p38MAPK, Rho/ROCK and PKC pathways are involved in influenza-induced cytoskeletal rearrangement and hyperpermeability in PMVEC via phosphorylating ERM.

    PubMed

    Zhang, Chenyue; Wu, Ying; Xuan, Zinan; Zhang, Shujing; Wang, Xudan; Hao, Yu; Wu, Jun; Zhang, Shu

    2014-11-04

    Severe influenza infections are featured by acute lung injury, a syndrome of pulmonary microvascular leak. A growing number of evidences have shown that the pulmonary microvascular endothelial cells (PMVEC) are critical target of influenza virus, promoting microvascular leak. It is reported that there are multiple mechanisms by which influenza virus could elicit increased pulmonary endothelial permeability, in both direct and indirect manners. Ezrin/radixin/moesin family proteins, the linkers between plasma membrane and actin cytoskeleton, have been reported to be involved in cell adhesion, motility and may modulate endothelial permeability. Studies have also shown that ERM is phosphorylated in response to various stimuli via p38MAPK, Rho/ROCK or PKC pathways. However, it is unclear that whether influenza infection could induce ERM phosphorylation and its relocalization. In the present study, we have found that there are cytoskeletal reorganization and permeability increases in the course of influenza virus infection, accompanied by upregulated levels of p-ERM. p-ERM's aggregation along the periphery of PMVEC upon influenza virus infection was detected via confocal microscopy. Furthermore, we sought to determine the role of p38MAPK, Rho/ROCK and PKC pathways in ERM phosphorylation as well as their involvement in influenza virus-induced endothelial malfunction. The activation of p38MAPK, Rho/ROCK and PKC pathways upon influenza virus stimulation were observed, as evidenced by the evaluation of phosphorylated p38 (p-p38), phosphorylated MKK (p-MKK) in p38MAPK pathway, ROCK1 in Rho/ROCK pathway and phosphorylated PKC (p-PKC) in PKC pathway. We also showed that virus-induced ERM phosphorylation was reduced by using p38MAPK inhibitor, SB203580 (20 μM), Rho/ROCK inhibitor, Y27632 (20 μM), PKC inhibitor, LY317615 (10 μM). Additionally, influenza virus-induced F-actin reorganization and hyperpermeability were attenuated by pretreatment with SB203580, Y27632 and LY317615

  7. Post-conditioning protects cardiomyocytes from apoptosis via PKC(epsilon)-interacting with calcium-sensing receptors to inhibit endo(sarco)plasmic reticulum-mitochondria crosstalk.

    PubMed

    Dong, Shiyun; Teng, Zongyan; Lu, Fang-Hao; Zhao, Ya-Jun; Li, Hulun; Ren, Huan; Chen, He; Pan, Zhen-Wei; Lv, Yan-Jie; Yang, Bao-Feng; Tian, Ye; Xu, Chang-Qing; Zhang, Wei-Hua

    2010-08-01

    The intracellular Ca(2+) concentration ([Ca(2+)](i)) is increased during cardiac ischemia/reperfusion injury (IRI), leading to endo(sarco)plasmic reticulum (ER) stress. Persistent ER stress, such as with the accumulation of [Ca(2+)](i), results in apoptosis. Ischemic post-conditioning (PC) can protect cardiomyocytes from IRI by reducing the [Ca(2+)](i) via protein kinase C (PKC). The calcium-sensing receptor (CaR), a G protein-coupled receptor, causes the production of inositol phosphate (IP(3)) to increase the release of intracellular Ca(2+) from the ER. This process can be negatively regulated by PKC through the phosphorylation of Thr-888 of the CaR. This study tested the hypothesis that PC prevents cardiomyocyte apoptosis by reducing the [Ca(2+)](i) through an interaction of PKC with CaR to alleviate [Ca(2+)](ER) depletion and [Ca(2+)](m) elevation by the ER-mitochondrial associated membrane (MAM). Cardiomyocytes were post-conditioned after 3 h of ischemia by three cycles of 5 min of reperfusion and 5 min of re-ischemia before 6 h of reperfusion. During PC, PKC(epsilon) translocated to the cell membrane and interacted with CaR. While PC led to a significant decrease in [Ca(2+)](i), the [Ca(2+)](ER) was not reduced and [Ca(2+)](m) was not increased in the PC and GdCl(3)-PC groups. Furthermore, there was no evident psi(m) collapse during PC compared with ischemia/reperfusion (I/R) or PKC inhibitor groups, as evaluated by laser confocal scanning microscopy. The apoptotic rates detected by TUNEL and Hoechst33342 were lower in PC and GdCl(3)-PC groups than those in I/R and PKC inhibitor groups. Apoptotic proteins, including m-calpain, BAP31, and caspase-12, were significantly increased in the I/R and PKC inhibitor groups. These results suggested that PKC(epsilon) interacting with CaR protected post-conditioned cardiomyocytes from programmed cell death by inhibiting disruption of the mitochondria by the ER as well as preventing calcium-induced signaling of the

  8. Expression and proliferation profiles of PKC, JNK and p38MAPK in physiologically stretched human bladder smooth muscle cells

    SciTech Connect

    Wazir, Romel; Luo, De-Yi; Dai, Yi; Yue, Xuan; Tian, Ye; Wang, Kun-Jie

    2013-08-30

    Highlights: •Stretch induces proliferation in human bladder smooth muscle cells (HBSMC). •5% Equibiaxial elongation produces maximum proliferation. •Physiologic stretch decreases apoptotic cell death. •PKC is involved in functional modulation of bladder. •JNK and p38 are not involved in proliferating HBSMC. -- Abstract: Objective: To determine protein kinase C (PKC), c-Jun NH2-Terminal Kinase (JNK) and P38 mitogen-activated protein kinases (p38MAPK) expression levels and effects of their respective inhibitors on proliferation of human bladder smooth muscle cells (HBSMCs) when physiologically stretched in vitro. Materials and methods: HBSMCs were grown on silicone membrane and stretch was applied under varying conditions; (equibiaxial elongation: 2.5%, 5%, 10%, 15%, 20%, 25%), (frequency: 0.05, 0.1, 0.2, 0.5, 1 Hz). Optimal physiological stretch was established by assessing proliferation with 5-Bromo-2-deoxyuridine (BrdU) assay and flow cytometry. PKC, JNK and p38 expression levels were analyzed by Western blot. Specificity was maintained by employing specific inhibitors; (GF109203X for PKC, SP600125 for JNK and SB203580 for p38MAPK), in some experiments. Results: Optimum proliferation was observed at 5% equibiaxial stretch (BrdU: 0.837 ± 0.026 (control) to 1.462 ± 0.023)%, (P < 0.05) and apoptotic cell death rate decreased from 16.4 ± 0.21% (control) to 4.5 ± 0.13% (P < 0.05) applied at 0.1 Hz. Expression of PKC was upregulated with slight increase in JNK and no change in p38MAPK after application of stretch. Inhibition had effects on proliferation (1.075 ± 0.024, P < 0.05 GF109203X); (1.418 ± 0.021, P > 0.05 SP600125) and (1.461 ± 0.01, P > 0.05 SB203580). These findings show that mechanical stretch can promote magnitude-dependent proliferative modulation through PKC and possibly JNK but not via p38MAPK in hBSMCs.

  9. AXL mediates resistance to PI3Kα inhibition by activating the EGFR/PKC/mTOR axis in head and neck and esophageal squamous cell carcinomas

    PubMed Central

    Elkabets, Moshe; Pazarentzos, Evangelos; Juric, Dejan; Sheng, Qing; Pelossof, Raphael A.; Brook, Samuel; Benzaken, Ana Oaknin; Rodon, Jordi; Morse, Natasha; Yan, Jenny Jiacheng; Liu, Manway; Das, Rita; Chen, Yan; Tam, Angela; Wang, Huiqin; Liang, Jinsheng; Gurski, Joseph M.; Kerr, Darcy A.; Rosell, Rafael; Teixidó, Cristina; Huang, Alan; Ghossein, Ronald A.; Rosen, Neal; Bivona, Trever G.; Scaltriti, Maurizio; Baselga, José

    2015-01-01

    Summary Phosphoinositide-3-kinase (PI3K)-α inhibitors have shown clinical activity in squamous carcinoma (SCC) of head and neck (H&N) bearing PIK3CA mutations or amplification. Studying models of therapeutic resistance we have observed that SCCs cells that become refractory to PI3Kα inhibition maintain PI3K-independent activation of the mammalian target of rapamycin (mTOR). This persistent mTOR activation is mediated by the tyrosine kinase receptor AXL. AXL is overexpressed in resistant tumors from both laboratory models and patients treated with the PI3Kα inhibitor BYL719. AXL dimerizes with and phosphorylates epidermal growth factor receptor (EGFR), resulting in activation of phospholipase Cγ (PLCγ)- protein kinase C (PKC), which in turn activates mTOR. Combined treatment with PI3Kα and either EGFR, AXL, or PKC inhibitors reverts this resistance. PMID:25873175

  10. The Xanthine Derivative KMUP-1 Attenuates Serotonin-Induced Vasoconstriction and K+-Channel Inhibitory Activity via the PKC Pathway in Pulmonary Arteries

    PubMed Central

    Dai, Zen-Kong; Liu, Yu-Wei; Hsu, Jong-Hau; Yeh, Jwu-Lai; Chen, Ing-Jun; Wu, Jiunn-Ren; Wu, Bin-Nan

    2015-01-01

    Serotonin (5-hydroxytryptamine, 5-HT) is a potent pulmonary vasoconstrictor that promotes pulmonary artery smooth muscle cell (PASMC) proliferation. 5-HT-induced K+ channel inhibition increases [Ca2+]i in PASMCs, which is a major trigger for pulmonary vasoconstriction and development of pulmonary arterial hypertension (PAH). This study investigated whether KMUP-1 reduces pulmonary vasoconstriction in isolated pulmonary arteries (PAs) and attenuates 5-HT-inhibited K+ channel activities in PASMCs. In endothelium-denuded PA rings, KMUP-1 (1 μM) dose-dependently reduced 5-HT (100 μM) mediated contractile responses. Responses to KMUP-1 were reversed by K+ channel inhibitors (TEA, 10 mM, 4-aminopyridine, 5 mM, and paxilline, 10 μM). In primary PASMCs, KMUP-1 also dose-dependently restored 5-HT-inhibited voltage-gated K+-channel (Kv1.5 and Kv2.1) and large-conductance Ca2+-activated K+-channel (BKCa) proteins, as confirmed by immunofluorescent staining. Furthermore, 5-HT (10 μM)-inhibited Kv1.5 protein was unaffected by the PKA inhibitor KT5720 (1 μM) and the PKC activator PMA (1 μM), but these effects were reversed by KMUP-1 (1 μM), 8-Br-cAMP (100 μM), chelerythrine (1 μM), and KMUP-1 combined with a PKA/PKC activator or inhibitor. Notably, KMUP-1 reversed 5-HT-inhibited Kv1.5 protein and this response was significantly attenuated by co-incubation with the PKC activator PMA, suggesting that 5-HT-mediated PKC signaling can be modulated by KMUP-1. In conclusion, KMUP-1 ameliorates 5-HT-induced vasoconstriction and K+-channel inhibition through the PKC pathway, which could be valuable to prevent the development of PAH. PMID:25999786

  11. Baccharis trimera inhibits reactive oxygen species production through PKC and down-regulation p47 (phox) phosphorylation of NADPH oxidase in SK Hep-1 cells.

    PubMed

    de Araújo, Glaucy Rodrigues; Rabelo, Ana Carolina Silveira; Meira, Janaína Serenato; Rossoni-Júnior, Joamyr Victor; Castro-Borges, William de; Guerra-Sá, Renata; Batista, Maurício Azevedo; Silveira-Lemos, Denise da; Souza, Gustavo Henrique Bianco de; Brandão, Geraldo Célio; Chaves, Míriam Martins; Costa, Daniela Caldeira

    2017-02-01

    Baccharis trimera, popularly known as "carqueja", is a native South-American plant possessing a high concentration of polyphenolic compounds and therefore high antioxidant potential. Despite the antioxidant potential described for B. trimera, there are no reports concerning the signaling pathways involved in this process. So, the aim of the present study was to assess the influence of B. trimera on the modulation of PKC signaling pathway and to characterize the effect of the nicotinamide adenine dinucleotide phosphate oxidase enzyme (NOX) on the generation of reactive oxygen species in SK Hep-1 cells. SK-Hep 1 cells were treated with B. trimera, quercetin, or rutin and then stimulated or not with PMA/ionomycin and labeled with carboxy H2DCFDA for detection of reactive oxygen species by flow cytometer. The PKC expression by Western blot and enzyme activity was performed to evaluate the influence of B. trimera and quercetin on PKC signaling pathway. p47 (phox) and p47 (phox) phosphorylated expression was performed by Western blot to evaluate the influence of B. trimera on p47 (phox) phosphorylation. The results showed that cells stimulated with PMA/ionomycin (activators of PKC) showed significantly increased reactive oxygen species production, and this production returned to baseline levels after treatment with DPI (NOX inhibitor). Both B. trimera and quercetin modulated reactive oxygen species production through the inhibition of PKC protein expression and enzymatic activity, also with inhibition of p47 (phox) phosphorylation. Taken together, these results suggest that B. trimera has a potential mechanism for inhibiting reactive oxygen species production through the PKC signaling pathway and inhibition subunit p47 (phox) phosphorylation of nicotinamide adenine dinucleotide phosphate oxidase.

  12. Catecholamines are produced by ascidian immune cells: The involvement of PKA and PKC in the adrenergic signaling pathway.

    PubMed

    de Abreu Mello, Andressa; Fernandes de Souza, Jéssica; Nunes da Fonseca, Rodrigo; Allodi, Silvana; Monteiro de Barros, Cintia

    2017-03-01

    The stress response is a complex mechanism, which includes changes in the immune system to enable organisms to maintain homeostasis. The neurohormones dopamine, noradrenaline (NA) and adrenalin are responsible for the physiological modulations that occur during acute stress. In the present study, we analyzed the effects of NA on the immune system specific to nitric-oxide (NO) production by subpopulations of immune cells (hemocytes) of the ascidian Phallusia nigra. We also investigated the capability of immune cells to produce catecholamine (CA). Finally, we tested the involvement of protein kinase A (PKA) and C (PKC) in the NA downstream signaling pathway. The results revealed that NA can reduce NO production by P. nigra hemocytes threefold, and that signet-ring cells, univacuolar refractile granulocytes and morula cells are the cell types most involved in this event. A challenge effected with Zymosan A induced CA production, and co-incubation with both inhibitors of the second messengers PKA and PKC revealed the involvement of these molecules in the adrenergic pathway of P. nigra hemocytes. Taken together, these results suggest that NO production can be down-regulated by NA through α- and β-adrenoceptors via the second messengers PKA and PKC.

  13. Nociceptor beta II, delta, and epsilon isoforms of PKC differentially mediate paclitaxel-induced spontaneous and evoked pain.

    PubMed

    He, Ying; Wang, Zaijie Jim

    2015-03-18

    As one of the most effective and frequently used chemotherapeutic agents, paclitaxel produces peripheral neuropathy (paclitaxel-induced peripheral neuropathy or PIPN) that negatively affects chemotherapy and persists after cancer therapy. The mechanisms underlying this dose-limiting side effect remain to be fully elucidated. This study aimed to investigate the role of nociceptor protein kinase C (PKC) isoforms in PIPN. Employing multiple complementary approaches, we have identified a subset of PKC isoforms, namely βII, δ, and ϵ, were activated by paclitaxel in the isolated primary afferent sensory neurons. Persistent activation of PKCβII, PKCδ, and PKCϵ was also observed in the dorsal root ganglion neurons after chronic treatment with paclitaxel in a mouse model of PIPN. Isoform-selective inhibitors of PKCβII, PKCδ, and PKCϵ given intrathecally dose-dependently attenuated paclitaxel-induced mechanical allodynia and heat hyperalgesia. Surprisingly, spinal inhibition of PKCβII and PKCδ, but not PKCϵ, blocked the spontaneous pain induced by paclitaxel. These data suggest that a subset of nociceptor PKC isoforms differentially contribute to spontaneous and evoked pain in PIPN, although it is not clear whether PKCϵ in other regions regulates spontaneous pain in PIPN. The findings can potentially offer new selective targets for pharmacological intervention of PIPN.

  14. A role for amygdaloid PKA and PKC in the acquisition of long-term conditional fear memories in rats.

    PubMed

    Goosens, K A; Holt, W; Maren, S

    2000-09-01

    Although there is great interest in the cellular mechanisms underlying Pavlovian conditioning, few studies have directly examined the contribution of intracellular signaling pathways in the amygdala to the acquisition and expression of conditional fear memories. In the present study, we examined this issue by infusing 1-(5'-isoquinolinesulfonyl)-2-methylpiperazine (H7), a potent inhibitor of both protein kinase C (PKC) and cAMP-dependent protein kinase (PKA), directly into the amygdala prior to fear conditioning or retention testing. We found that infusion of H7 prior to training attenuated long-term conditional fear in a dose-dependent manner (Experiment 1), but short-term fear memories were spared. The contribution of protein kinases to conditional fear was region-specific within the amygdala: infusion of H7 into the basolateral amygdala (BLA) but not the central nucleus of the amygdala (CEA) resulted in attenuated freezing (Experiment 2). Moreover, the deficits in fear conditioning produced by PKA/PKC inhibition were not modality-specific, insofar as intra-BLA H7 reduced both contextual and auditory fear. The effects of H7 on conditional freezing were not attributable to either state-dependency or performance deficits (Experiment 3). Together, these experiments suggest that amygdaloid PKA and PKC play an important role in the acquisition of fear memories.

  15. PKC and AKT Modulate cGMP/PKG Signaling Pathway on Platelet Aggregation in Experimental Sepsis

    PubMed Central

    Lopes-Pires, M. Elisa; Naime, Ana C. Antunes; Almeida Cardelli, Nádia J.; Anjos, Débora J.; Antunes, Edson; Marcondes, Sisi

    2015-01-01

    Sepsis severity has been positively correlated with platelet dysfunction, which may be due to elevations in nitric oxide (NO) and cGMP levels. Protein kinase C, Src kinases, PI3K and AKT modulate platelet activity in physiological conditions, but no studies evaluated the role of these enzymes in platelet aggregation in sepsis. In the present study we tested the hypothesis that in sepsis these enzymes positively modulate upstream the NO-cGMP pathway resulting in platelet inhibition. Rats were injected with lipopolysaccharide (LPS, 1 mg/kg, i.p.) and blood was collected after 6 h. Platelet aggregation was induced by ADP (10 μM). Western blotting assays were carried out to analyze c-Src and AKT activation in platelets. Intraplatelet cGMP levels were determined by enzyme immunoassay kit. Phosphorylation of c-SRC at Tyr416 was the same magnitude in platelets of control and LPS group. Incubation of the non-selective Src inhibitor PP2 (10 μM) had no effect on platelet aggregation of LPS-treated rats. LPS increased intraplatelet cGMP levels by 5-fold compared with control group, which was accompanied by 76% of reduction in ADP-induced platelet aggregation. The guanylyl cyclase inhibitor ODQ (25 μM) and the PKG inhibitor Rp-8-Br-PET-cGMPS (25 μM) fully reversed the inhibitory effect of LPS on platelet aggregation. Likewise, the PKC inhibitor GF109203X (10 μM) reversed the inhibition by LPS of platelet aggregation and decreased cGMP levels in platelets. AKT phosphorylation at Thr308 was significantly higher in platelets of LPS compared with control group, which was not reduced by PI3K inhibition. The AKT inhibitor API-1 (20 μM) significantly increased aggregation and reduced cGMP levels in platelets of LPS group. However, the PI3K inhibitor wortmannin and LY29004 had no effect on platelet aggregation of LPS-treated rats. Therefore, inhibition of ADP-induced platelet aggregation after LPS injection is mediated by cGMP/PKG-dependent mechanisms, and PKC and AKT act

  16. PKC-dependent extracellular signal-regulated kinase 1/2 pathway is involved in the inhibition of Ib on AngiotensinII-induced proliferation of vascular smooth muscle cells

    SciTech Connect

    Wang Yu; Yan Tianhua; Wang Qiujuan Wang Wei; Xu Jinyi; Wu Xiaoming; Ji Hui

    2008-10-10

    AngiotensinII (AngII) induces vascular smooth muscle cell (VSMC) proliferation, which plays an important role in the development and progression of hypertension. AngII-induced cellular events have been implicated, in part, in the activation of protein kinase C (PKC) and extracellular signal-regulated kinases 1/2 (ERK1/2). In the present study, we investigated the effect of Ib, a novel nonpeptide AngII receptor type 1 (AT{sub 1}) antagonist, on the activation of PKC and ERK1/2 in VSMC proliferation induced by AngII. MTT, and [{sup 3}H]thymidine incorporation assay showed that AngII-induced VSMC proliferation was inhibited significantly by Ib. The specific binding of [{sup 125}I]AngII to AT{sub 1} receptors was blocked by Ib in a concentration-dependent manner with IC{sub 50} value of 0.96 nM. PKC activity assay and Western blot analysis demonstrated that Ib significantly inhibited the activation of PKC and phosphorylation of ERK1/2 induced by AngII, respectively. Furthermore, AngII-induced ERK1/2 activation was obviously blocked by GF109203X, a PKC inhibitor. These findings suggest that the suppression of Ib on AngII-induced VSMC proliferation may be attributed to its inhibitory effect on PKC-dependent ERK1/2 pathway.

  17. Locomotion in Lymphocytes is Altered by Differential PKC Isoform Expression

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    1999-01-01

    Lymphocyte locomotion is critical for proper elicitation of the immune response. Locomotion of immune cells via the interstitium is essential for optimal immune function during wound healing, inflammation and infection. There are conditions which alter lymphocyte locomotion and one of them is spaceflight. Lymphocyte locomotion is severely inhibited in true spaceflight (true microgravity) and in rotating wall vessel culture (modeled microgravity). When lymphocytes are activated prior to culture in modeled microgravity, locomotion is not inhibited and the levels are comparable to those of static cultured lymphocytes. When a phorbol ester (PMA) is used in modeled microgravity, lymphocyte locomotion is restored by 87%. This occurs regardless if PMA is added after culture in the rotating wall vessel or during culture. Inhibition of DNA synthesis also does not alter restoration of lymphocyte locomotion by PMA. PMA is a direct activator of (protein kinase C) PKC . When a calcium ionophore, ionomycin is used it does not possess any restorative properties towards locomotion either alone or collectively with PMA. Since PMA brings about restoration without help from calcium ionophores (ionomycin), it is infer-red that calcium independent PKC isoforms are involved. Changes were perceived in the protein levels of PKC 6 where levels of the protein were downregulated at 24,72 and 96 hours in untreated rotated cultures (modeled microgravity) compared to untreated static (1g) cultures. At 48 hours there is an increase in the levels of PKC & in the same experimental set up. Studies on transcriptional and translational patterns of calcium independent isoforms of PKC such as 8 and E are presented in this study.

  18. Protease-activated receptor-2 stimulates intestinal epithelial chloride transport through activation of PLC and selective PKC isoforms.

    PubMed

    van der Merwe, Jacques Q; Moreau, France; MacNaughton, Wallace K

    2009-06-01

    Serine proteases play important physiological roles through their activity at G protein-coupled protease-activated receptors (PARs). We examined the roles that specific phospholipase (PL) C and protein kinase (PK) C (PKC) isoforms play in the regulation of PAR(2)-stimulated chloride secretion in intestinal epithelial cells. Confluent SCBN epithelial monolayers were grown on Snapwell supports and mounted in modified Ussing chambers. Short-circuit current (I(sc)) responses to basolateral application of the selective PAR(2) activating peptide, SLIGRL-NH(2), were monitored as a measure of net electrogenic ion transport caused by PAR(2) activation. SLIGRL-NH(2) induced a transient I(sc) response that was significantly reduced by inhibitors of PLC (U73122), phosphoinositol-PLC (ET-18), phosphatidylcholine-PLC (D609), and phosphatidylinositol 3-kinase (PI3K; LY294002). Immunoblot analysis revealed the phosphorylation of both PLCbeta and PLCgamma following PAR(2) activation. Pretreatment of the cells with inhibitors of PKC (GF 109203X), PKCalpha/betaI (Gö6976), and PKCdelta (rottlerin), but not PKCzeta (selective pseudosubstrate inhibitor), also attenuated this response. Cellular fractionation and immunoblot analysis, as well as confocal immunocytochemistry, revealed increases of PKCbetaI, PKCdelta, and PKCepsilon, but not PKCalpha or PKCzeta, in membrane fractions following PAR(2) activation. Pretreatment of the cells with U73122, ET-18, or D609 inhibited PKC activation. Inhibition of PI3K activity only prevented PKCdelta translocation. Immunoblots revealed that PAR(2) activation induced phosphorylation of both cRaf and ERK1/2 via PKCdelta. Inhibition of PKCbetaI and PI3K had only a partial effect on this response. We conclude that basolateral PAR(2)-induced chloride secretion involves activation of PKCbetaI and PKCdelta via a PLC-dependent mechanism resulting in the stimulation of cRaf and ERK1/2 signaling.

  19. Distinct PKC isoforms mediate the activation of cPLA2 and adenylyl cyclase by phorbol ester in RAW264.7 macrophages

    PubMed Central

    Lin, Wan-W; Chen, Bin C

    1998-01-01

    The modulatory effects of protein kinase C (PKC) on the activation of cytosolic phospholipase A2 (cPLA2) and adenylyl cyclase (AC) have recently been described. Since the signalling cascades associated with these events play critical roles in various functions of macrophages, we set out to investigate the crosstalk between PKC and the cPLA2 and AC pathways in mouse RAW 264.7 macrophages and to determine the involvement of individual PKC isoforms. The cPLA2 and AC pathways were studied by measuring the potentiation by the phorbol ester PMA of ionomycin-induced arachidonic acid (AA) release and prostagladin E1 (PGE1)-stimulated cyclic AMP production, respectively.PMA at 1 μM caused a significant increase in AA release both in the presence (371%) and absence (67%) of ionomycin induction, while exposure of RAW 264.7 cells to PMA increased PGE1 stimulation of cyclic AMP levels by 208%.Treatment of cells with staurosporine and Ro 31-8220 inhibited the PMA-induced potentiation of both AA release and cyclic AMP accumulation, while Go 6976 (an inhibitor of classical PKC isoforms) and LY 379196 (a specific inhibitor of PKCβ) inhibited the AA response but failed to affect the enhancement of the cyclic AMP response by PMA.Long term pretreatment of cells with PMA abolished the subsequent effect of PMA in potentiating AA release, but only inhibited the cyclic AMP response by 42%.Neither PD 98059, an inhibitor of MEK, nor genistein, an inhibitor of tyrosine kinases, had any effect on the ability of PMA to potentiate AA or cyclic AMP production.The potentiation of AA release, but not of cyclic AMP formation, by PMA was sensitive to inhibition by wortmannin. This effect was unrelated to the inhibition of PKC activation as deduced from the translocation of PKC activity to the cell membrane.Western blot analysis revealed the presence of eight PKC isoforms (α, βI, βII, δ, ε, μ λ and ξ) in RAW 264.7 cells and PMA was shown to induce the translocation of the α, βI, βII,

  20. Tissue angiotensin II during progression or ventricular hypertrophy to heart failure in hypertensive rats; differential effects on PKC epsilon and PKC beta.

    PubMed

    Inagaki, Koichi; Iwanaga, Yoshitaka; Sarai, Nobuaki; Onozawa, Yoko; Takenaka, Hiroyuki; Mochly-Rosen, Daria; Kihara, Yasuki

    2002-10-01

    The protein kinase C (PKC) family has been implicated as second messengers in mechanosensitive modulation of cardiac hypertrophy. However, little information is available on the role of expression and activation of specific cardiac PKC isozymes during development of left ventricular hypertrophy (LVH) and failure (LVF). Dahl salt-sensitive rats fed an 8% salt diet developed systemic hypertension and concentric LVH at 11 weeks of age that is followed by left ventricle (LV) dilatation and global hypokinesis at 17 weeks. Among several PKC isozymes expressed in the LV myocardium, only PKC epsilon showed a 94% increase at the LVH stage. At the LVF stage, however, PKC epsilon returned to the control level, whereas PKC beta I and beta II increased by 158% and 155%, respectively. Hearts were studied at each stage using the Langendorff set-up, and a LV balloon was inflated to achieve an equivalent diastolic wall stress. Following mechanical stretch, PKC epsilon was significantly activated in LVH myocardium in which tissue angiotensin II levels were increased by 59%. Pre-treatment with valsartan, an AT(1)-receptor blocker, abolished the stretch-mediated PKC epsilon activation. Mechanical stretch no longer induced PKC epsilon activation in LVF. Chronic administration of valsartan blunted the progression of LVF and inhibited the increase in PKC beta. Mechanosensitive PKC epsilon activation is augmented and therefore may contribute to the development of compensatory hypertrophy. This effect was dependent on activation of tissue angiotensin II. However, this compensatory mechanism becomes inactive in LVF, where PKC beta may participate in the progression to cardiac dysfunction and LV remodeling.

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

    PubMed Central

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

    1996-01-01

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

  2. Stimulation of non-amyloidogenic processing of amyloid-β protein precursor by cryptotanshinone involves activation and translocation of ADAM10 and PKC-α.

    PubMed

    Durairajan, Siva Sundara Kumar; Liu, Liang-Feng; Lu, Jia-Hong; Koo, Irene; Maruyama, Kei; Chung, Sookja K; Huang, Jian-Dong; Li, Min

    2011-01-01

    Cerebral deposition of amyloid-β peptide (Aβ) plaques is now considered the central feature of Alzheimer's disease. Recent studies suggest that cryptotanshinone (CTS) extracted from the root of Salvia miltiorrhiza Bunge could be used for the prevention and treatment of Alzheimer's disease. In this study, we investigated the role of CTS on non-amyloidogenic processing of amyloid-β protein precursor (AβPP) as well as its regulation by protein kinase C (PKC). Treatment with CTS dose-dependently and significantly reduced both intracellular and secreted levels of Aβ40 and Aβ42 in N2a mouse neuroblastoma cells stably expressing human SwedishAβPP (N2a-SwedAβPP). Using N2a-SwedAβPP and human neuroblastoma SHSY5Y cells, it was demonstrated that CTS significantly and dose-dependently increased the production of sAβPPα and C-terminal fragment-α (CTF-α) from AβPP. At the same time, CTS specifically increased the maturation of "a disintegrin and metalloproteinase-10" (ADAM10), an α-secretase candidate. The increase of sAβPPα secretion by CTS was blocked by the hydroxamate-based inhibitors GI254023X and GW280264X, and by the PKCinhibitor GÖ6976, suggesting involvement of the ADAM10 and PKC-α in CTS-induced α-secretase cleavage. In other experiments, CTS induced the phosphorylation of PKC-α indicating that PKC-α is involved in CTS-induced sAβPPα secretion. Furthermore, treatment of neuroblastoma cells with CTS induced the co-translocation of ADAM10 and PKC-α to the cell membrane, the site at which AβPP was cleaved, and this translocation was significantly reduced by GÖ6976. These results suggest that CTS-induced sAβPPα secretion is regulated by a PKC-α and ADAM10 cascade in neuroblastoma cells and may be involved in the lowering of Aβ production.

  3. Different effect of chronic stress on learning and memory in BALB/c and C57BL/6 inbred mice: Involvement of hippocampal NO production and PKC activity.

    PubMed

    Palumbo, María Laura; Zorrilla Zubilete, María Aurelia; Cremaschi, Graciela Alicia; Genaro, Ana María

    2009-07-01

    Nitric oxide (NO) has been involved in many pathophysiological brain processes. Recently, we showed that neuronal nitric oxide synthase (nNOS)-mediated decrease in NO production is involved in memory impairment induced by chronic mild stress (CMS) in BALB/c mice. Two genetically different inbred murine strains, C57BL/6 and BALB/c, show distinct behavioral responses, neurodevelopmental and neurochemical parameters. Here, we perform a comparative study on CMS effects upon learning and memory in both strains, analyzing the role of NO production and its regulation by protein kinase C (PKC). Stressed BALB/c, but not C57Bl/6 mice, showed a poor learning performance in both the open field and passive avoidance inhibitory tasks. Also, CMS induced a diminished NO production by nNOS, associated with an increment in gamma and zeta PKC isoenzymes in BALB/c mice. In C57BL/6 mice, CMS had no effect on NO production, but increased delta and decreased betaI PKC isoforms. In vivo administration of a NOS inhibitor induced behavioral alterations in both strains. These results suggest a differential effect of stress, with BALB/c being more vulnerable to stress than C57BL/6 mice. This effect could be related to a differential regulation of NOS and PKC isoenzymes, pointing to an important role of NO in learning and memory.

  4. Suppression of macrophage-mediated phagocytosis of apoptotic cells by soluble β-glucan due to a failure of PKC-βII translocation.

    PubMed

    Sekiguchi, Suzuno; Tomisawa, Yui; Ohki, Tomomi; Tsuboi, Kumiko; Nagata, Kisaburo; Kobayashi, Yoshiro

    2016-02-01

    If apoptotic cells are not removed efficiently, they may proceed to the stage of secondary necrosis, which would cause inflammation. Therefore, identification of cause(s) and agent(s) for down-modulating phagocytosis of apoptotic cells would help understand the pathologies. In this study we found that macrophage-mediated phagocytosis of apoptotic cells was suppressed by both soluble and particulate β-glucan. This suppression was not observed when secondary necrotic cells were used. The adhesion of apoptotic cells to macrophages was not suppressed by soluble β-glucan, suggesting that soluble β-glucan suppresses phagocytosis at a post-adhesion step. Experiments involving PKC inhibitors suggested that PKC-βII is required for phagocytosis of apoptotic cells but not secondary necrotic ones by macrophages. Translocation of GFP-PKC-βII from the cytoplasm to membranes occurred upon interaction with apoptotic cells but not secondary necrotic ones. Such translocation was inhibited by soluble β-glucan. Overall, this study suggests that suppression of macrophage-mediated phagocytosis of apoptotic cells by soluble β-glucan is due to a failure of PKC-βII translocation.

  5. PKC Epsilon: A Novel Oncogenic Player in Prostate Cancer

    DTIC Science & Technology

    2013-09-01

    levels. (A) RWPE-1 cells were infected with ViraPower lentiviruses containing a human PKCε construct cloned into pLenti6/V-5 DESTRWPE-1. Expression...kinase C epsilon (PKCε), a member of the PKC family of phorbol ester/diacylglycerol receptors, is up-regulated in many human cancers, including prostate...In prostate cancer cells PKCε mediates survival through Bad -dependent and Bad -independent mechanisms and is involved in the transition to androgen

  6. PKC Epsilon: A Novel Oncogenic Player in Prostate Cancer

    DTIC Science & Technology

    2015-11-01

    determine if COX-2 inhibition could also affect the signaling event in adenocarcinoma formed in the compound PKC;Pten mice. Our results showed that PKC...responsive probasin (PB) promoter[ 29 ]. Interestingly, only PB-PKC mice developed dysplastic changes characteristic of prostatic intraepithelial neoplasia...alteration in human prostate cancer, resulted in a compound mutant mice (PB-PKC;Pten+/- mice) that developed fully invasive adenocarcinoma. We recently

  7. A Kinase Inhibitor Screen Reveals Protein Kinase C-dependent Endocytic Recycling of ErbB2 in Breast Cancer Cells*

    PubMed Central

    Bailey, Tameka A.; Luan, Haitao; Tom, Eric; Bielecki, Timothy Alan; Mohapatra, Bhopal; Ahmad, Gulzar; George, Manju; Kelly, David L.; Natarajan, Amarnath; Raja, Srikumar M.; Band, Vimla; Band, Hamid

    2014-01-01

    ErbB2 overexpression drives oncogenesis in 20–30% cases of breast cancer. Oncogenic potential of ErbB2 is linked to inefficient endocytic traffic into lysosomes and preferential recycling. However, regulation of ErbB2 recycling is incompletely understood. We used a high-content immunofluorescence imaging-based kinase inhibitor screen on SKBR-3 breast cancer cells to identify kinases whose inhibition alters the clearance of cell surface ErbB2 induced by Hsp90 inhibitor 17-AAG. Less ErbB2 clearance was observed with broad-spectrum PKC inhibitor Ro 31-8220. A similar effect was observed with Go 6976, a selective inhibitor of classical Ca2+-dependent PKCs (α, β1, βII, and γ). PKC activation by PMA promoted surface ErbB2 clearance but without degradation, and ErbB2 was observed to move into a juxtanuclear compartment where it colocalized with PKC-α and PKC-δ together with the endocytic recycling regulator Arf6. PKC-α knockdown impaired the juxtanuclear localization of ErbB2. ErbB2 transit to the recycling compartment was also impaired upon PKC-δ knockdown. PMA-induced Erk phosphorylation was reduced by ErbB2 inhibitor lapatinib, as well as by knockdown of PKC-δ but not that of PKC-α. Our results suggest that activation of PKC-α and -δ mediates a novel positive feedback loop by promoting ErbB2 entry into the endocytic recycling compartment, consistent with reported positive roles for these PKCs in ErbB2-mediated tumorigenesis. As the endocytic recycling compartment/pericentrion has emerged as a PKC-dependent signaling hub for G-protein-coupled receptors, our findings raise the possibility that oncogenesis by ErbB2 involves previously unexplored PKC-dependent endosomal signaling. PMID:25225290

  8. CTRP3 Stimulates Proliferation and Anti-Apoptosis of Prostate Cells through PKC Signaling Pathways.

    PubMed

    Hou, Qi; Lin, Jinyan; Huang, Wentao; Li, Maoyin; Feng, Jianhua; Mao, Xiangming

    2015-01-01

    C1q/TNF-related protein-3 (CTRP3) is a novel adipokine with roles in multiple cellular processes. However, little is known about its function in prostate cells. This study investigated the effects and mechanisms of CTRP3 in prostate cells. We first generated and purified CTRP3 protein in HEK 293T cells. Proliferation of RWPE-1 prostate cells was evaluated by MTT analyses under treatment with different concentrations of CTRP3 for various exposure times. The results revealed maximum enhancement of proliferation with 10 μg/mL CTRP3 for 72 h. Cell apoptosis and cell cycle were determined by TUNEL staining and flow cytometry analysis. TUNEL assay showed decreased TUNEL-positive cells in RWPE-1 prostate cells treated with CTRP3, and flow cytometry showed significantly decreased apoptotic cells upon CTRP3 treatment (treated cells, 8.34±1.175 vs. controls, 20.163±0.35) (P < 0.01). Moreover, flow cytometry analysis also showed a significant decrease of cells in the G1 phase and an increase of cells in the S and G2 phase upon CTRP3 treatment (treated cells, 42.85±1.40 vs. control, 52.77±0.90; 28.41±0.57 vs. 23.49±1.13; 27.08±1.97 vs. 22.20±1.32, respectively) (all P < 0.05). Two-dimensional gel electrophoresis and mass spectrometry identified differentially expressed proteins, including cytokeratin-19, GLRX3 and DDAH1, which were upregulated in CTRP3 treated cells, and cytokeratin-17 and 14-3-3 sigma, which were downregulated. GLRX3, DDAH1 and 14-3-3 sigma were confirmed using western blot analysis. A PKC inhibitor, staurosporine, was used to inhibit PKC activity in CTRP3 treated RWPE-1 cells. Staurosporine completely abolished the CTRP3-induced increased phosphorylation of intracellular PKC substrates and CTRP3-stimulated effect by RWPE-1 cells. Our results provide the first evidence for a physiological role of the novel adipokine, CTRP3, in prostate cells. Our findings suggest that CTRP3 could improve proliferation and anti-apoptosis of prostate cells through

  9. Glutamine contributes to maintenance of mouse embryonic stem cell self-renewal through PKC-dependent downregulation of HDAC1 and DNMT1/3a

    PubMed Central

    Ryu, Jung Min; Lee, Sang Hun; Seong, Je Kyung; Han, Ho Jae

    2015-01-01

    Although glutamine (Gln) is not an essential amino acid, it is considered a critical substrate in many key metabolic processes that control a variety of physiological functions and are involved in regulating early embryonic development. Thus, we investigated the effect of Gln on regulation of mouse embryonic stem cell (mESC) self-renewal and related signaling pathways. Gln deprivation decreased Oct4 expression as well as expression of cell cycle regulatory proteins. However, Gln treatment retained the expression of cell cycle regulatory proteins and the Oct4 in mESCs, which were blocked by compound 968 (a glutaminase inhibitor). In addition, Gln stimulated PI3K/Akt pathway, which subsequently elicited PKCε translocation to membrane without an influx of intracellular Ca2+. Inhibition of Akt and PKC blocked Gln-induced Oct4 expression and proliferation. Gln also stimulated mTOR phosphorylation in a time-dependent manner, which abolished by PKC inhibition. Furthermore, Gln increased the cellular population of both Oct4 and bromodeoxyuridine positive cells, suggesting that Gln regulates self-renewal ability of mESCs. Gln induced a decrease in HDAC1, but not in HDAC2, which were blocked by PKC inhibitors. Gln treatment resulted in an increase in global histone acetylation and methylation. In addition, Gln significantly reduced methylation of the Oct4 promoter region through decrease in DNMT1 and DNMT3a expression, which were blocked by PKC and HDAC inhibitors. In conclusion, Gln stimulates mESC proliferation and maintains mESC undifferentiation status through transcription regulation via the Akt, PKCε, and mTOR signaling pathways. PMID:26375799

  10. Probiotics ameliorate the hydrogen peroxide-induced epithelial barrier disruption by a PKC- and MAP kinase-dependent mechanism

    PubMed Central

    Seth, A.; Yan, Fang; Polk, D.Brent; Rao, R. K.

    2009-01-01

    Probiotics promote intestinal epithelial integrity and reduce infection and diarrhea. We evaluated the effect of Lactobacillus rhamnosus GG-produced soluble proteins (p40 and p75) on the hydrogen peroxide-induced disruption of tight junctions and barrier function in Caco-2 cell monolayers. Pretreatment of cell monolayers with p40 or p75 attenuated the hydrogen peroxide-induced decrease in transepithelial resistance and increase in inulin permeability in a time- and dose-dependent manner. p40 and p75 also prevented hydrogen peroxide-induced redistribution of occludin, ZO-1, E-cadherin, and β-catenin from the intercellular junctions and their dissociation from the detergent-insoluble fractions. Both p40 and p75 induced a rapid increase in the membrane translocation of PKCβI and PKCε. The attenuation of hydrogen peroxide-induced inulin permeability and redistribution of tight junction proteins by p40 and p75 was abrogated by Ro-32-0432, a PKC inhibitor. p40 and p75 also rapidly increased the levels of phospho-ERK1/2 in the detergent-insoluble fractions. U0126 (a MAP kinase inhibitor) attenuated the p40- and p75-mediated reduction of hydrogen peroxide-induced tight junction disruption and inulin permeability. These studies demonstrate that probiotic-secretory proteins protect the intestinal epithelial tight junctions and the barrier function from hydrogen peroxide-induced insult by a PKC- and MAP kinase-dependent mechanism. PMID:18292183

  11. Probiotics ameliorate the hydrogen peroxide-induced epithelial barrier disruption by a PKC- and MAP kinase-dependent mechanism.

    PubMed

    Seth, A; Yan, Fang; Polk, D Brent; Rao, R K

    2008-04-01

    Probiotics promote intestinal epithelial integrity and reduce infection and diarrhea. We evaluated the effect of Lactobacillus rhamnosus GG-produced soluble proteins (p40 and p75) on the hydrogen peroxide-induced disruption of tight junctions and barrier function in Caco-2 cell monolayers. Pretreatment of cell monolayers with p40 or p75 attenuated the hydrogen peroxide-induced decrease in transepithelial resistance and increase in inulin permeability in a time- and dose-dependent manner. p40 and p75 also prevented hydrogen peroxide-induced redistribution of occludin, ZO-1, E-cadherin, and beta-catenin from the intercellular junctions and their dissociation from the detergent-insoluble fractions. Both p40 and p75 induced a rapid increase in the membrane translocation of PKCbetaI and PKCepsilon. The attenuation of hydrogen peroxide-induced inulin permeability and redistribution of tight junction proteins by p40 and p75 was abrogated by Ro-32-0432, a PKC inhibitor. p40 and p75 also rapidly increased the levels of phospho-ERK1/2 in the detergent-insoluble fractions. U0126 (a MAP kinase inhibitor) attenuated the p40- and p75-mediated reduction of hydrogen peroxide-induced tight junction disruption and inulin permeability. These studies demonstrate that probiotic-secretory proteins protect the intestinal epithelial tight junctions and the barrier function from hydrogen peroxide-induced insult by a PKC- and MAP kinase-dependent mechanism.

  12. Increased extracellular pressure stimulates tumor proliferation by a mechanosensitive calcium channel and PKC-β.

    PubMed

    Basson, Marc D; Zeng, Bixi; Downey, Christina; Sirivelu, Madhu P; Tepe, Jetze J

    2015-02-01

    Large tumors exhibit high interstitial pressure heightened by growth against the constraining stroma. Such pressures could stimulate tumor proliferation via a mechanosensitive ion channel. We studied the effects of 0-80 mmHg increased extracellular pressure for 24 h on proliferation of SW620, Caco-2, and CT-26 colon; MCF-7 breast; and MLL and PC3 prostate cancer cells, and delineated its mechanism in SW620 cells with specific inhibitors and siRNA. Finally, we compared NF-kB, phospho-IkB and cyclin D1 immunoreactivity in the high pressure centers and low pressure peripheries of human tumors. Pressure-stimulated proliferation in all cells. Pressure-driven SW620 proliferation required calcium influx via the T-type Ca(2+) channel Cav3.3, which stimulated PKC-β to invoke the IKK-IkB-NF-kB pathway to increase proliferation and S-phase fraction. The mitotic index and immunoreactivity of NF-kB, phospho-IkB, and cyclin D1 in the center of 28 large human colon, lung, and head and neck tumors exceeded that in tumor peripheries. Extracellular pressure increases [Ca(2+)]i via Cav3.3, driving a PKC-β- IKK- IkB-NF-kB pathway that stimulates cancer cell proliferation. Rapid proliferation in large stiff tumors may increase intratumoral pressure, activating this pathway to stimulate further proliferation in a feedback cycle that potentiates tumor growth. Targeting this pathway may inhibit proliferation in large unresectable tumors.

  13. Lipid emulsion inhibits vasodilation induced by a toxic dose of bupivacaine by suppressing bupivacaine-induced PKC and CPI-17 dephosphorylation but has no effect on vasodilation induced by a toxic dose of mepivacaine

    PubMed Central

    Cho, Hyunhoo; Ok, Seong Ho; Kwon, Seong Chun; Lee, Soo Hee; Baik, Jiseok; Kang, Sebin; Oh, Jiah

    2016-01-01

    Background The goal of this in vitro study was to investigate the effect of lipid emulsion on vasodilation caused by toxic doses of bupivacaine and mepivacaine during contraction induced by a protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), in an isolated endothelium-denuded rat aorta. Methods The effects of lipid emulsion on the dose-response curves induced by bupivacaine or mepivacaine in an isolated aorta precontracted with PDBu were assessed. In addition, the effects of bupivacaine on the increased intracellular calcium concentration ([Ca2+]i) and contraction induced by PDBu were investigated using fura-2 loaded aortic strips. Further, the effects of bupivacaine, the PKC inhibitor GF109203X and lipid emulsion, alone or in combination, on PDBu-induced PKC and phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) was examined by western blotting. Results Lipid emulsion attenuated the vasodilation induced by bupivacaine, whereas it had no effect on that induced by mepivacaine. Lipid emulsion had no effect on PDBu-induced contraction. The magnitude of bupivacaine-induced vasodilation was higher than that of the bupivacaine-induced decrease in [Ca2+]i. PDBu promoted PKC and CPI-17 phosphorylation in aortic VSMCs. Bupivacaine and GF109203X attenuated PDBu-induced PKC and CPI-17 phosphorylation, whereas lipid emulsion attenuated bupivacaine-mediated inhibition of PDBu-induced PKC and CPI-17 phosphorylation. Conclusions These results suggest that lipid emulsion attenuates the vasodilation induced by a toxic dose of bupivacaine via inhibition of bupivacaine-induced PKC and CPI-17 dephosphorylation. This lipid emulsion-mediated inhibition of vasodilation may be partly associated with the lipid solubility of local anesthetics. PMID:27738501

  14. Requirements for PKC-augmented JNK activation by MKK4/7

    PubMed Central

    Lopez-Bergami, Pablo; Ronai, Ze'ev

    2008-01-01

    The c-Jun N-terminal kinases (JNKs) are activated in response to stress, DNA damage, and cytokines by MKK4 and MKK7. We recently demonstrated that PKC can augment the degree of JNK activation by phosphorylating JNK, which requires the adaptor protein RACK1. Here we report on the conditions required for PKC-dependent JNK activation. In vitro kinase assays reveal that PKC phosphorylation of JNK is not sufficient for its activation but rather augments JNK activation by canonical JNK upstream kinases MKK4 or MKK7 alone or in combination. Further, to enhance JNK activity, PKC phosphorylation of JNK should precede its phosphorylation by MKK4/7. Inhibition of PKC phosphorylation of JNK affects both early and late phases of JNK activation following UV-irradiation and reduces the apoptotic response mediated by JNK. These data provide important insight into the requirements for PKC activation of JNK signaling. PMID:18182317

  15. Virtual screening of protein kinase C inhibitors from natural product library to modulate general anaesthetic effects.

    PubMed

    Zhao, Junhui; Zhou, Chuixian

    2015-01-01

    Protein kinase C (PKC) plays a key role in neurotransmission in the central nervous system, and targeting PKC domain is considered as a strategy to modulate the anaesthetic effects. In this study, we described a synthetic pipeline to perform high-throughput virtual screening against a large library of 3D structural natural products released recently in order to discover those potential PKC modulators. A total of 100 natural products with top scores were raised, from which 12 promising candidates were tested to determine their inhibitory potencies against PKC. As might be expected, the promiscuous kinase inhibitor staurosporine showed a high PKC inhibitory activity (IC50 = 64 nM), and other two tested compounds, i.e. fisetin and tetrahydropapaverine, were also highly potent with their activities at nanomolar level (IC50 = 370 and 190, respectively).

  16. Zinc pyrithione induces ERK- and PKC-dependent necrosis distinct from TPEN-induced apoptosis in prostate cancer cells.

    PubMed

    Carraway, Robert E; Dobner, Paul R

    2012-02-01

    Zinc dyshomeostasis can induce cell death. However, the mechanisms involved have not been fully elucidated in prostate cancer (PCa) cells, which differ dramatically from normal cells in their zinc handling ability. Here, we studied the effects of the ionophore Zn-pyrithione (ZP) and the chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Both compounds induced cell death at micromolar concentrations when incubated with androgen-dependent (LNCaP), androgen-independent (PC3, DU145) and androgen-sensitive (C4-2) PCa cell-lines. Compared to PCa cells, RWPE1 prostate epithelial cells were less sensitive to ZP and more sensitive to TPEN, but total cellular zinc levels were changed similarly. ZnSO4 enhanced the toxicity of ZP, but inhibited the effects of TPEN as expected. The morphological/biochemical responses to ZP and TPEN differed. ZP decreased ATP levels and stimulated ERK, AKT and PKC phosphorylation. DNA laddering was observed only at low doses of ZP but all doses of TPEN. TPEN activated caspase 3/7 and induced PARP-cleavage, DNA-fragmentation, ROS-formation and apoptotic bodies. PKC and ERK-pathway inhibitors, and antioxidants protected against ZP-induced but not TPEN-induced death. Inhibitors of MPTP-opening protected both. Cell death in response to TPEN (but not ZP) was diminished by a calpain inhibitor and largely prevented by a caspase 3 inhibitor. Overall, the results indicated primarily a necrotic cell death for ZP and an apoptotic cell death for TPEN. The enhanced sensitivity of PCa cells to ZP and the apparent ability of ZP and TPEN to kill quiescent and rapidly dividing cells in a p53-independent manner suggest that ZP/TPEN might be used to develop adjunct treatments for PCa.

  17. Regulation of aPKC activity by Nup358 dependent SUMO modification

    PubMed Central

    Yadav, Santosh Kumar; Magre, Indrasen; Singh, Aditi; Khuperkar, Deepak; Joseph, Jomon

    2016-01-01

    Atypical PKC (aPKC) family members are involved in regulation of diverse cellular processes, including cell polarization. aPKCs are known to be activated by phosphorylation of specific threonine residues in the activation loop and turn motif. They can also be stimulated by interaction with Cdc42~GTP-Par6 complex. Here we report that PKCζ, a member of the aPKC family, is activated by SUMOylation. We show that aPKC is endogenously modified by SUMO1 and the nucleoporin Nup358 acts as its SUMO E3 ligase. Results from in vitro SUMOylation and kinase assays showed that the modification enhances the kinase activity of PKCζ by ~10-fold. By monitoring the phosphorylation of Lethal giant larvae (Lgl), a downstream target of aPKC, we confirmed these findings in vivo. Consistent with the function of Nup358 as a SUMO E3 ligase for aPKC, depletion of Nup358 attenuated the extent of SUMOylation and the activity of aPKC. Moreover, overexpression of the C-terminal fragment of Nup358 that possesses the E3 ligase activity enhanced SUMOylation of endogenous aPKC and its kinase activity. Collectively, our studies reveal a role for Nup358-dependent SUMOylation in the regulation of aPKC activity and provide a framework for understanding the role of Nup358 in cell polarity. PMID:27682244

  18. A high throughput system for the evaluation of protein kinase C inhibitors based on Elk1 transcriptional activation in human astrocytoma cells.

    PubMed

    Sharif, T R; Sharif, M

    1999-02-01

    Protein kinase C (PKC) designates a family of kinases that regulate many essential functions including cell growth and differentiation. The tight regulation of PKC activity is crucial for maintaining normal cellular proliferation and excessive activity leads to abnormal or uncontrolled cell growth. Recent reports indicate that malignant glioma cell lines express 100 to 1000-fold higher PKC activity when compared to non-neoplastic astrocytes. This high activity correlates well with the proliferation of tumor cells in vitro. We recently reported on the anti-proliferative properties of selective PKC inhibitors on the growth of U-373MG human astrocytoma cell line, and their ability to block mitogen-activated protein (MAP) kinase pathway activated by substance P (SP) neuropeptide receptor signaling via a PKC-dependent mechanism. Therefore, inhibiting PKC activity by selective PKC inhibitors may present a promising approach for improving astroglial brain tumor therapy. For this purpose, we constructed a high throughput model cell system to evaluate the efficacy of PKC inhibitors. This system is based on the measurement of light production in U-373MG cells stably transfected with the luciferase reporter gene whose expression depends on the transcriptional activation of GAL4-Elk1 fusion protein by enzyme components of the MAP kinase pathway and the upstream activation of PKC (PKC activation-->MAP kinases-->GAL4-Elk1 phosphorylation-->luciferase expression-->luciferase activity). In brief, we have demonstrated that the PKC activator 12-O-tetradecanoyl phorbol 13-acetate (TPA)-induced luciferase activity in this cell system is mediated via the MAP kinase pathway and can be blocked in the presence of MEK1 selective inhibitors (PD 098059 or U0126). We also demonstrated that TPA-induced luciferase activity in U-373MG stable clones can be blocked by PKC inhibitors (CGP 41251, Go 6976, and GF 109203X) in a concentration dependent manner. In contrast, epidermal growth factor (EGF

  19. Maturation and long-term hypoxia-induced acclimatization responses in PKC-mediated signaling pathways in ovine cerebral arterial contractility.

    PubMed

    Goyal, Ravi; Mittal, Ashwani; Chu, Nina; Arthur, Rebecca Afiba; Zhang, Lubo; Longo, Lawrence D

    2010-11-01

    In the developing fetus, cerebral arteries (CA) show striking differences in signal transduction mechanisms compared with the adult, and these differences are magnified in response to high-altitude long-term hypoxia (LTH). In addition, in the mature organism, cerebrovascular acclimatization to LTH may be associated with several clinical problems, the mechanisms of which are unknown. Because PKC plays a key role in regulating CA contractility, in fetal and adult cerebral arteries, we tested the hypothesis that LTH differentially regulates the PKC-mediated Ca(2+) sensitization pathways and contractility. In four groups of sheep [fetal normoxic (FN), fetal hypoxic (FH), adult normoxic (AN), and adult hypoxic (AH)], we examined, simultaneously, responses of CA tension and intracellular Ca(2+) concentration and measured CA levels of PKC, ERK1/2, RhoA, 20-kDa myosin light chain, and the 17-kDa PKC-potentiated myosin phosphatase inhibitor CPI-17. The PKC activator phorbol 12,13-dibutyrate (PDBu) produced robust contractions in all four groups. However, PDBu-induced contractions were significantly greater in AH CA than in the other groups. In all CA groups except AH, in the presence of MEK inhibitor (U-0126), the PDBu-induced contractions were increased a further 20-30%. Furthermore, in adult CA, PDBu led to increased phosphorylation of ERK1, but not ERK2; in fetal CA, the reverse was the case. PDBu-stimulated ERK2 phosphorylation also was significantly greater in FH than FN CA. Also, although RhoA/Rho kinase played a significant role in PDBu-mediated contractions of FN CA, this was not the case in FH or either adult group. Also, whereas CPI-17 had a significant role in adult CA contractility, this was not the case for the fetus. Overall, in ovine CA, the present study demonstrates several important maturational and LTH acclimatization changes in PKC-induced contractile responses and downstream pathways. The latter may play a key role in the pathophysiologic disorders

  20. Complex modulation of the expression of PKC isoforms in the rat brain during chronic type 1 diabetes mellitus.

    PubMed

    Vetri, Francesco; Chavez, Rafael; Xu, Hao-Liang; Paisansathan, Chanannait; Pelligrino, Dale A

    2013-01-15

    We previously demonstrated that chronic hyperglycemia has a detrimental influence on neurovascular coupling in the brain-an effect linked to an alteration in the protein kinase C (PKC)-mediated phosphorylation pattern. Moreover, the activity of PKC was increased, in diabetic rat brain, in a tissue fraction composed primarily of the superficial glia limitans and pial vessels, but trended toward a decrease in cerebral cortical gray matter. However, that study did not examine the expression patterns of PKC isoforms in the rat brain. Thus, in a rat model of streptozotocin (STZ)-induced chronic type 1 diabetes mellitus (T1DM), and in non-diabetic (ND) controls, two hypotheses were addressed. First, chronic T1DM is accompanied by changes in the expression of PKC-α, βII, γ, δ, and ε Second, those changes differ when comparing cerebral cortex and glio-pial tissue. In addition, we analyzed the expression of a form of PKC-γ, phosphorylated on threonine 514 (pT514-PKC-γ), as well as the receptor for activated C kinase 1 (RACK1). The expression pattern of different PKC isoforms was altered in a complex and tissue-specific manner during chronic hyperglycemia. Notably, in the gray matter, PKC-α expression significantly decreased, while pT514-PKC-γ expression increased. However, PKC-βII, -γ, -δ, -ε, and RACK1 expressions did not change. Conversely, in glio-pial tissue, PKC-α and RACK1 were upregulated, whereas PKC-γ, pT514-PKC-γ, and PKC-ε were downregulated. PKC-βII, and PKC-δ, were unchanged. These findings suggest that the PKC activity increase previously seen in the glio-pial tissue of diabetic rats may be due to the selective upregulation of PKC-α, and ultimately lead to the impairment of neurovascular coupling.

  1. Implication of PKC in the seasonal variation of the immune response of the hemocytes of Mytilus galloprovincialis Lmk. and its role in interleukin-2-induced nitric oxide synthesis.

    PubMed

    Novas, Ana; Barcia, Ramiro; Ramos-Martínez, Juan Ignacio

    2007-10-01

    The hemocytes are the cells responsible for the immune response in marine mollusks. The role of NO in processes related to the activation of the hemocytes has turned out evident over the late years. In the case of the mussel Mytilus galloprovincialis Lmk., hemocyte NO basal production varies throughout the year, showing a maximum in summer and a minimum in winter. IL-2 reverts the low winter NO basal production through a process mediated by cAMP-dependent protein kinase and by an apparent side effect of protein kinase C. The seasonal variation of NO production in the presence of the PKC inhibitor bisindolylmaleimide (BSM) allows suggesting a model in which PKC would modulate the activity of the enzymes responsible for nitric oxide production.

  2. Prostaglandin E2 stimulates β1-integrin expression in hepatocellular carcinoma through the EP1 receptor/PKC/NF-κB pathway

    PubMed Central

    Bai, Xiaoming; Wang, Jie; Guo, Yan; Pan, Jinshun; Yang, Qinyi; Zhang, Min; Li, Hai; Zhang, Li; Ma, Juan; Shi, Feng; Shu, Wei; Wang, Yipin; Leng, Jing

    2014-01-01

    Prostaglandin E2 (PGE2) has been implicated in cell invasion in hepatocellular carcinoma (HCC), via increased β1-integrin expression and cell migration; however, the mechanism remains unclear. PGE2 exerts its effects via four subtypes of the E prostanoid receptor (EP receptor 1–4). The present study investigated the effect of EP1 receptor activation on β1-integrin expression and cell migration in HCC. Cell migration increased by 60% in cells treated with 17-PT-PGE2 (EP1 agonist), which was suppressed by pretreatment with a β1-integrin polyclonal antibody. PGE2 increased β1-integrin expression by approximately 2-fold. EP1 receptor transfection or treatment with 17-PT-PGE2 mimicked the effect of PGE2 treatment. EP1 siRNA blocked PGE2-mediated β1-integrin expression. 17-PT-PGE2 treatment induced PKC and NF-κB activation; PKC and NF-κB inhibitors suppressed 17-PT-PGE2-mediated β1-integrin expression. FoxC2, a β1-integrin transcription factor, was also upregulated by 17-PT-PGE2. NF-κB inhibitor suppressed 17-PT-PGE2-mediated FoxC2 upregulation. Immunohistochemistry showed p65, FoxC2, EP1 receptor and β1-integrin were all highly expressed in the HCC cases. This study suggested that PGE2 upregulates β1-integrin expression and cell migration in HCC cells by activating the PKC/NF-κB signaling pathway. Targeting PGE2/EP1/PKC/NF-κB/FoxC2/β1-integrin pathway may represent a new therapeutic strategy for the prevention and treatment of this cancer. PMID:25289898

  3. 20-HETE increases NADPH oxidase-derived ROS production and stimulates the L-type Ca2+ channel via a PKC-dependent mechanism in cardiomyocytes

    PubMed Central

    Han, Yong; Bao, Yuyan; Li, Wei; Li, Xingting; Shen, Xin; Wang, Xu; Yao, Fanrong; O'Rourke, Stephen T.; Sun, Chengwen

    2010-01-01

    The production of 20-hydroxyeicosatetraenoic acid (20-HETE) is increased during ischemia-reperfusion, and inhibition of 20-HETE production has been shown to reduce infarct size caused by ischemia. This study was aimed to discover the molecular mechanism underlying the action of 20-HETE in cardiac myocytes. The effect of 20-HETE on L-type Ca2+ currents (ICa,L) was examined in rat isolated cardiomyocytes by patch-clamp recording in the whole cell mode. Superfusion of cardiomyocytes with 20-HETE (10–100 nM) resulted in a concentration-dependent increase in ICa,L, and this action of 20-HETE was attenuated by a specific NADPH oxidase inhibitor, gp91ds-tat (5 μM), or a superoxide scavenger, polyethylene glycol-superoxide dismutase (25 U/ml), suggesting that NADPH-oxidase-derived superoxide is involved in the stimulatory action of 20-HETE on ICa,L. Treatment of cardiomyocytes with 20-HETE (100 nM) increased both NADPH oxidase activity and superoxide production by approximately twofold. To study the molecular mechanism mediating the 20-HETE-induced increase in NADPH oxidase activity, PKC activity was measured in cardiomyocytes. Incubation of the cells with 20-HETE (100 nM) significantly increased PKC activity, and pretreatment of cardiomyocytes with a selective PKC inhibitor, GF-109203 (1 μM), attenuated the 20-HETE-induced increases in ICa,L and in NADPH oxidase activity. In summary, 20-HETE stimulates NADPH oxidase-derived superoxide production, which activates L-type Ca2+ channels via a PKC-dependent mechanism in cardiomyocytes. 20-HETE and 20-HETE-producing enzymes could be novel targets for the treatment of cardiac ischemic diseases. PMID:20675568

  4. Roles of PKC and phospho‑adducin in transepithelial fluid secretion by Malpighian tubules of the yellow fever mosquito

    PubMed Central

    Miyauchi, Jeremy T.; Piermarini, Peter M.; Yang, Jason D.; Gilligan, Diana M.; Beyenbach, Klaus W.

    2013-01-01

    The diuretic hormone aedeskinin‑III is known to increase the paracellular Cl- conductance in Malpighian (renal) tubules of the mosquito Aedes aegypti via a G protein-coupled receptor. The increase serves the blood-meal-initiated diuresis and is associated with elevated levels of Ca2+ and phosphorylated adducin in the cytosol of tubule. In the present study we have cloned adducin in Aedes Malpighian tubules and investigated its physiological roles. Immunolabeling experiments are consistent with the association of adducin with the cortical cytoskeleton, especially near the apical brush border of the tubule. An antibody against phosphorylated adducin revealed the transient phosphorylation of adducin 2 min after stimulating tubules with aedeskinin‑III. The PKC inhibitor bisindolylmaleimide‑I blocked the phosphorylation of adducin as well as the electrophysiological and diuretic effects of aedeskinin‑III. Bisindolylmaleimide‑I also inhibited fluid secretion in control tubules. Phorbol 12‑myristate 13‑acetate increased phosphorylated adducin levels in Malpighian tubules, but it inhibited fluid secretion. Thus, the phosphorylation of adducin by PKC alone is insufficient to trigger diuretic rates of fluid secretion; elevated levels of intracellular Ca2+ may also be required. The above results suggest that the phosphorylation of adducin, which is known to destabilize the cytoskeleton, may (1) facilitate the traffic of transporters into the apical brush border supporting diuretic rates of cation secretion and (2) destabilize proteins in the septate junction thereby enabling paracellular anion (Cl‑) secretion at diuretic rates. Moreover, PKC and the phosphorylation of adducin play a central role in control and diuretic tubules, consistent with the dynamic behavior of both transcellular and paracellular transport pathways. PMID:24062972

  5. Intra-lesional injection of the novel PKC activator EBC-46 rapidly ablates tumors in mouse models.

    PubMed

    Boyle, Glen M; D'Souza, Marjorie M A; Pierce, Carly J; Adams, Ryan A; Cantor, Aaron S; Johns, Jenny P; Maslovskaya, Lidia; Gordon, Victoria A; Reddell, Paul W; Parsons, Peter G

    2014-01-01

    Intra-lesional chemotherapy for treatment of cutaneous malignancies has been used for many decades, allowing higher local drug concentrations and less toxicity than systemic agents. Here we describe a novel diterpene ester, EBC-46, and provide preclinical data supporting its use as an intra-lesional treatment. A single injection of EBC-46 caused rapid inflammation and influx of blood, followed by eschar formation and rapid tumor ablation in a range of syngeneic and xenograft models. EBC-46 induced oxidative burst from purified human polymorphonuclear cells, which was prevented by the Protein Kinase C inhibitor bisindolylmaleimide-1. EBC-46 activated a more specific subset of PKC isoforms (PKC-βI, -βII, -α and -γ) compared to the structurally related phorbol 12-myristate 13-acetate (PMA). Although EBC-46 showed threefold less potency for inhibiting cell growth than PMA in vitro, it was more effective for cure of tumors in vivo. No viable tumor cells were evident four hours after injection by ex vivo culture. Pharmacokinetic profiles from treated mice indicated that EBC-46 was retained preferentially within the tumor, and resulted in significantly greater local responses (erythema, oedema) following intra-lesional injection compared with injection into normal skin. The efficacy of EBC-46 was reduced by co-injection with bisindolylmaleimide-1. Loss of vascular integrity following treatment was demonstrated by an increased permeability of endothelial cell monolayers in vitro and by CD31 immunostaining of treated tumors in vivo. Our results demonstrate that a single intra-lesional injection of EBC-46 causes PKC-dependent hemorrhagic necrosis, rapid tumor cell death and ultimate cure of solid tumors in pre-clinical models of cancer.

  6. Intra-Lesional Injection of the Novel PKC Activator EBC-46 Rapidly Ablates Tumors in Mouse Models

    PubMed Central

    Pierce, Carly J.; Adams, Ryan A.; Cantor, Aaron S.; Johns, Jenny P.; Maslovskaya, Lidia; Gordon, Victoria A.; Reddell, Paul W.; Parsons, Peter G.

    2014-01-01

    Intra-lesional chemotherapy for treatment of cutaneous malignancies has been used for many decades, allowing higher local drug concentrations and less toxicity than systemic agents. Here we describe a novel diterpene ester, EBC-46, and provide preclinical data supporting its use as an intra-lesional treatment. A single injection of EBC-46 caused rapid inflammation and influx of blood, followed by eschar formation and rapid tumor ablation in a range of syngeneic and xenograft models. EBC-46 induced oxidative burst from purified human polymorphonuclear cells, which was prevented by the Protein Kinase C inhibitor bisindolylmaleimide-1. EBC-46 activated a more specific subset of PKC isoforms (PKC-βI, -βII, -α and -γ) compared to the structurally related phorbol 12-myristate 13-acetate (PMA). Although EBC-46 showed threefold less potency for inhibiting cell growth than PMA in vitro, it was more effective for cure of tumors in vivo. No viable tumor cells were evident four hours after injection by ex vivo culture. Pharmacokinetic profiles from treated mice indicated that EBC-46 was retained preferentially within the tumor, and resulted in significantly greater local responses (erythema, oedema) following intra-lesional injection compared with injection into normal skin. The efficacy of EBC-46 was reduced by co-injection with bisindolylmaleimide-1. Loss of vascular integrity following treatment was demonstrated by an increased permeability of endothelial cell monolayers in vitro and by CD31 immunostaining of treated tumors in vivo. Our results demonstrate that a single intra-lesional injection of EBC-46 causes PKC-dependent hemorrhagic necrosis, rapid tumor cell death and ultimate cure of solid tumors in pre-clinical models of cancer. PMID:25272271

  7. Angiotensin II stimulates renal proximal tubule Na(+)-ATPase activity through the activation of protein kinase C.

    PubMed

    Rangel, L B A; Caruso-Neves, C; Lara, L S; Lopes, A G

    2002-08-31

    Recently, our group described an AT(1)-mediated direct stimulatory effect of angiotensin II (Ang II) on the Na(+)-ATPase activity of proximal tubules basolateral membranes (BLM) [Am. J. Physiol. 248 (1985) F621]. Data in the present report suggest the participation of a protein kinase C (PKC) in the molecular mechanism of Ang II-mediated stimulation of the Na(+)-ATPase activity due to the following observations: (i) the stimulation of protein phosphorylation in BLM, induced by Ang II, is mimicked by the PKC activator TPA, and is completely reversed by the specific PKC inhibitor, calphostin C; (ii) the Na(+)-ATPase activity is stimulated by Ang II and TPA in the same magnitude, being these effects abolished by the use of the PKC inhibitors, calphostin C and sphingosine; (iii) the Na(+)-ATPase activity is activated by catalytic subunit of PKC (PKC-M), in a similar and nonadditive manner to Ang II; and (iv) Ang II stimulates the phosphorylation of MARCKS, a specific substrate for PKC.

  8. Hedgehog-regulated atypical PKC promotes phosphorylation and activation of Smoothened and Cubitus interruptus in Drosophila

    PubMed Central

    Jiang, Kai; Liu, Yajuan; Fan, Junkai; Epperly, Garretson; Gao, Tianyan; Jiang, Jin; Jia, Jianhang

    2014-01-01

    Smoothened (Smo) is essential for transduction of the Hedgehog (Hh) signal in both insects and vertebrates. Cell surface/cilium accumulation of Smo is thought to play an important role in Hh signaling, but how the localization of Smo is controlled remains poorly understood. In this study, we demonstrate that atypical PKC (aPKC) regulates Smo phosphorylation and basolateral accumulation in Drosophila wings. Inactivation of aPKC by either RNAi or a mutation inhibits Smo basolateral accumulation and attenuates Hh target gene expression. In contrast, expression of constitutively active aPKC elevates basolateral accumulation of Smo and promotes Hh signaling. The aPKC-mediated phosphorylation of Smo at Ser680 promotes Ser683 phosphorylation by casein kinase 1 (CK1), and these phosphorylation events elevate Smo activity in vivo. Moreover, aPKC has an additional positive role in Hh signaling by regulating the activity of Cubitus interruptus (Ci) through phosphorylation of the Zn finger DNA-binding domain. Finally, the expression of aPKC is up-regulated by Hh signaling in a Ci-dependent manner. Our findings indicate a direct involvement of aPKC in Hh signaling beyond its role in cell polarity. PMID:25349414

  9. aPKC Phosphorylates p27Xic1, Providing a Mechanistic Link between Apicobasal Polarity and Cell-Cycle Control

    PubMed Central

    Sabherwal, Nitin; Thuret, Raphael; Lea, Robert; Stanley, Peter; Papalopulu, Nancy

    2014-01-01

    Summary During the development of the nervous system, apicobasally polarized stem cells are characterized by a shorter cell cycle than nonpolar progenitors, leading to a lower differentiation potential of these cells. However, how polarization might be directly linked to the kinetics of the cell cycle is not understood. Here, we report that apicobasally polarized neuroepithelial cells in Xenopus laevis have a shorter cell cycle than nonpolar progenitors, consistent with mammalian systems. We show that the apically localized serine/threonine kinase aPKC directly phosphorylates an N-terminal site of the cell-cycle inhibitor p27Xic1 and reduces its ability to inhibit the cyclin-dependent kinase 2 (Cdk2), leading to shortening of G1 and S phases. Overexpression of activated aPKC blocks the neuronal differentiation-promoting activity of p27Xic1. These findings provide a direct mechanistic link between apicobasal polarity and the cell cycle, which may explain how proliferation is favored over differentiation in polarized neural stem cells. PMID:25490266

  10. Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways

    SciTech Connect

    Matsuoka, Hiroshi; Tsubaki, Masanobu; Yamazoe, Yuzuru; Ogaki, Mitsuhiko; Satou, Takao; Itoh, Tatsuki; Kusunoki, Takashi; Nishida, Shozo

    2009-07-15

    In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities of matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKC{alpha} and PKC{delta} phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.

  11. L-type calcium channel gating is modulated by bradykinin with a PKC-dependent mechanism in NG108-15 cells.

    PubMed

    Toselli, Mauro; Taglietti, Vanni

    2005-05-01

    Bradykinin (BK) excites dorsal root ganglion cells, leading to the sensation of pain. The actions of BK are thought to be mediated by heterotrimeric G protein-regulated pathways. Indeed there is strong evidence that in different cell types BK is involved in phosphoinositide breakdown following activation of G(q/11). In the present study we show that the Ca(2+) current flowing through L-type voltage-gated Ca(2+) channels in NG108-15 cells (differentiated in vitro to acquire a neuronal phenotype), measured using the whole-cell patch clamp configuration, is reversibly inhibited by BK in a voltage-independent fashion, suggesting a cascade process where a second messenger system is involved. This inhibitory action of BK is mimicked by the application of 1,2-oleoyl-acetyl glycerol (OAG), an analog of diacylglycerol that activates PKC. Interestingly, OAG occluded the effects of BK and both effects were blocked by selective PKC inhibitors. The down modulation of single L-type Ca(2+) channels by BK and OAG was also investigated in cell-attached patches. Our results indicate that the inhibitory action of BK involves activation of PKC and mainly shows up in a significant reduction of the probability of channel opening, caused by an increase and clustering of null sweeps in response to BK.

  12. Enhancement of NK cell-mediated lysis of non-small lung cancer cells by nPKC activator, ingenol 3,20 dibenzoate.

    PubMed

    Gong, Chenyuan; Yao, Chao; Xu, Zihang; Ni, Zhongya; Zhu, Xiaowen; Wang, Lixin; Yan, Xuewei; Zhou, Wuxiong; Zhu, Shiguo

    2017-03-01

    The IFN-γ production is crucial for NK cell-mediated lysis of cancer cells. Thus increasing the IFN-γ production by NK cells may be an ideal strategy to improve their tumoricidal effect. Since the focus on new drug development has shifted towards natural products, limited information is out there about natural products that enhance the IFN-γ production by NK cells. In this study, through a high-throughput screening, we have identified a natural product ingenol 3,20 dibenzoate (IDB), an activator of tumor suppressor protein kinase C (PKC) isozymes, could increase the IFN-γ production and degranulation by NK cells, especially when NK cells were stimulated by non-small lung cancer (NSCLC) cells. IDB also significantly enhanced the NK cell-mediated lysis of NSCLC cells. Furthermore, PKC inhibitor, sotrastaurin abrogated IDB-induced IFN-γ production, degranulation and cytotoxicity, but did not affect IFN-γ production by NK cells without IDB treatment and NSCLC cell stimulation. The IFN-γ neutralization reversed the IDB-induced enhancement of NK cell mediated killing. In conclusion, our study indicated that IDB enhanced NK cell-mediated lysis of NSCLC cells is dependent on specific PKC mediated IFN-γ production and degranulation. Thus, IDB may have a promising application in clinic for NK cell-based cancer immunotherapy.

  13. Acrylonitrile-induced extracellular signal-regulated kinase (ERK) activation via protein kinase C (PKC) in SK-N-SH neuroblastoma cells.

    PubMed

    Chantara, Wantika; Watcharasit, Piyajit; Thiantanawat, Apinya; Satayavivad, Jutamaad

    2006-01-01

    Acrylonitrile (ACN) is classified by IARC as a probable carcinogen. Chronic exposure to ACN increases the incidence of tumors in various organs of test animals, including the brain and lung. ERK1/2 activation plays crucial roles in cell proliferation and is involved in many steps of tumor progression. Therefore, this study examined whether ACN altered the activation state of ERK1/2 in human neuroblastoma SK-N-SH cells. Treatment of these cells with ACN greatly increased phosphorylation of ERK1/2 in dose- and time-dependent manners. This effect was inhibited by PD 98059 and U 0126, specific inhibitors of MEK, indicating that MEK, an upstream activator of ERK1/2, was directly involved in ACN-induced ERK1/2 activation. Furthermore, the activation of ERK1/2 by ACN was attenuated by inhibition of PKC with GF 109203X, rottlerin and prolonged incubation with PMA (phorbol 12-myristate 13-acetate). This demonstrated the participation of PKC in the ACN-stimulated activation of ERK1/2. Taken together, our results indicate that ACN-induced ERK1/2 activation involves PKC through a MEK-dependent pathway.

  14. Cortical PKC inhibition promotes axonal regeneration of the corticospinal tract and forelimb functional recovery after cervical dorsal spinal hemisection in adult rats.

    PubMed

    Wang, Xiaofei; Hu, Jianguo; She, Yun; Smith, George M; Xu, Xiao-Ming

    2014-11-01

    Our previous study shows that conventional protein kinases C (cPKCs) are key signaling mediators that are activated by extracellular inhibitory molecules. Inhibition of cPKC by intrathecal infusion of a cPKC inhibitor, GÖ6976, into the site of dorsal hemisection (DH) induces regeneration of lesioned dorsal column sensory, but not corticospinal tract (CST), axons. Here, we investigated whether a direct cortical delivery of GÖ6976 into the soma of corticospinal neurons promotes regeneration of CST and the recovery of forelimb function in rats with cervical spinal cord injuries. We report that cortical delivery of GÖ6976 reduced injury-induced activation of conventional PKCα and PKCβ1 in CST neurons, promoted regeneration of CST axons through and beyond a cervical DH at C4, formed new synapses on target neurons caudal to the injury, and enhanced forelimb functional recovery in adult rats. When combined with lenti-Chondroitinase ABC treatment, cortical administration of GÖ6976 promoted even greater CST axonal regeneration and recovery of forelimb function. Thus, this study has demonstrated a novel strategy that can promote anatomical regeneration of damaged CST axons and partial recovery of forelimb function. Importantly, such an effect is critically dependent on the efficient blockage of injury-induced PKC activation in the soma of layer V CST neurons.

  15. Wnt5a Increases Properties of Lung Cancer Stem Cells and Resistance to Cisplatin through Activation of Wnt5a/PKC Signaling Pathway

    PubMed Central

    Yang, Jiali; Zhang, Kangjian; Wu, Jing; Shi, Juan; Xue, Jing; Li, Jing; Zhu, Yongzhao; Wei, Jun

    2016-01-01

    The development of chemoresistance to cisplatin regimens causes a poor prognosis in patients with advanced NSCLC. The role of noncanonical Wnt signaling in the regulation of properties of lung cancer stem cells and chemoresistance was interrogated, by accessing capacities of cell proliferation, migration, invasion, and clonogenicity as well as the apoptosis in A549 cell lines and cisplatin-resistant A549 cells treated with Wnt5a conditional medium or protein kinase C (PKC) inhibitor GF109203X. Results showed that the noncanonical Wnt signaling ligand, Wnt5a, could promote the proliferation, migration, invasion, and colony formation in A549 lung adenocarcinoma cells and cisplatin-resistant A549/DDP cells and increase the fraction of ALDH-positive cell in A549/DDP cells. An exposure of cells to Wnt5a led to a significant reduction of A549/DDP cell apoptosis but not A549 cells. An addition of GF109203X could both strikingly increase the baseline apoptosis and resensitize the Wnt5a-inhibited cell apoptosis. Interestingly, an inhibition of Wnt/PKC signaling pathway could reduce properties of lung cancer stem cells, promote cell apoptosis, and resensitize cisplatin-resistant cells to cisplatin via a caspase/AIF-dependent pathway. These data thus suggested that the Wnt5a could promote lung cancer cell mobility and cisplatin-resistance through a Wnt/PKC signaling pathway and a blockage of this signaling may be an alternative therapeutic strategy for NSCLC patients with resistance to chemotherapies. PMID:27895670

  16. Ferroptosis, a newly characterized form of cell death in Parkinson's disease that is regulated by PKC.

    PubMed

    Do Van, Bruce; Gouel, Flore; Jonneaux, Aurélie; Timmerman, Kelly; Gelé, Patrick; Pétrault, Maud; Bastide, Michèle; Laloux, Charlotte; Moreau, Caroline; Bordet, Régis; Devos, David; Devedjian, Jean-Christophe

    2016-10-01

    Parkinson's disease (PD) is a complex illness characterized by progressive dopaminergic neuronal loss. Several mechanisms associated with the iron-induced death of dopaminergic cells have been described. Ferroptosis is an iron-dependent, regulated cell death process that was recently described in cancer. Our present work show that ferroptosis is an important cell death pathway for dopaminergic neurons. Ferroptosis was characterized in Lund human mesencephalic cells and then confirmed ex vivo (in organotypic slice cultures) and in vivo (in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model). Some of the observed characteristics of ferroptosis differed from those reported previously. For example, ferroptosis may be initiated by PKCα activation, which then activates MEK in a RAS-independent manner. The present study is the first to emphasize the importance of ferroptosis dysregulation in PD. In neurodegenerative diseases like PD, iron chelators, Fer-1 derivatives and PKC inhibitors may be strong drug candidates to pharmacologically modulate the ferroptotic signaling cascade.

  17. PKC-epsilon activation is required for recognition memory in the rat.

    PubMed

    Zisopoulou, Styliani; Asimaki, Olga; Leondaritis, George; Vasilaki, Anna; Sakellaridis, Nikos; Pitsikas, Nikolaos; Mangoura, Dimitra

    2013-09-15

    Activation of PKCɛ, an abundant and developmentally regulated PKC isoform in the brain, has been implicated in memory throughout life and across species. Yet, direct evidence for a mechanistic role for PKCɛ in memory is still lacking. Hence, we sought to evaluate this in rats, using short-term treatments with two PKCɛ-selective peptides, the inhibitory ɛV1-2 and the activating ψɛRACK, and the novel object recognition task (NORT). Our results show that the PKCɛ-selective activator ψɛRACK, did not have a significant effect on recognition memory. In the short time frames used, however, inhibition of PKCɛ activation with the peptide inhibitor ɛV1-2 significantly impaired recognition memory. Moreover, when we addressed at the molecular level the immediate proximal signalling events of PKCɛ activation in acutely dissected rat hippocampi, we found that ψɛRACK increased in a time-dependent manner phosphorylation of MARCKS and activation of Src, Raf, and finally ERK1/2, whereas ɛV1-2 inhibited all basal activity of this pathway. Taken together, these findings present the first direct evidence that PKCɛ activation is an essential molecular component of recognition memory and point toward the use of systemically administered PKCɛ-regulating peptides as memory study tools and putative therapeutic agents.

  18. Novel, potent and selective inhibitors of protein kinase C show oral anti-inflammatory activity.

    PubMed

    Nixon, J S; Bishop, J; Bradshaw, D; Davis, P D; Hill, C H; Elliott, L H; Kumar, H; Lawton, G; Lewis, E J; Mulqueen, M

    1991-01-01

    Clarification of the precise role of protein kinase C (PKC) in cellular functional responses has been hampered by a lack of potent, selective inhibitors. The structural lead provided by staurosporine, a potent but non-selective protein kinase (PK) inhibitor, was used to derive a series of bis(indolyl)maleimides of which the most potent, Ro 31-8425 (I50: PKC = 8 nM) showed 350-fold selectivity for PKC over cAMP-dependent protein kinase. Ro 31-8425 antagonised cellular processes triggered by phorbol esters (potent, specific PKC activators) and inhibited the allogeneic mixed lymphocyte reaction, suggesting a role for PKC in T-cell activation. Methylation of the primary amine in Ro 31-8425 produced an analogue. Ro 31-8830 which, when administered orally, produced a dose-dependent inhibition of a phorbol ester-induced paw oedema in mice (minimum effective dose = 15 mg/kg). Ro 31-8830 also selectively inhibited the secondary inflammation in a developing adjuvant arthritis model in the rat. The results presented here suggest that these selective inhibitors of PKC may have therapeutic value in the treatment of T-cell-mediated autoimmune diseases.

  19. Protein Kinase C Inhibitors as Modulators of Vascular Function and Their Application in Vascular Disease

    PubMed Central

    Khalil, Raouf A.

    2013-01-01

    Blood pressure (BP) is regulated by multiple neuronal, hormonal, renal and vascular control mechanisms. Changes in signaling mechanisms in the endothelium, vascular smooth muscle (VSM) and extracellular matrix cause alterations in vascular tone and blood vessel remodeling and may lead to persistent increases in vascular resistance and hypertension (HTN). In VSM, activation of surface receptors by vasoconstrictor stimuli causes an increase in intracellular free Ca2+ concentration ([Ca2+]i), which forms a complex with calmodulin, activates myosin light chain (MLC) kinase and leads to MLC phosphorylation, actin-myosin interaction and VSM contraction. Vasoconstrictor agonists could also increase the production of diacylglycerol which activates protein kinase C (PKC). PKC is a family of Ca2+-dependent and Ca2+-independent isozymes that have different distributions in various blood vessels, and undergo translocation from the cytosol to the plasma membrane, cytoskeleton or the nucleus during cell activation. In VSM, PKC translocation to the cell surface may trigger a cascade of biochemical events leading to activation of mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK), a pathway that ultimately increases the myofilament force sensitivity to [Ca2+]i, and enhances actin-myosin interaction and VSM contraction. PKC translocation to the nucleus may induce transactivation of various genes and promote VSM growth and proliferation. PKC could also affect endothelium-derived relaxing and contracting factors as well as matrix metalloproteinases (MMPs) in the extracellular matrix further affecting vascular reactivity and remodeling. In addition to vasoactive factors, reactive oxygen species, inflammatory cytokines and other metabolic factors could affect PKC activity. Increased PKC expression and activity have been observed in vascular disease and in certain forms of experimental and human HTN. Targeting of vascular PKC using PKC inhibitors may function in concert with

  20. Oxidized low-density lipoprotein attenuated desmoglein 1 and desmocollin 2 expression via LOX-1/Ca(2+)/PKC-β signal in human umbilical vein endothelial cells.

    PubMed

    Li, Yuan-Bin; Zhang, Qing-Hai; Chen, Zhuang; He, Zhi-Jun; Yi, Guang-Hui

    Numerous studies have reported the presence of oxidized LDL (ox-LDL) and expression of its lectin-like receptor, LOX-1, have been shown in atherosclerotic regions. The present study aims to investigate the effects of ox-LDL on expression of desmoglein 1 (DSG1) and desmocollin 2 (DSC2) in endothelial cells, and to explore the role of LOX-1 mediated signal in the permeability injury associated with DSG1 and DSC2 disruption induced by oxidized lipoprotein. RT-PCR and Western blotting were applied to determine the mRNA and protein expression levels of DSG1 and DSC2 in human umbilical vein endothelial cells (HUVECs) respectively. Immunoreactivities of DSG1 and DSC2 were detected by laser scanning confocal microscope (LSCM). HUVEC monolayers permeability was evaluated by FITC-labeled LDL in transwell assay system. The possible signal was assessed using in vitro blocking LOX-1 or Ca(2+) channel or PKC. The DSG1 and DSC2 expression were decreased by ox-LDL in concentration- and time-dependent manner. The effects of ox-LDL were mediated by its endothelial receptor, LOX-1. In parallel experiments, ox-LDL increased the influx of extracellular calcium, activation of protein kinase C (PKC) and permeability to LDL, which was inhibited by the LOX-1blocking antibody (10 μg/ml), Ca(2+) channel blocker (Diltiazem, 50 μmol/L) and PKCinhibitor (hispidin, 4 μmol/L). These results suggested that ox-LDL-induced decrease in DSG1 and DSC2 expression and monolayer barrier injury via calcium uptake and PKC-β activation following up-regulation of LOX-1 is one of the mechanisms of inducing greater permeability in HUVECs.

  1. Modulation of transglutaminase 2 activity in H9c2 cells by PKC and PKA signalling: a role for transglutaminase 2 in cytoprotection

    PubMed Central

    Almami, Ibtesam; Dickenson, John M; Hargreaves, Alan J; Bonner, Philip L R

    2014-01-01

    BACKGROUND AND PURPOSE Tissue transglutaminase (TG2) has been shown to mediate cell survival in many cell types. In this study, we investigated whether the role of TG2 in cytoprotection was mediated by the activation of PKA and PKC in cardiomyocyte-like H9c2 cells. EXPERIMENTAL APPROACH H9c2 cells were extracted following stimulation with phorbol-12-myristate-13-acetate (PMA) and forskolin. Transglutaminase activity was determined using an amine incorporating and a protein crosslinking assay. The presence of TG isoforms (TG1, 2, 3) was determined using Western blot analysis. The role of TG2 in PMA- and forskolin-induced cytoprotection was investigated by monitoring H2O2-induced oxidative stress in H9c2 cells. KEY RESULTS Western blotting showed TG2 >> TG1 protein expression but no detectable TG3. The amine incorporating activity of TG2 in H9c2 cells increased in a time and concentration-dependent manner following stimulation with PMA and forskolin. PMA and forskolin-induced TG2 activity was blocked by PKC (Ro 31-8220) and PKA (KT 5720 and Rp-8-Cl-cAMPS) inhibitors respectively. The PMA- and forskolin-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Immunocytochemistry revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (β-tubulin) and novel (α-actinin) protein substrates for TG2. Pretreatment with PMA and forskolin reversed H2O2-induced decrease in MTT reduction and release of LDH. TG2 inhibitors R283 and Z-DON blocked PMA- and forskolin-induced cytoprotection. CONCLUSIONS AND IMPLICATIONS TG2 activity was stimulated via PKA- and PKC-dependent signalling pathways in H9c2 cells These results suggest a role for TG2 in cytoprotection induced by these kinases. PMID:24821315

  2. Salvianolic acid B protects against acetaminophen hepatotoxicity by inducing Nrf2 and phase II detoxification gene expression via activation of the PI3K and PKC signaling pathways.

    PubMed

    Lin, Musen; Zhai, Xiaohan; Wang, Guangzhi; Tian, Xiaofeng; Gao, Dongyan; Shi, Lei; Wu, Hang; Fan, Qing; Peng, Jinyong; Liu, Kexin; Yao, Jihong

    2015-02-01

    Acetaminophen (APAP) is used drugs worldwide for treating pain and fever. However, APAP overdose is the principal cause of acute liver failure in Western countries. Salvianolic acid B (SalB), a major water-soluble compound extracted from Radix Salvia miltiorrhiza, has well-known antioxidant and anti-inflammatory actions. We aimed to evaluate the ability of SalB to protect against APAP-induced acute hepatotoxicity by inducing nuclear factor-erythroid-2-related factor 2 (Nrf2) expression. SalB pretreatment ameliorated acute liver injury caused by APAP, as indicated by blood aspartate transaminase levels and histological findings. Moreover, SalB pretreatment increased the expression of Nrf2, Heme oxygenase-1 (HO-1) and glutamate-l-cysteine ligase catalytic subunit (GCLC). Furthermore, the HO-1 inhibitor zinc protoporphyrin and the GCLC inhibitor buthionine sulfoximine reversed the protective effect of SalB. Additionally, siRNA-mediated depletion of Nrf2 reduced the induction of HO-1 and GCLC by SalB, and SalB pretreatment activated the phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC) signaling pathways. Both inhibitors (PI3K and PKC) blocked the protective effect of SalB against APAP-induced cell death, abolishing the SalB-induced Nrf2 activation and decreasing HO-1 and GCLC expression. These results indicated that SalB induces Nrf2, HO-1 and GCLC expression via activation of the PI3K and PKC pathways, thereby protecting against APAP-induced liver injury.

  3. PKC{alpha} expression regulated by Elk-1 and MZF-1 in human HCC cells

    SciTech Connect

    Hsieh, Y.-H.; Wu, T.-T.; Tsai, J.-H.; Huang, C.-Y.; Hsieh, Y.-S.; Liu, J.-Y. . E-mail: jyl@csmu.edu.tw

    2006-01-06

    Our previous study found that PKC{alpha} was highly expressed in the poor-differentiated human HCC cells and associated with cell migration and invasion. In this study, we further investigated the gene regulation of this enzyme. We showed that PKC{alpha} expression enhancement in the poor-differentiated human HCC cells was found neither by DNA amplification nor by increasing mRNA stability using differential PCR and mRNA decay assays. After screening seven transcription factors in the putative cis-acting regulatory elements of human PKC{alpha} promoters, only Elk-1 and MZF-1 antisense oligonucleotide showed a significant reduction in the PKC{alpha} mRNA level. They also reduced cell proliferation, cell migratory and invasive capabilities, and DNA binding activities in the PKC{alpha} promoter region. Over-expression assay confirmed that the PKC{alpha} expression may be modulated by these two factors at the transcriptional level. Therefore, these results may provide a novel mechanism for PKC{alpha} expression regulation in human HCC cells.

  4. Phorbol ester stimulation of RasGRP1 regulates the sodium-chloride cotransporter by a PKC-independent pathway

    PubMed Central

    Ko, Benjamin; Joshi, Leena M.; Cooke, Leslie L.; Vazquez, Norma; Musch, Mark W.; Hebert, Steven C.; Gamba, Gerardo; Hoover, Robert S.

    2007-01-01

    The sodium-chloride cotransporter (NCC) is the principal salt-absorptive pathway in the mammalian distal convoluted tubule (DCT) and is the site of action of one of the most effective classes of antihypertensive medications, thiazide diuretics. We developed a cell model system to assess NCC function in a mammalian cell line that natively expresses NCC, the mouse DCT (mDCT) cell line. We used this system to study the complex regulation of NCC by the phorbol ester (PE) 12-O-tetradecanoylphorbol-13-acetate (TPA), a diacylglycerol (DAG) analog. It has generally been thought that PEs mediate their effects on transporters through the activation of PKC. However, there are at least five other DAG/PE targets. Here we describe how one of those alternate targets of DAG/PE effects, Ras guanyl-releasing protein 1 (RasGRP1), mediates the PE-induced suppression of function and the surface expression of NCC. Functional assessment of NCC by using thiazide-sensitive 22Na+ uptakes revealed that TPA completely suppresses NCC function. Biotinylation experiments demonstrated that this result was primarily because of decreased surface expression of NCC. Although inhibitors of PKC had no effect on this suppression, MAPK inhibitors completely prevented the TPA effect. RasGRP1 activates the MAPK pathway through activation of the small G protein Ras. Gene silencing of RasGRP1 prevented the PE-mediated suppression of NCC activity, the activation of the H-Ras isoform of Ras, and the activation of ERK1/2 MAPK. This finding confirmed the critical role of RasGRP1 in mediating the PE-induced suppression of NCC activity through the stimulation of the MAPK pathway. PMID:18077438

  5. Activation of PKC{beta}{sub II} and PKC{theta} is essential for LDL-induced cell proliferation of human aortic smooth muscle cells via Gi-mediated Erk1/2 activation and Egr-1 upregulation

    SciTech Connect

    Heo, Kyung-Sun; Kim, Dong-Uk; Kim, Lila; Nam, Miyoung; Baek, Seung-Tae; Park, Song-Kyu; Park, Youngwoo; Myung, Chang-Seon; Hwang, Sung-Ook Hoe, Kwang-Lae

    2008-03-28

    Native LDL may be a mitogenic stimulus of VSMC proliferation in lesions where endothelial disruption occurs. Recent studies have demonstrated that the mitogenic effects of LDL are accompanied by Erk1/2 activation via an unknown G-protein-coupled receptor (GPCR). In this article, we report that LDL translocated PKC{beta}{sub II} and PKC{theta} from cytosol to plasma membrane, and inhibition of PKC{beta}{sub II} and PKC{theta} decreased LDL effects via the deactivation of Erk1/2. Moreover, pertussis toxin, but not cholera toxin or heparin, inhibited LDL-induced translocation of PKC{beta}{sub II} and PKC{theta}, suggesting that Gi protein plays a role in LDL effects. Of LPA, S1P, and LDL, whose signaling is conveyed via Gi/o proteins, only LDL induced translocation of PKC{beta}{sub II} and PKC{theta}. Inhibition of PKC{beta}{sub II} or PKC{theta}, as well as of Erk1/2 and GPCR, decreases LDL-induced upregulation of Egr-1, which is critical for cell proliferation. This is the first report, to our knowledge, that the participation of PKC{theta} in VSMC proliferation is unique.

  6. Immunoreactivity of PKC gammalambda and RACK1 in baker's yeast, lobster and wheat germ.

    PubMed

    Kuo, W N; Jones, D L; Ku, T W; Weeks, K D; Jordon, P M; Dopson, N C

    1995-08-01

    Varied patterns of immunoreactive bands of protein kinase C gamma (PKC gamma) and receptor for activated C-kinase-1 (RACK1) were detected by analysis of Western blots in crude extracts of wheat germ, lobster tail meat, and three strains of baker's yeast. Anti-PKC lambda also reacted with wheat germ and yeast extracts, but failed to react with the lobster extract. The findings may implicate a regulatory role and an evolutionary conservation of these PKC isoenzymes and their receptor proteins in eukaryotes.

  7. Live-imaging of PKC translocation in Sf9 cells and in aplysia sensory neurons.

    PubMed

    Farah, Carole A; Sossin, Wayne S

    2011-04-06

    Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (α, βI, βII and γ), novel type I PKCs (ε and η), novel type II PKCs (δ and θ), and atypical PKCs (ζ and ι). The classical PKCs are activated by Ca(2+) and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca(2+)-independent. The atypical PKCs are activated by neither Ca(2+) nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation (1-2). Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail (3). In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article (4), expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using

  8. Long-term pioglitazone treatment augments insulin sensitivity and PKC-epsilon and PKC-theta activation in skeletal muscles in sucrose fed rats.

    PubMed

    Marková, I; Zídek, V; Musilová, A; Simáková, M; Mlejnek, P; Kazdová, L; Pravenec, M

    2010-01-01

    It has been suggested that thiazolidinediones (TZDs) ameliorate insulin resistance in muscle tissue by suppressing muscle lipid storage and the activity of novel protein kinase C (nPKC) isoforms. To test this hypothesis, we analyzed long-term metabolic effects of pioglitazone and the activation of nPKC-epsilon and -theta isoforms in an animal model of the metabolic syndrome, the spontaneously hypertensive rat (a congenic SHR strain with wild type Cd36 gene) fed a diet with 60 % sucrose from the age of 4 to 8 months. Compared to untreated controls, pioglitazone treatment was associated with significantly increased basal (809+/-36 vs 527+/-47 nmol glucose/g/2h, P<0.005) and insulin-stimulated glycogenesis (1321+/-62 vs 749+/-60 nmol glucose/g/2h, P<0.0001) in isolated gastrocnemius muscles despite increased concentrations of muscle triglycerides (3.83+/-0.33 vs 2.25+/-0.12 micromol/g, P<0.005). Pioglitazone-treated rats exhibited significantly increased membrane/total (cytosolic plus membrane) ratio of both PKC-epsilon and PKC-theta isoforms compared to untreated controls. These results suggest that amelioration of insulin resistance after long-term pioglitazone treatment is associated with increased activation of PKC-epsilon and -theta isoforms in spite of increased lipid concentration in skeletal muscles.

  9. Crosstalk between Wnt signaling and Phorbol ester-mediated PKC signaling in MCF-7 human breast cancer cells.

    PubMed

    Kim, Soyoung; Chun, So-Young; Kwon, Yun-Suk; Nam, Kyung-Soo

    2016-02-01

    Although many studies have implicated the crosstalk between the Wnt and PKC signaling pathways in tumor initiation and progression, the molecular roles of PKC isoforms in the Wnt signaling pathway remain poorly understood. In this study, we explored the contribution of PKC isoforms to canonical and noncanonical Wnt signaling pathway in mediating cell migration and an epithelial-mesenchymal transition (EMT). When MCF-7 cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for up to 3 weeks, the effect of TPA on Wnt signaling pathway was dramatically different depending on the exposure time. The short term exposure (3 days) of MCF-7 cells to TPA exhibited significant induction of Wnt5a expression, along with the enhanced expression of PKC-α, to promote cell migration, which suggested that activation of noncanonical Wnt signaling pathway is associated with PKC-α. However, the chronic exposure (3 weeks) of cells to TPA completely suppressed Wnt5a expression and the expression of PKC-η and PKC-δ, whereas the expression of Wnt3a and PKC-θ were up-regulated to activate the canonical Wnt signaling pathway. Moreover, the loss of epithelial markers, including E-cadherin and GATA-3, suggested that chronic exposure of TPA stimulates EMT. Taken together, our data suggest that PKC-θ positively regulates the canonical Wnt signaling pathway, and that PKC-η and PKC-δ negatively modulate this signaling pathway.

  10. Correction of metabolic abnormalities in a rodent model of obesity, metabolic syndrome, and type 2 diabetes mellitus by inhibitors of hepatic protein kinase C-ι.

    PubMed

    Sajan, Mini P; Nimal, Sonali; Mastorides, Stephen; Acevedo-Duncan, Mildred; Kahn, C Ronald; Fields, Alan P; Braun, Ursula; Leitges, Michael; Farese, Robert V

    2012-04-01

    Excessive activity of hepatic atypical protein kinase (aPKC) is proposed to play a critical role in mediating lipid and carbohydrate abnormalities in obesity, the metabolic syndrome, and type 2 diabetes mellitus. In previous studies of rodent models of obesity and type 2 diabetes mellitus, adenoviral-mediated expression of kinase-inactive aPKC rapidly reversed or markedly improved most if not all metabolic abnormalities. Here, we examined effects of 2 newly developed small-molecule PKC-ι/λ inhibitors. We used the mouse model of heterozygous muscle-specific knockout of PKC-λ, in which partial deficiency of muscle PKC-λ impairs glucose transport in muscle and thereby causes glucose intolerance and hyperinsulinemia, which, via hepatic aPKC activation, leads to abdominal obesity, hepatosteatosis, hypertriglyceridemia, and hypercholesterolemia. One inhibitor, 1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)methyl]cyclopentyl-[1R-(1a,2b,3b,4a)], binds to the substrate-binding site of PKC-λ/ι, but not other PKCs. The other inhibitor, aurothiomalate, binds to cysteine residues in the PB1-binding domains of aPKC-λ/ι/ζ and inhibits scaffolding. Treatment with either inhibitor for 7 days inhibited aPKC, but not Akt, in liver and concomitantly improved insulin signaling to Akt and aPKC in muscle and adipocytes. Moreover, both inhibitors diminished excessive expression of hepatic, aPKC-dependent lipogenic, proinflammatory, and gluconeogenic factors; and this was accompanied by reversal or marked improvements in hyperglycemia, hyperinsulinemia, abdominal obesity, hepatosteatosis, hypertriglyceridemia, and hypercholesterolemia. Our findings highlight the pathogenetic importance of insulin signaling to hepatic PKC-ι in obesity, the metabolic syndrome, and type 2 diabetes mellitus and suggest that 1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)methyl]cyclopentyl-[1R-(1a,2b,3b,4a)] and aurothiomalate or similar agents that

  11. Topical application of a protein kinase C inhibitor reduces skin and hair pigmentation.

    PubMed

    Park, Hee-Young; Lee, Jin; González, Salvador; Middelkamp-Hup, Maritza A; Kapasi, Sameer; Peterson, Shaun; Gilchrest, Barbara A

    2004-01-01

    To determine whether inhibition of PKC-beta activity decreases pigmentation, paired cultures of primary human melanocytes were first pretreated with bisindolylmaleimide (Bis), a selective PKC inhibitor, or vehicle alone for 30 min, and then treated with TPA for an additional 90 min to activate PKC in the presence of Bis. Bis blocked the expected induction of tyrosinase activity by activation of PKC. Addition of a peptide corresponding to amino acids 501-511 of tyrosinase containing its PKC-beta phosphorylation site, a presumptive PKC-beta pseudosubstrate, gave similar results. To determine whether Bis reduces pigmentation in vivo, the backs of four shaved and depilated pigmented guinea pigs were UV irradiated with a solar simulator for 2 wk excluding weekends. Compared to vehicle alone, Bis (300 microM), applied twice daily to paired sites for various periods encompassing the irradiation period, decreased tanning. Bis also, although less strikingly, reduced basal epidermal melanin when topically applied twice daily, 5 d per wk, for 3 wk to shaved and depilated unirradiated skin. Moreover, topical application of Bis (100 microM) once daily for 9 d to the freshly depilated backs of 8-wk-old mice markedly lightened the color of regrowing hair. These results demonstrate that inhibiting PKC activity in vivo selectively blocks tanning and reduces basal pigmentation in the epidermis and in anagen hair shafts.

  12. Short-term Mg deficiency upregulates protein kinase C isoforms in cardiovascular tissues and cells; relation to NF-kB, cytokines, ceramide salvage sphingolipid pathway and PKC-zeta: hypothesis and review.

    PubMed

    Altura, Burton M; Shah, Nilank C; Shah, Gatha J; Zhang, Aimin; Li, Wenyan; Zheng, Tao; Perez-Albela, Jose Luis; Altura, Bella T

    2014-01-01

    Numerous recent,epidemiological studies reveal that Western populations are growing more and more deficient in daily Mg intake which have been linked to etiology of cardiovascular (CV) diseases. A growing body of evidence suggests that a major missing link to this dilemma may reside within the sphingolipid-ceramide pathways. For the past 25 years , our labs have been focusing on these pathways in Mg-deficient mammals. The objective of this paper is two-fold: 1) to test various hypotheses and 2) to review the current status of the field and how protein kinase C isoforms may be pivotal to solving some of the CV attributes of Mg deficiency. Below, we test the hypotheses that: 1) short-term dietary deficiency of magnesium (MgD) would result in the upregulation of protein kinase C (PKC) isoforms in left ventricular (LV) and aortic smooth muscle (ASM) and serum; 2) MgD would result in a release of select cytokines and an upregulation of NF-kB in LV and ASM, and in primary cultured aortic smooth muscle cells (PCASMC); 3) MgD would result in an activation of the sphingolipid salvage pathway in LV and ASM, and in PCASMC; 4) MgD would result in a synthesis of sphingosine, but not sphinganine, in PCASMC which could be inhibited by fumonisin B1 (FB) an inhibitor of ceramide synthase (CS), but not scyphostatin an inhibitor of neutral sphingomyelinase (N-SMase); 5) incubation of PCASMC (in low Mg(2+)) with the PKC-mimic PMA would result in release and synthesis of NF-kB, cytokines, and ceramide but not sphingosine. The new data indicate that short-term MgD (10% normal dietary intake) result in an upregulation of all three classes of PKC isoforms in LV, aortic muscle and in serum coupled to the upregulation of ceramide, NF-kB activation, and cytokines. High degrees of linear correlation were found to exist between upregulation of PKC isoforms, p65 and cytokine release, suggesting cross-talk between these molecules and molecular pathways. Our experiments with PCASMCs demonstrated

  13. Isoform-specific dynamic translocation of PKC by α1-adrenoceptor stimulation in live cells

    PubMed Central

    O-Uchi, Jin; Sorenson, Jaime; Jhun, Bong Sook; Mishra, Jyotsna; Hurst, Stephen; Williams, Kaleef; Sheu, Shey-Shing; Lopes, Coeli M.B.

    2015-01-01

    Protein kinase C (PKC) plays key roles in the regulation of signal transduction and cellular function in various cell types. At least ten PKC isoforms have been identified and intracellular localization and trafficking of these individual isoforms are important for regulation of enzyme activity and substrate specificity. PKC can be activated at downstream of Gq-protein coupled receptor (GqPCR) signaling and translocated to the various cellular compartments including plasma membrane (PM). Recent reports suggested that a different type of GqPCRs would activate different PKC isoforms (classic, novel and atypical PKCs) with different trafficking patterns. However, the knowledge of isoform-specific activation of PKC by each GqPCR is limited. α1-Adrenoceptor (α1-AR) is the one of the GqPCR highly expressed in the cardiovascular system. In this study, we examined the isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-AR stimulation (α1-ARS). Rat PKCα, βI, βII, δ, ε and ζ fused with GFP at C-term were co-transfected with human α1A-AR into HEK293T cells. The isoform-specific dynamic translocation of PKC in living HEK293T cells by α1-ARS using phenylephrine was measured by confocal microscopy. Before stimulation, GFP-PKCs were localized at cytosolic region. α1-ARS strongly and rapidly translocated a classical PKC (cPKC), PKCα, (< 30s) to PM, with PKCα returning diffusively into the cytosol within 5 min. α1-ARS rapidly translocated other cPKCs, PKCβI and PKCβII, to the PM (<30s), with sustained membrane localization. One of novel PKCs (nPKCs), PKCε, but not another nPKC, PKCδ, was translocated by α1-AR stimulation to the PM (<30s) and its membrane localization was also sustained. Finally, α1-AR stimulation did not cause a diacylglycerol-insensitive atypical PKC, PKCζ translocation. Our data suggest that PKCα, β and ε activation may underlie physiological and pathophysiological responses of α1-AR signaling for the

  14. Ellagic acid inhibits PKC signaling by improving antioxidant defense system in murine T cell lymphoma.

    PubMed

    Mishra, Sudha; Vinayak, Manjula

    2014-07-01

    Antioxidants protect the cells from the damaging effects of reactive oxygen species (ROS). Production of ROS during cellular metabolism is balanced by their removal by antioxidants. Any condition leading to increased levels of ROS results in oxidative stress, which participates in multistage carcinogenesis by causing oxidative DNA damage, mutations in the proto-oncogenes and tumor suppressor genes. Antioxidant defense system is required to overcome the process of carcinogenesis generated by ROS. Antioxidant enzymes are major contributors to endogenous antioxidant defense system. Protein kinase C (PKC) is generally involved in cell proliferation and its over expression leads to abnormal tumor growth. Out of three classes of PKC, classical PKC is mainly involved in cell proliferation and tumor growth. Classical PKC initiates signaling pathway and leads to activation of a number of downstream protein via activation of NF-κB. Therefore any agent which can promotes the endogenous antioxidant defense system should be able to down regulate PKC and NF-κB activation and thus may be useful in reducing cancer progression. To investigate this hypothesis we have tested the effect of antioxidant ellagic acid on antioxidant enzymes and PKC signaling in Dalton's lymphoma bearing (DL) mice. DL mice were treated with three different doses of ellagic acid. The treatment significantly increases the activity and expression of antioxidant enzymes and down regulates the expression of classical isozymes of PKC as well as the activation of NF-κB, indicating that ellagic acid improves antioxidant defense system and PKC signaling via NF-κB which may contribute to its cancer preventive role.

  15. PKC{eta} is a negative regulator of AKT inhibiting the IGF-I induced proliferation

    SciTech Connect

    Shahaf, Galit; Rotem-Dai, Noa; Koifman, Gabriela; Raveh-Amit, Hadas; Frost, Sigal A.; Livneh, Etta

    2012-04-15

    The PI3K-AKT pathway is frequently activated in human cancers, including breast cancer, and its activation appears to be critical for tumor maintenance. Some malignant cells are dependent on activated AKT for their survival; tumors exhibiting elevated AKT activity show sensitivity to its inhibition, providing an Achilles heel for their treatment. Here we show that the PKC{eta} isoform is a negative regulator of the AKT signaling pathway. The IGF-I induced phosphorylation on Ser473 of AKT was inhibited by the PKC{eta}-induced expression in MCF-7 breast adenocarcinoma cancer cells. This was further confirmed in shRNA PKC{eta}-knocked-down MCF-7 cells, demonstrating elevated phosphorylation on AKT Ser473. While PKC{eta} exhibited negative regulation on AKT phosphorylation it did not alter the IGF-I induced ERK phosphorylation. However, it enhanced ERK phosphorylation when stimulated by PDGF. Moreover, its effects on IGF-I/AKT and PDGF/ERK pathways were in correlation with cell proliferation. We further show that both PKC{eta} and IGF-I confer protection against UV-induced apoptosis and cell death having additive effects. Although the protective effect of IGF-I involved activation of AKT, it was not affected by PKC{eta} expression, suggesting that PKC{eta} acts through a different route to increase cell survival. Hence, our studies show that PKC{eta} provides negative control on AKT pathway leading to reduced cell proliferation, and further suggest that its presence/absence in breast cancer cells will affect cell death, which could be of therapeutic value.

  16. ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells

    PubMed Central

    Díaz-Vegas, Alexis; Campos, Cristian A.; Contreras-Ferrat, Ariel; Casas, Mariana; Buvinic, Sonja; Jaimovich, Enrique; Espinosa, Alejandra

    2015-01-01

    During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y1 receptor agonist, induced a large signal while UTPyS (P2Y2 agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y1. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC. PMID:26053483

  17. ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells.

    PubMed

    Díaz-Vegas, Alexis; Campos, Cristian A; Contreras-Ferrat, Ariel; Casas, Mariana; Buvinic, Sonja; Jaimovich, Enrique; Espinosa, Alejandra

    2015-01-01

    During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y1 receptor agonist, induced a large signal while UTPyS (P2Y2 agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y1. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.

  18. DNA damage targets PKC{eta} to the nuclear membrane via its C1b domain

    SciTech Connect

    Tamarkin, Ana; Zurgil, Udi; Braiman, Alex; Hai, Naama; Krasnitsky, Ella; Maissel, Adva; Ben-Ari, Assaf; Yankelovich, Liat; Livneh, Etta

    2011-06-10

    Translocation to cellular membranes is one of the hallmarks of PKC activation, occurring as a result of the generation of lipid secondary messengers in target membrane compartments. The activation-induced translocation of PKCs and binding to membranes is largely directed by their regulatory domains. We have previously reported that PKC{eta}, a member of the novel subfamily and an epithelial specific isoform, is localized at the cytoplasm and ER/Golgi and is translocated to the plasma membrane and the nuclear envelope upon short-term activation by PMA. Here we show that PKC{eta} is shuttling between the cytoplasm and the nucleus and that upon etoposide induced DNA damage is tethered at the nuclear envelope. Although PKC{eta} expression and its phosphorylation on the hydrophobic motif (Ser675) are increased by etoposide, this phosphorylation is not required for its accumulation at the nuclear envelope. Moreover, we demonstrate that the C1b domain is sufficient for translocation to the nuclear envelope. We further show that, similar to full-length PKC{eta}, the C1b domain could also confer protection against etoposide-induced cell death. Our studies demonstrate translocation of PKC{eta} to the nuclear envelope, and suggest that its spatial regulation could be important for its cellular functions including effects on cell death.

  19. Synapse formation changes the rules for desensitization of PKC translocation in Aplysia.

    PubMed

    Farah, Carole A; Naqib, Faisal; Weatherill, Daniel B; Pack, Christopher C; Sossin, Wayne S

    2015-02-01

    Protein kinase Cs (PKCs) are activated by translocating from the cytoplasm to the membrane. We have previously shown that serotonin-mediated translocation of PKC to the plasma membrane in Aplysia sensory neurons was subject to desensitization, a decrease in the ability of serotonin to induce translocation after previous application of serotonin. In Aplysia, changes in the strength of the sensory-motor neuron synapse are important for behavioral sensitization and PKC regulates a number of important aspects of this form of synaptic plasticity. We have previously suggested that the desensitization of PKC translocation in Aplysia sensory neurons may partially explain the differences between spaced and massed training, as spaced applications of serotonin, a cellular analog of spaced training, cause greater desensitization of PKC translocation than one massed application of serotonin, a cellular analog of massed training. Our previous studies were performed in isolated sensory neurons. In the present study, we monitored translocation of fluorescently-tagged PKC to the plasma membrane in living sensory neurons that were co-cultured with motor neurons to allow for synapse formation. We show that desensitization now becomes similar during spaced and massed applications of serotonin. We had previously modeled the signaling pathways that govern desensitization in isolated sensory neurons. We now modify this mathematical model to account for the changes observed in desensitization dynamics following synapse formation. Our study shows that synapse formation leads to significant changes in the molecular signaling networks that underlie desensitization of PKC translocation.

  20. The roles of maternal Vangl2 and aPKC in Xenopus oocyte and embryo patterning

    PubMed Central

    Cha, Sang-Wook; Tadjuidje, Emmanuel; Wylie, Christopher; Heasman, Janet

    2011-01-01

    The Xenopus oocyte contains components of both the planar cell polarity and apical-basal polarity pathways, but their roles are not known. Here, we examine the distribution, interactions and functions of the maternal planar cell polarity core protein Vangl2 and the apical-basal complex component aPKC. We show that Vangl2 is distributed in animally enriched islands in the subcortical cytoplasm in full-grown oocytes, where it interacts with a post-Golgi v-SNARE protein, VAMP1, and acetylated microtubules. We find that Vangl2 is required for the stability of VAMP1 as well as for the maintenance of the stable microtubule architecture of the oocyte. We show that Vangl2 interacts with atypical PKC, and that both the acetylated microtubule cytoskeleton and the Vangl2-VAMP1 distribution are dependent on the presence of aPKC. We also demonstrate that aPKC and Vangl2 are required for the cell membrane asymmetry that is established during oocyte maturation, and for the asymmetrical distribution of maternal transcripts for the germ layer and dorsal/ventral determinants VegT and Wnt11. This study demonstrates the interaction and interdependence of Vangl2, VAMP1, aPKC and the stable microtubule cytoskeleton in the oocyte, shows that maternal Vangl2 and aPKC are required for specific oocyte asymmetries and vertebrate embryonic patterning, and points to the usefulness of the oocyte as a model to study the polarity problem. PMID:21813572

  1. Different Associations of CD45 Isoforms with STAT3, PKC and ERK Regulate IL-6-Induced Proliferation in Myeloma

    PubMed Central

    Zheng, Xu; Li, Allison S.; Zheng, Huanyu; Zhao, Dongmei; Guan, Dagang; Zou, Huawei

    2015-01-01

    In response to interleukin 6 (IL-6) stimulation, both CD45RO and CD45RB, but not CD45RA, translocate to lipid rafts. However, the significance of this distinct translocation and the downstream signals in CD45 isoforms-participated IL-6 signal are not well understood. Using sucrose fractionation, we found that phosphorylated signal transducer and activator of transcription (STAT)3 and STAT1 were mainly localized in lipid rafts in response to IL-6 stimulation, despite both STAT3 and STAT1 localizing in raft and non-raft fractions in the presence or absence of IL-6. On the other hand, extracellular signal-regulated kinase (ERK), and phosphorylated ERK were localized in non-raft fractions regardless of the existence of IL-6. The rafts inhibitor significantly impeded the phosphorylation of STAT3 and STAT1 and nuclear translocation, but had little effect on (and only postponing) the phosphorylation of ERK. This data suggests that lipid raft-dependent STAT3 and STAT1 pathways are dominant pathways of IL-6 signal in myeloma cells. Interestingly, the phosphorylation level of STAT3 but not STAT1 in CD45+ cells was significantly higher compared to that of CD45- cells, while the phosphorylation level of ERK in CD45+ myeloma cells was relatively low. Furthermore, exogenously expressed CD45RO/RB significantly enhanced STAT3, protein kinase C (PKC) and downstream NF-κB activation; however, CD45RA/RB inhibited IL-6-induced ERK phosphorylation. CD45 also enhanced the nuclear localization of STAT3 but not that of STAT1. In response to IL-6 stimulation, CD45RO moved into raft compartments and formed a complex with STAT3 and PKC in raft fraction, while CD45RA remained outside of lipid rafts and formed a complex with ERK in non-raft fraction. This data suggests a different role of CD45 isoforms in IL-6-induced signaling, indicating that while CD45RA/RB seems inhibit the rafts-unrelated ERK pathway, CD45RO/RB may actually work to enhance the rafts-related STAT3 and PKC/NF-κB pathways

  2. PKC-mediated modulation of L-type calcium channels may contribute to fat-induced insulin resistance.

    PubMed

    McCarty, Mark F

    2006-01-01

    Increased intracellular free calcium [Ca2+]i has been noted in adipocytes, platelets, and leukocytes of subjects with insulin resistance syndrome or allied disorders. In rodent studies, measures which increase [Ca2+]i in adipocytes and skeletal muscle are associated with impaired insulin signaling, attributable at least in part to diminished ability of insulin to activate phosphoserine phosphatase-1 (PP-1). In fat-fed insulin resistant rats, pre-treatment with a drug that selectively chelates intracellular calcium eliminates about half of the decrement in insulin-stimulated glucose uptake induced by fat feeding; since this chelator does not influence the insulin sensitivity of chow-fed rats, it is reasonable to suspect that fat feeding boosts [Ca2+]i in skeletal muscle, and that this effect is partially responsible for the associated reduction in insulin sensitivity. Clinical insulin resistance is associated with increased levels of triglycerides and other fatty acid metabolites in muscle fibers; this can give rise to diacylglycerol-mediated activation of PKC, which in turn compromises insulin signaling by triggering kinase cascades that phosphorylate IRS-1 on key serine residues. Yet there is also evidence that, in skeletal muscle, PKC activity up-regulates the function of L-type calcium channels, increasing their maximal conductance while left-shifting their voltage dependence. Thus, the PKC activation associated with fat overexposure might be expected to boost basal [Ca2+]i in skeletal muscle, potentially impeding insulin-mediated activation of PP-1. This hypothesis is consistent with several clinical studies demonstrating that long-acting inhibitors of L-type calcium channels can improve insulin sensitivity in overweight hypertensives; it should be readily testable in rodent models of fat-induced insulin resistance. Since parathyroid hormone can act on adipocytes and muscle to boost [Ca2+]i, mild secondary hyperparathyroidism associated with low calcium intakes

  3. Plasma inhibitory activity (PIA): a pharmacodynamic assay reveals insights into the basis for cytotoxic response to FLT3 inhibitors

    PubMed Central

    Levis, Mark; Brown, Patrick; Smith, B. Douglas; Stine, Adam; Pham, Rosalyn; Stone, Richard; DeAngelo, Daniel; Galinsky, Ilene; Giles, Frank; Estey, Elihu; Kantarjian, Hagop; Cohen, Pamela; Wang, Yanfeng; Roesel, Johannes; Karp, Judith E.; Small, Donald

    2006-01-01

    We have developed a useful surrogate assay for monitoring the efficacy of FLT3 inhibition in patients treated with oral FLT3 inhibitors. The plasma inhibitory activity (PIA) for FLT3 correlates with clinical activity in patients treated with CEP-701 and PKC412. Using the PIA assay, along with in vitro phosphorylation and cytotoxicity assays in leukemia cells, we compared PKC412 and its metabolite, CGP52421, with CEP-701. While both drugs could effectively inhibit FLT3 in vitro, CEP-701 was more cytotoxic to primary samples at comparable levels of FLT3 inhibition. PKC412 appears to be more selective than CEP-701 and therefore less effective at inducing cytotoxicity in primary acute myeloid leukemia (AML) samples in vitro. However, the PKC412 metabolite CGP52421 is less selective than its parent compound, PKC412, and is more cytotoxic against primary blast samples at comparable levels of FLT3 inhibition. The plasma inhibitory activity assay represents a useful correlative tool in the development of small-molecule inhibitors. Our application of this assay has revealed that the metabolite CGP52421 may contribute a significant portion of the antileukemia activity observed in patients receiving oral PKC412. Additionally, our results suggest that nonselectivity may constitute an important component of the cytotoxic effect of FLT3 inhibitors in FLT3-mutant AML. PMID:16857987

  4. Corticotropin-releasing hormone stimulates mitotic kinesin-like protein 1 expression via a PLC/PKC-dependent signaling pathway in hippocampal neurons.

    PubMed

    Sheng, Hui; Xu, Yongjun; Chen, Yanming; Zhang, Yanmin; Ni, Xin

    2012-10-15

    Corticotropin-releasing hormone (CRH) has been shown to modulate dendritic development in hippocampus. Mitotic kinesin-like protein 1 (MKLP1) plays key roles in dendritic differentiation. In the present study, we examined the effects of CRH on MKLP1 expression in cultured hippocampal neurons and determine subsequent signaling pathways involved. CRH dose-dependently increased MKLP1 mRNA and protein expression. This effect can be reversed by CRHR1 antagonist but not by CRHR2 antagonist. CRHR1 knockdown impaired this effect of CRH. CRH stimulated GTP-bound Gαs protein and phosphorylated phospholipase C (PLC)-β3 expression, which were blocked by CRHR1 antagonist. Transfection of GP antagonist-2A, an inhibitory peptide of Gαq protein, blocked CRH-induced phosphorylated PLC-β3 expression. PLC and PKC inhibitors completely blocked whereas adenylyl cyclase (AC) and PKA inhibitors did not affect CRH-induced MKLP1 expression. Our results indicate that CRH act on CRHR1 to induce MKLP1 expression via PLC/PKC signaling pathway. CRH may regulate MKLP1 expression, thereby modulating dendritic development.

  5. Enantioselective Synthesis of a PKC Inhibitor via Catalytic C-HBond Activation

    SciTech Connect

    Wilson, Rebecca M.; Thalji, Reema K.; Bergman, Robert G.; Ellman,Jonathan A.

    2006-02-26

    The syntheses of two biologically active molecules possessing dihydropyrroloindole cores (1 and 2) were completed using rhodium-catalyzed imine-directed C-H bond functionalization, with the second of these molecules containing a stereocenter that can be set with 90% ee during cyclization using chiral nonracemic phosphoramidite ligands. Catalytic decarbonylation and direct indole/maleimide coupling provide efficient access to 2.

  6. High-molecular weight hyaluronan reduced renal PKC activation in genetically diabetic mice.

    PubMed

    Campo, Giuseppe M; Avenoso, Angela; Micali, Antonio; Nastasi, Giancarlo; Squadrito, Francesco; Altavilla, Domenica; Bitto, Alessandra; Polito, Francesca; Rinaldi, Maria Grazia; Calatroni, Alberto; D'Ascola, Angela; Campo, Salvatore

    2010-11-01

    The cluster determinant (CD44) seems to play a key role in tissues injured by diabetes type 2. CD44 stimulation activates the protein kinase C (PKC) family which in turn activates the transcriptional nuclear factor kappa B (NF-κB) responsible for the expression of the inflammation mediators such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-18 (IL-18), inducible nitric oxide synthase (iNOS), and matrix metalloproteinases (MMPs). Regulation of CD44 interaction with its ligands depends greatly upon PKC. We investigated the effect of the treatment with high-molecular weight hyaluronan (HA) on diabetic nephropathy in genetically diabetic mice. BKS.Cg-m+/+Lepr(db) mice had elevated plasma insulin from 15 days of age and high blood sugar levels at 4 weeks. The severe nephropathy that developed was characterized by a marked increased in CD44 receptors, protein kinase C betaI, betaII, and epsilon (PKC(βI), PKC(βII), and PKCε) mRNA expression and the related protein products in kidney tissue. High levels of mRNA and related protein levels were also detected in the damaged kidney for NF-κB, TNF-α, IL-6, IL-18, MMP-7, and iNOS. Chronic daily administration of high-molecular mass HA for 2 weeks significantly reduced CD44, PKC(βI), PKC(βII), and PKCα gene expression and the related protein production in kidney tissue and TNF-α, IL-6, IL-18, MMP-7, and iNOS expression and levels also decreased. Histological analysis confirmed the biochemical data. However, blood parameters of diabetes were unchanged. These results suggest that the CD44 and PKC play an important role in diabetes and interaction of high-molecular weight HA with these proteins may reduce inflammation and secondary pathologies due to this disease.

  7. Bisindolylmaleimide protein-kinase-C inhibitors delay the decline in DNA synthesis in mouse hair follicle organ cultures.

    PubMed

    Harmon, C S; Nevins, T D; Ducote, J; Lutz, D

    1997-01-01

    We have used a series of bisindolylmaleimide selective protein-kinase C (PKC) inhibitors to investigate the role of this enzyme in the regulation of cell proliferation in mouse hair follicle organ cultures. Mouse whisker follicles were isolated by microdissection, and rates of DNA synthesis during culture were determined from 3H-thymidine incorporation. The bisindolylmaleimides Ro 31-7549, Ro 31-8161, Ro 31-8425 and Ro 31-8830 inhibit isolated brain PKC with IC50 values of 8-80 nM, are > 60-fold less potent against protein kinase A, and inhibit PKC-mediated protein phosphorylation in platelets with IC50 values in the range 0.25-4.4 microM. These PKC inhibitors were found to increase levels of mouse hair follicle DNA synthesis, with EC50 values in the range 1-4 microM and maximal levels in the range 151-197% of control. Ro 31-7549 had an IC50 value 50-fold lower than that of minoxidil, while the maximal level of DNA synthesis for the PKC inhibitor was 86% higher. Incubation of mouse hair follicles with Ro 31-7549 resulted in a delay of approximately 24 h in the onset of decline in follicular DNA synthesis rates. Ro 31-6045 and Ro 31-7208, bisindolylmaleimides without activity in the platelet PKC assay, did not affect mouse hair follicle DNA synthesis rates. Taken together, these findings show that PKC mediates, at least in part, the rapid loss of proliferative activity that occurs in mouse whisker follicles in culture, and provide further evidence that PKC plays a role as a negative proliferative signal in hair follicles.

  8. Upregulation of cystathionine β-synthetase in the arcuate nucleus produces pain hypersensitivity via PKC upregulation and GluN2B phosphorylation in rats with chronic pancreatitis.

    PubMed

    Zheng, Hang; Zhu, Hong-Yan; Zhang, Xiao-Yu; Wang, Meng; Xiao, Ying; Xu, Guang-Yin; Jiang, Xing-Hong

    2016-10-25

    Hydrogen sulfide (H2S) contributes to visceral hyperalgesia in primary sensory neurons, but its role in central nervous system remains largely unknown. This study was to investigate the roles and underlying mechanisms of H2S and its endogenous synthesis enzymes in the arcuate nucleus (ARC) in rat pancreatic hyperalgesia. Chronic pancreatitis (CP) was induced in male adult Sprague-Dawley rats by intra-pancreatic ductal injection of trinitrobenzene sulfonic acid (TNBS). Abdominal hyperalgesia was assessed by referred somatic behaviors to mechanical stimulation of rat abdomen. Western blot analysis was performed to detect protein expression in the ARC. CP markedly upregulated cystathionine β-synthetase (CBS) expression but did not alter cystathionine-γ-lyase level in the ARC at 4 weeks after TNBS injection. Although the expression of total GluN2B was not altered, CP greatly enhanced the phosphorylation level of GluN2B in the ARC when compared with age- and sex-matched control rats. CP also significantly increased expression of protein kinase Cγ (PKCγ) in the ARC. Arcuate microinjection of O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA, an inhibitor of CBS) significantly attenuated abdominal pain in CP rats in a dose-dependent manner and reversed the CP-induced upregulation of p-GluN2B and PKCγ in the ARC. Furthermore, the GluN2B inhibitor or specific PKC inhibitor chelerythrine significantly attenuated abdominal hyperalgesia in CP rats. The p-GluN2B expression was also suppressed by PKC inhibitor. Taken together, our results suggest that the upregulation of CBS in the ARC leads to an activation of GluN2B via PKCγ, which may play an important role in generation of pain hypersensitivity of CP.

  9. The structure of a dual-specificity tyrosine phosphorylation-regulated kinase 1A-PKC412 complex reveals disulfide-bridge formation with the anomalous catalytic loop HRD(HCD) cysteine.

    PubMed

    Alexeeva, Marina; Åberg, Espen; Engh, Richard A; Rothweiler, Ulli

    2015-05-01

    Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a protein kinase associated with neuronal development and brain physiology. The DYRK kinases are very unusual with respect to the sequence of the catalytic loop, in which the otherwise highly conserved arginine of the HRD motif is replaced by a cysteine. This replacement, along with the proximity of a potential disulfide-bridge partner from the activation segment, implies a potential for redox control of DYRK family activities. Here, the crystal structure of DYRK1A bound to PKC412 is reported, showing the formation of the disulfide bridge and associated conformational changes of the activation loop. The DYRK kinases represent emerging drug targets for several neurological diseases as well as cancer. The observation of distinct activation states may impact strategies for drug targeting. In addition, the characterization of PKC412 binding offers new insights for DYRK inhibitor discovery.

  10. PKC-Theta in Regulatory and Effector T-cell Functions

    PubMed Central

    Brezar, Vedran; Tu, Wen Juan; Seddiki, Nabila

    2015-01-01

    One of the major goals in immunology research is to understand the regulatory mechanisms that underpin the rapid switch on/off of robust and efficient effector (Teffs) or regulatory (Tregs) T-cell responses. Understanding the molecular mechanisms underlying the regulation of such responses is critical for the development of effective therapies. T-cell activation involves the engagement of T-cell receptor and co-stimulatory signals, but the subsequent recruitment of serine/threonine-specific protein Kinase C-theta (PKC-θ) to the immunological synapse (IS) is instrumental for the formation of signaling complexes, which ultimately lead to a transcriptional network in T cells. Recent studies demonstrated that major differences between Teffs and Tregs occurred at the IS where its formation induces altered signaling pathways in Tregs. These pathways are characterized by reduced recruitment of PKC-θ, suggesting that PKC-θ inhibits Tregs suppressive function in a negative feedback loop. As the balance of Teffs and Tregs has been shown to be central in several diseases, it was not surprising that some studies revealed that PKC-θ plays a major role in the regulation of this balance. This review will examine recent knowledge on the role of PKC-θ in T-cell transcriptional responses and how this protein can impact on the function of both Tregs and Teffs. PMID:26528291

  11. Role of PKC isozymes in low-power light-stimulated proliferation of cultured skin cells

    NASA Astrophysics Data System (ADS)

    Grossman, Nili; Kleitman, Vered; Meller, Julia; Kaufmann, Roland; Akgun, Nermin; Ruck, Angelika; Livneh, Etta; Lubart, Rachel

    2000-11-01

    Exposure of cultured skin cells to low power visible light leads to a transiently stimulated proliferation. Facilitation of this response requires the presence of active PKC, elevation of intracellular calcium, and involves reactive oxygen species. In the present study, the role of PKC(alpha) and PCK(eta) was examined using paired murine fibroblasts, differing in the level of these isozymes expression. The ability of the cells to respond to low power UVA light or HeNe laser by stimulated proliferation was correlated with an active state or overexpression of PKC(alpha) , but not PKC(eta) . A parallel response was obtained in cells that were loaded with A1PcS4 before photosensitization. Whenever this latter treatment caused a light-stimulated inhibition, it was accompanied by the intracellular calcium and photosensitizer dynamics typical of the effect of PDT on rate epithelial cells. Accordingly, added antioxidants that suppressed light-stimulated proliferation also suppressed this light-stimulated inhibition. The model systems employed in this study are the first to demonstrate the specific effect of PKC isozymes on light-stimulated proliferation, in relation to oxidative stress, and indicate their dual role in light-tissue interaction.

  12. Increased IGFBP-1 phosphorylation in response to leucine deprivation is mediated by CK2 and PKC.

    PubMed

    Malkani, Niyati; Biggar, Kyle; Shehab, Majida Abu; Li, Shawn Shun-Cheng; Jansson, Thomas; Gupta, Madhulika B

    2016-04-15

    Insulin-like growth factor binding protein-1 (IGFBP-1), secreted by fetal liver, is a key regulator of IGF-I bioavailability and fetal growth. IGFBP-1 phosphorylation decreases IGF-I bioavailability and diminishes its growth-promoting effects. Growth-restricted fetuses have decreased levels of circulating essential amino acids. We recently showed that IGFBP-1 hyperphosphorylation (pSer101/119/169) in response to leucine deprivation is regulated via activation of the amino acid response (AAR) in HepG2 cells. Here we investigated nutrient-sensitive protein kinases CK2/PKC/PKA in mediating IGFBP-1 phosphorylation in leucine deprivation. We demonstrated that leucine deprivation stimulated CK2 activity (enzymatic assay) and induced IGFBP-1 phosphorylation (immunoblotting/MRM-MS). Inhibition (pharmacological/siRNA) of CK2/PKC, but not PKA, prevented IGFBP-1 hyperphosphorylation in leucine deprivation. PKC inhibition also prevented leucine deprivation-stimulated CK2 activity. Functionally, leucine deprivation decreased IGF-I-induced-IGF-1R autophosphorylation when CK2/PKC were not inhibited. Our data strongly support that PKC promotes leucine deprivation-induced IGFBP-1 hyperphosphorylation via CK2 activation, mechanistically linking decreased amino acid availability and reduced fetal growth.

  13. Slmb antagonises the aPKC/Par-6 complex to control oocyte and epithelial polarity.

    PubMed

    Morais-de-Sá, Eurico; Mukherjee, Avik; Lowe, Nick; St Johnston, Daniel

    2014-08-01

    The Drosophila anterior-posterior axis is specified when the posterior follicle cells signal to polarise the oocyte, leading to the anterior/lateral localisation of the Par-6/aPKC complex and the posterior recruitment of Par-1, which induces a microtubule reorganisation that localises bicoid and oskar mRNAs. Here we show that oocyte polarity requires Slmb, the substrate specificity subunit of the SCF E3 ubiquitin ligase that targets proteins for degradation. The Par-6/aPKC complex is ectopically localised to the posterior of slmb mutant oocytes, and Par-1 and oskar mRNA are mislocalised. Slmb appears to play a related role in epithelial follicle cells, as large slmb mutant clones disrupt epithelial organisation, whereas small clones show an expansion of the apical domain, with increased accumulation of apical polarity factors at the apical cortex. The levels of aPKC and Par-6 are significantly increased in slmb mutants, whereas Baz is slightly reduced. Thus, Slmb may induce the polarisation of the anterior-posterior axis of the oocyte by targeting the Par-6/aPKC complex for degradation at the oocyte posterior. Consistent with this, overexpression of the aPKC antagonist Lgl strongly rescues the polarity defects of slmb mutant germline clones. The role of Slmb in oocyte polarity raises an intriguing parallel with C. elegans axis formation, in which PAR-2 excludes the anterior PAR complex from the posterior cortex to induce polarity, but its function can be substituted by overexpressing Lgl.

  14. Activation of protein synthesis in mouse uterine epithelial cells by estradiol-17β is mediated by a PKC-ERK1/2-mTOR signaling pathway.

    PubMed

    Wang, Yuxiang; Zhu, Liyin; Kuokkanen, Satu; Pollard, Jeffrey W

    2015-03-17

    The uterine epithelium of mice and humans undergoes cyclical waves of cell proliferation and differentiation under the regulation of estradiol-17β (E2) and progesterone (P4). These epithelial cells respond to E2 with increased protein and DNA synthesis, whereas P4 inhibits only the E2-induced DNA synthetic response. Here we show that E2 regulates protein synthesis in these epithelial cells through activating PKC that in turn stimulates ERK1/2 to phosphorylate and thereby activate the central regulator of protein synthesis mechanistic target of rapamycin (mTOR). This mTOR pathway is not inhibited by P4. Inhibitor studies with an estrogen receptor (ESR1) antagonist showed the dependence of this mTOR pathway on ESR1 but that once activated, a phosphorylation cascade independent of ESR1 propagates the pathway. E2 also stimulates an IGF1 receptor (IGF1R) to PI3 kinase to AKT to GSK-3β pathway required for activation of the canonical cell cycle machinery that is inhibited by P4. PKC activation did not stimulate this pathway nor does inhibition of PKC or ERK1/2 affect it. These studies therefore indicate a mechanism whereby DNA and protein synthesis are regulated by two ESR1-activated pathways that run in parallel with only the one responsible for the initiation of DNA synthesis blocked by P4. Inhibition of mTOR by rapamycin in vivo resulted in inhibition of E2-induced protein and DNA synthesis. Proliferative diseases of the endometrium such as endometriosis and cancer are common and E2 dependent. Thus, defining this mTOR pathway suggests that local (intrauterine or peritoneal) rapamycin administration might be a therapeutic option for these diseases.

  15. Increased PKC activity and altered GSK3β/NMDAR function drive behavior cycling in HINT1-deficient mice: bipolarity or opposing forces.

    PubMed

    Garzón-Niño, Javier; Rodríguez-Muñoz, María; Cortés-Montero, Elsa; Sánchez-Blázquez, Pilar

    2017-02-27

    Mice with histidine triad nucleotide-binding protein 1 (HINT1) deletion exhibit manic-like symptoms that evolve into depressive-like behavior in response to stressful paradigms. Molecular and electrophysiological studies have indicated that HINT1(-/-) mice exhibit increased PKC, PKA, and GSK3β activities, as well as glutamate N-methyl-D-aspartate receptor (NMDAR)/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) and NR2B/NR2A subunit ratios. Pharmacological interventions stabilized their behavior but through different mechanisms. GSK3β inhibitors and valproate directly attenuated the expression of the manic-like symptoms, whereas PKC inhibition, lamotrigine, or risperidone promoted NMDAR-mediated depressive-like behaviors that counterbalanced the preexisting manic-like symptoms. Naïve HINT1(-/-) mice exposed to stressful paradigms rapidly manifested depressive-like behaviors in subsequent stressful situations, a capacity that persisted for a couple of weeks thereafter. During the depressive-like phase, citalopram, amitriptyline and MK801 precipitated manic-like behaviors in stressed HINT1(-/-) mice. Notably, the antagonism of NMDARs prevented HINT1(-/-) mice from alternating behaviors in response to stress. A comparison with "manic" Black Swiss mice indicated that in HINT1(-/-) mice, PKC supports manic-like symptoms and reduces the expression of depressive-like behaviors via activation of GSK3β and regulation of NR2B-enriched NMDARs. HINT1(-/-) mice represent a suitable model for studying human BPD and may facilitate the identification of novel targets and drugs to treat this mental disorder.

  16. Increased PKC activity and altered GSK3β/NMDAR function drive behavior cycling in HINT1-deficient mice: bipolarity or opposing forces

    PubMed Central

    Garzón-Niño, Javier; Rodríguez-Muñoz, María; Cortés-Montero, Elsa; Sánchez-Blázquez, Pilar

    2017-01-01

    Mice with histidine triad nucleotide-binding protein 1 (HINT1) deletion exhibit manic-like symptoms that evolve into depressive-like behavior in response to stressful paradigms. Molecular and electrophysiological studies have indicated that HINT1−/− mice exhibit increased PKC, PKA, and GSK3β activities, as well as glutamate N-methyl-D-aspartate receptor (NMDAR)/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) and NR2B/NR2A subunit ratios. Pharmacological interventions stabilized their behavior but through different mechanisms. GSK3β inhibitors and valproate directly attenuated the expression of the manic-like symptoms, whereas PKC inhibition, lamotrigine, or risperidone promoted NMDAR-mediated depressive-like behaviors that counterbalanced the preexisting manic-like symptoms. Naïve HINT1−/− mice exposed to stressful paradigms rapidly manifested depressive-like behaviors in subsequent stressful situations, a capacity that persisted for a couple of weeks thereafter. During the depressive-like phase, citalopram, amitriptyline and MK801 precipitated manic-like behaviors in stressed HINT1−/− mice. Notably, the antagonism of NMDARs prevented HINT1−/− mice from alternating behaviors in response to stress. A comparison with “manic” Black Swiss mice indicated that in HINT1−/− mice, PKC supports manic-like symptoms and reduces the expression of depressive-like behaviors via activation of GSK3β and regulation of NR2B-enriched NMDARs. HINT1−/− mice represent a suitable model for studying human BPD and may facilitate the identification of novel targets and drugs to treat this mental disorder. PMID:28240305

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

    PubMed

    Shimada, M; Maeda, T; Terada, T

    2001-04-01

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

  18. The selective protein kinase C inhibitor, Ro-31-8220, inhibits mitogen-activated protein kinase phosphatase-1 (MKP-1) expression, induces c-Jun expression, and activates Jun N-terminal kinase.

    PubMed

    Beltman, J; McCormick, F; Cook, S J

    1996-10-25

    The role of protein kinase C (PKC) in inflammation, mitogenesis, and differentiation has been deduced in part through the use of a variety of PKC inhibitors. Two widely used inhibitors are the structurally related compounds GF109203X and Ro-31-8220, both of which potently inhibit PKC activity and are believed to be highly selective. While using GF109203X and Ro-31-8220 to address the role of PKC in immediate early gene expression, we observed striking differential effects by each of these two compounds. Growth factors induce the expression of the immediate early gene products MAP kinase phosphatase-1 (MKP-1), c-Fos and c-Jun. Ro-31-8220 inhibits growth factor-stimulated expression of MKP-1 and c-Fos but strongly stimulated c-Jun expression, even in the absence of growth factors. GF109203X displays none of these properties. These data suggest that Ro-31-8220 may have other pharmacological actions in addition to PKC inhibition. Indeed, Ro-31-8220 strongly stimulates the stress-activated protein kinase, JNK1. Furthermore, Ro-31-8220 apparently activates JNK in a PKC-independent manner. Neither the down-regulation of PKC by phorbol esters nor the inhibition of PKC by GF109203X affected the ability of Ro-31-8220 to activate JNK1. These data suggest that, in addition to potently inhibiting PKC, Ro-31-8220 exhibits novel pharmacological properties which are independent of its ability to inhibit PKC.

  19. Kainate receptor activation induces glycine receptor endocytosis through PKC deSUMOylation

    PubMed Central

    Sun, Hao; Lu, Li; Zuo, Yong; Wang, Yan; Jiao, Yingfu; Zeng, Wei-Zheng; Huang, Chao; Zhu, Michael X.; Zamponi, Gerald W.; Zhou, Tong; Xu, Tian-Le; Cheng, Jinke; Li, Yong

    2014-01-01

    Surface expression and regulated endocytosis of glycine receptors (GlyRs) play a critical function in balancing neuronal excitability. SUMOylation (SUMO modification) is of critical importance for maintaining neuronal function in the central nervous system. Here we show that activation of kainate receptors (KARs) causes GlyR endocytosis in a calcium- and protein kinase C (PKC)-dependent manner, leading to reduced GlyR-mediated synaptic activity in cultured spinal cord neurons and the superficial dorsal horn of rat spinal cord slices. This effect requires SUMO1/sentrin-specific peptidase 1 (SENP1)-mediated deSUMOylation of PKC, indicating that the crosstalk between KARs and GlyRs relies on the SUMOylation status of PKC. SENP1-mediated deSUMOylation of PKC is involved in the kainate-induced GlyR endocytosis and thus plays an important role in the anti-homeostatic regulation between excitatory and inhibitory ligand-gated ion channels. Altogether, we have identified a SUMOylation-dependent regulatory pathway for GlyR endocytosis, which may have important physiological implications for proper neuronal excitability. PMID:25236484

  20. PKC{delta}-mediated IRS-1 Ser24 phosphorylation negatively regulates IRS-1 function

    SciTech Connect

    Greene, Michael W. . E-mail: michael.greene@bassett.org; Ruhoff, Mary S.; Roth, Richard A.; Kim, Jeong-a; Quon, Michael J.; Krause, Jean A.

    2006-10-27

    The IRS-1 PH and PTB domains are essential for insulin-stimulated IRS-1 Tyr phosphorylation and insulin signaling, while Ser/Thr phosphorylation of IRS-1 disrupts these signaling events. To investigate consensus PKC phosphorylation sites in the PH-PTB domains of human IRS-1, we changed Ser24, Ser58, and Thr191 to Ala (3A) or Glu (3E), to block or mimic phosphorylation, respectively. The 3A mutant abrogated the inhibitory effect of PKC{delta} on insulin-stimulated IRS-1 Tyr phosphorylation, while reductions in insulin-stimulated IRS-1 Tyr phosphorylation, cellular proliferation, and Akt activation were observed with the 3E mutant. When single Glu mutants were tested, the Ser24 to Glu mutant had the greatest inhibitory effect on insulin-stimulated IRS-1 Tyr phosphorylation. PKC{delta}-mediated IRS-1 Ser24 phosphorylation was confirmed in cells with PKC{delta} catalytic domain mutants and by an RNAi method. Mechanistic studies revealed that IRS-1 with Ala and Glu point mutations at Ser24 impaired phosphatidylinositol-4,5-bisphosphate binding. In summary, our data are consistent with the hypothesis that Ser24 is a negative regulatory phosphorylation site in IRS-1.

  1. Tyrosinase kinetics in epidermal melanocytes: analysis of DAG-PKC-dependent signaling pathway

    NASA Astrophysics Data System (ADS)

    Stolnitz, Mikhail M.; Peshkova, Anna Y.

    2001-05-01

    Tyrosinase is the key enzyme of melanogenesis with unusual enzyme kinetics. Protein kinase C plays an important role in regulating of tyrosinase activity. In the paper the mathematical model of PKC-DAG-dependent signal transduction pathway for UV-radiation is presented.

  2. Saccharomyces Cerevisiae Hoc1, a Suppressor of Pkc1, Encodes a Putative Glycosyltransferase

    PubMed Central

    Neiman, A. M.; Mhaiskar, V.; Manus, V.; Galibert, F.; Dean, N.

    1997-01-01

    The Saccharomyces cerevisiae gene PKC1 encodes a protein kinase C isozyme that regulates cell wall synthesis. Here we describe the characterization of HOC1, a gene identified by its ability to suppress the cell lysis phenotype of pkc1-371 cells. The HOC1 gene (Homologous to OCH1) is predicted to encode a type II integral membrane protein that strongly resembles Och1p, an α-1,6-mannosyltransferase. Immunofluorescence studies localized Hoc1p to the Golgi apparatus. While overexpression of HOC1 rescued the pkc1-371 temperature-sensitive cell lysis phenotype, disruption of HOC1 lowered the restrictive temperature of the pkc1-371 allele. Disruption of HOC1 also resulted in hypersensitivity to Calcofluor White and hygromycin B, phenotypes characteristic of defects in cell wall integrity and protein glycosylation, respectively. The function of HOC1 appears to be distinct from that of OCH1. Taken together, these results suggest that HOC1 encodes a Golgi-localized putative mannosyltransferase required for the proper construction of the cell wall. PMID:9055074

  3. Bidirectional control of BK channel open probability by CAMKII and PKC in medial vestibular nucleus neurons

    PubMed Central

    van Welie, Ingrid

    2011-01-01

    Large conductance K+ (BK) channels are a key determinant of neuronal excitability. Medial vestibular nucleus (MVN) neurons regulate eye movements to ensure image stabilization during head movement, and changes in their intrinsic excitability may play a critical role in plasticity of the vestibulo-ocular reflex. Plasticity of intrinsic excitability in MVN neurons is mediated by kinases, and BK channels influence excitability, but whether endogenous BK channels are directly modulated by kinases is unknown. Double somatic patch-clamp recordings from MVN neurons revealed large conductance potassium channel openings during spontaneous action potential firing. These channels displayed Ca2+ and voltage dependence in excised patches, identifying them as BK channels. Recording isolated single channel currents at physiological temperature revealed a novel kinase-mediated bidirectional control in the range of voltages over which BK channels are activated. Application of activated Ca2+/calmodulin-dependent kinase II (CAMKII) increased BK channel open probability by shifting the voltage activation range towards more hyperpolarized potentials. An opposite shift in BK channel open probability was revealed by inhibition of phosphatases and was occluded by blockade of protein kinase C (PKC), suggesting that active PKC associated with BK channel complexes in patches was responsible for this effect. Accordingly, direct activation of endogenous PKC by PMA induced a decrease in BK open probability. BK channel activity affects excitability in MVN neurons and bidirectional control of BK channels by CAMKII, and PKC suggests that cellular signaling cascades engaged during plasticity may dynamically control excitability by regulating BK channel open probability. PMID:21307321

  4. Induction of human leukemia cell differentiation via PKC/MAPK pathways by arsantin, a sesquiterpene lactone from Artemisia santolina.

    PubMed

    Kweon, Sin Ho; Song, Ju Han; Kim, Hee Jin; Kim, Tae Sung; Choi, Bo Gil

    2015-11-01

    Sesquiterpene lactone compounds have received considerable attention in pharmacological research due to their therapeutic effects including anti-cancer and anti-inflammatory activities. In this report, we investigated the effect of arsantin, a sesquiterpene lactone compound present in Artemisia santolina, on cellular differentiation in the human promyelocytic leukemia HL-60 cell culture system. Arsantin significantly induced HL-60 cell differentiation in a concentration-dependent manner. Cytofluorometric analysis indicated that arsantin induced HL-60 cell differentiation predominantly into granulocytes. Both PKC and MAPK inhibitors suppressed the HL-60 cell differentiation induced by arsantin. Moreover, treatment with arsantin increased protein levels of PKCα and PKCβII isoforms, and also induced increased protein levels and phosphorylation form of MAPKs in HL-60 cells. Importantly, arsantin synergistically enhanced differentiation of HL-60 cells in a dose-dependent manner when combined with either low doses of 1,25-(OH)2D3 or ATRA. The ability to enhance the differentiation potential of 1,25-(OH)2D3 or ATRA by arsantin may improve outcomes in the therapy of acute promyelocytic leukemia.

  5. Prostaglandin E{sub 2} regulates melanocyte dendrite formation through activation of PKC{zeta}

    SciTech Connect

    Scott, Glynis Fricke, Alex; Fender, Anne; McClelland, Lindy; Jacobs, Stacey

    2007-11-01

    Prostaglandins are lipid signaling intermediates released by keratinocytes in response to ultraviolet irradiation (UVR) in the skin. The main prostaglandin released following UVR is PGE{sub 2}, a ligand for 4 related G-protein-coupled receptors (EP{sub 1}, EP{sub 2}, EP{sub 3} and EP{sub 4}). Our previous work established that PGE{sub 2} stimulates melanocyte dendrite formation through activation of the EP{sub 1} and EP{sub 3} receptors. The purpose of the present report is to define the signaling intermediates involved in EP{sub 1}- and EP{sub 3}-dependent dendrite formation in human melanocytes. We recently showed that activation of the atypical PKC{zeta} isoform stimulates melanocyte dendricity in response to treatment with lysophosphatidylcholine. We therefore examined the potential contribution of PKC{zeta} activation on EP{sub 1}- and EP{sub 3}-dependent dendrite formation in melanocytes. Stimulation of the EP{sub 1} and EP{sub 3} receptors by selective agonists activated PKC{zeta}, and inhibition of PKC{zeta} activation abrogated EP{sub 1}- and EP{sub 3}-receptor-mediated melanocyte dendricity. Because of the importance of Rho-GTP binding proteins in the regulation of melanocyte dendricity, we also examined the effect of EP{sub 1} and EP{sub 3} receptor activation on Rac and Rho activity. Neither Rac nor Rho was activated upon treatment with EP{sub 1,3}-receptor agonists. We show that melanocytes express only the EP{sub 3A1} isoform, but not the EP{sub 3B} receptor isoform, previously associated with Rho activation, consistent with a lack of Rho stimulation by EP{sub 3} agonists. Our data suggest that PKC{zeta} activation plays a predominant role in regulation of PGE{sub 2}-dependent melanocyte dendricity.

  6. Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity

    PubMed Central

    de Jong, Arthur P. H.; Meijer, Marieke; Saarloos, Ingrid; Cornelisse, Lennart Niels; Toonen, Ruud F. G.; Sørensen, Jakob B.; Verhage, Matthijs

    2016-01-01

    Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAG-induced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1T112A), which prevents its PKC-dependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons. This mutant also reduces potentiation of spontaneous release, but only if alternative Ca2+ sensors, Doc2A/B proteins, are absent. However, unlike mutations in Munc13-1 or Munc18-1 that prevent DAG-induced potentiation, the synaptotagmin-1 mutation does not affect paired-pulse facilitation. Furthermore, experiments to probe vesicle priming (recovery after train stimulation and dual application of hypertonic solutions) also reveal no abnormalities. Expression of synaptotagmin-2, which lacks a seven amino acid sequence that contains the phosphorylation site in synaptotagmin-1, or a synaptotagmin-1 variant with these seven residues removed (Syt1Δ109–116), supports normal DAG-induced potentiation. These data suggest that this seven residue sequence in synaptotagmin-1 situated in the linker between the transmembrane and C2A domains is inhibitory in the unphosphorylated state and becomes permissive of potentiation upon phosphorylation. We conclude that synaptotagmin-1 phosphorylation is an essential step in PKC-dependent potentiation of synaptic transmission, acting downstream of the two other essential DAG/PKC substrates, Munc13-1 and Munc18-1. PMID:27091977

  7. Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity.

    PubMed

    de Jong, Arthur P H; Meijer, Marieke; Saarloos, Ingrid; Cornelisse, Lennart Niels; Toonen, Ruud F G; Sørensen, Jakob B; Verhage, Matthijs

    2016-05-03

    Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAG-induced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1(T112A)), which prevents its PKC-dependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons. This mutant also reduces potentiation of spontaneous release, but only if alternative Ca(2+) sensors, Doc2A/B proteins, are absent. However, unlike mutations in Munc13-1 or Munc18-1 that prevent DAG-induced potentiation, the synaptotagmin-1 mutation does not affect paired-pulse facilitation. Furthermore, experiments to probe vesicle priming (recovery after train stimulation and dual application of hypertonic solutions) also reveal no abnormalities. Expression of synaptotagmin-2, which lacks a seven amino acid sequence that contains the phosphorylation site in synaptotagmin-1, or a synaptotagmin-1 variant with these seven residues removed (Syt1(Δ109-116)), supports normal DAG-induced potentiation. These data suggest that this seven residue sequence in synaptotagmin-1 situated in the linker between the transmembrane and C2A domains is inhibitory in the unphosphorylated state and becomes permissive of potentiation upon phosphorylation. We conclude that synaptotagmin-1 phosphorylation is an essential step in PKC-dependent potentiation of synaptic transmission, acting downstream of the two other essential DAG/PKC substrates, Munc13-1 and Munc18-1.

  8. Ellagic acid checks lymphoma promotion via regulation of PKC signaling pathway.

    PubMed

    Mishra, Sudha; Vinayak, Manjula

    2013-02-01

    Protein Kinase C (PKC) isozymes are key components involved in cell proliferation and their over activation leads to abnormal tumor growth. PKC follows signalling pathway by activation of downstream gene NF-kB and early transcription factor c-Myc. Over activation of NF-kB and c-Myc gene are also linked with unregulated proliferation of cancer cells. Therefore any agent which can inhibit the activation of Protein kinase C, NF-kB and c-Myc may be useful in reducing cancer progression. To investigate this hypothesis we have tested the effect of ellagic acid on these genes in Dalton's lymphoma bearing (DL). The role of ellagic acid was also tested in regulation of tumor suppressor gene Transforming growth factor-β1 (TGF-β1). DL mice were treated with three different doses (40, 60 and 80 mg/kg body weight) of ellagic acid. Ascites cells of mice were used for the experiments. Ellagic acid administration to DL mice decreased oxidative stress by reducing lipid peroxidation. Ellagic acid also down regulates the expression of classical isozymes of PKC i.e. PKCα, PKCβ, and PKCγ as well as activity of total PKC and NF-kB, indicating its antitumor action. The anticarcinogenic action of ellagic acid was also confirmed by up regulation of TGF-β1 and down regulation of c-Myc. Lymphoma prevention by ellagic acid is further supported by decrease in cell proliferation, cell viability, ascites fluid accumulation and increase in life span of DL mice. All these findings suggest that ellagic acid prevents the cancer progression by down regulation of PKC signaling pathway leading to cell proliferation.

  9. Wnt5a through Noncanonical Wnt/JNK or Wnt/PKC Signaling Contributes to the Differentiation of Mesenchymal Stem Cells into Type II Alveolar Epithelial Cells In Vitro

    PubMed Central

    Cai, Shixia; Dong, Liang; Liu, Le; Yang, Yi; Guo, Fengmei; Lu, Xiaomin; He, Hongli; Chen, Qihong; Hu, Shuling; Qiu, Haibo

    2014-01-01

    The differentiation of mesenchymal stem cells (MSCs) into type II alveolar epithelial (AT II) cells is critical for reepithelization and recovery in acute respiratory distress syndrome (ARDS), and Wnt signaling was considered to be the underlying mechanisms. In our previous study, we found that canonical Wnt pathway promoted the differentiation of MSCs into AT II cells, however the role of the noncanonical Wnt pathway in this process is unclear. It was disclosed in this study that noncanonical Wnt signaling in mouse bone marrow–derived MSCs (mMSCs) was activated during the differentiation of mMSCs into AT II cells in a modified co-culture system with murine lung epithelial-12 cells and small airway growth media. The levels of surfactant protein (SP) C, SPB and SPD, the specific markers of AT II cells, increased in mMSCs when Wnt5a was added to activate noncanonical Wnt signaling, while pretreatment with JNK or PKC inhibitors reversed the promotion of Wnt5a. The differentiation rate of mMSCs also depends on their abilities to accumulate and survive in inflammatory tissue. We found that the Wnt5a supplement promoted the vertical and horizontal migration of mMSCs, ameliorated the cell death and the reduction of Bcl-2/Bax induced by H2O2. The effect of Wnt5a on the migration of mMSCs and their survival after H2O2 exposure were partially inhibited with PKC or JNK blockers. In conclusion, Wnt5a through Wnt/JNK signaling alone or both Wnt/JNK and Wnt/PKC signaling promoted the differentiation of mMSCs into AT II cells and the migration of mMSCs; through Wnt/PKC signaling, Wnt5a increased the survival of mMSCs after H2O2 exposure in vitro. PMID:24658098

  10. Spinal D-Serine Increases PKC-Dependent GluN1 Phosphorylation Contributing to the Sigma-1 Receptor-Induced Development of Mechanical Allodynia in a Mouse Model of Neuropathic Pain.

    PubMed

    Choi, Sheu-Ran; Moon, Ji-Young; Roh, Dae-Hyun; Yoon, Seo-Yeon; Kwon, Soon-Gu; Choi, Hoon-Seong; Kang, Suk-Yun; Han, Ho-Jae; Beitz, Alvin J; Lee, Jang-Hern

    2017-04-01

    We have recently shown that spinal sigma-1 receptor (Sig-1R) activation facilitates nociception via an increase in phosphorylation of the N-methyl-D-aspartate (NMDA) receptor GluN1 subunit (pGluN1). The present study was designed to examine whether the Sig-1R-induced facilitative effect on NMDA-induced nociception is mediated by D-serine, and whether D-serine modulates spinal pGluN1 expression and the development of neuropathic pain after chronic constriction injury (CCI) of the sciatic nerve. Intrathecal administration of the D-serine degrading enzyme, D-amino acid oxidase attenuated the facilitation of NMDA-induced nociception induced by the Sig-1R agonist, 2-(4-morpholinethyl)1-phenylcyclohexane carboxylate. Exogenous D-serine increased protein kinase C (PKC)-dependent (Ser896) pGluN1 expression and facilitated NMDA-induced nociception, which was attenuated by preteatment with the PKC inhibitor, chelerythrine. In CCI mice, administration of the serine racemase inhibitor, L-serine O-sulfate potassium salt or D-amino acid oxidase on postoperative days 0 to 3 suppressed CCI-induced mechanical allodynia (MA) and pGluN1 expression on day 3 after CCI surgery. Intrathecal administration of D-serine restored MA as well as the GluN1 phosphorylation on day 3 after surgery that was suppressed by the Sig-1R antagonist, N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine dihydrobromide or the astrocyte inhibitor, fluorocitrate. In contrast, D-serine had no effect on CCI-induced thermal hyperalgesia or GluN1 expression. These results indicate that spinal D-serine: 1) mediates the facilitative effect of Sig-1R on NMDA-induced nociception, 2) modulates PKC-dependent pGluN1 expression, and 3) ultimately contributes to the induction of MA after peripheral nerve injury.

  11. Activation of protein kinase C as a modulator of potentiated UK-14304-induced contractions in dog mesenteric artery and vein.

    PubMed

    Shimamoto, H; Shimamoto, Y; Kwan, C Y; Daniel, E E

    1995-12-01

    We assessed the role of protein kinase C (PKC) in the mechanism responsible for the potentiation of UK-14304-induced contractions produced when isolated dog mesenteric vascular rings were pretreated with threshold concentrations of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), KCl, or endothelin-1 (ET-1). In dog mesenteric artery. UK-14304 produced a biphasic concentration-response curve in the presence of TPA, KCl, or ET-1, with the curve portion at lower concentrations being alpha 2-adrenoceptor dependent and the portion at higher concentrations being alpha 1-adrenoceptor dependent. Calphostin C (10(-6)M), a PKC inhibitor, abolished amplified UK-14304-induced contraction in the TPA-pretreated tissues. In the KCl- and ET-1-pretreated tissues. 10(-6)M calphostin C antagonized amplified UK-14304-induced contractions by approximately 20% in both parts of the concentration-response curve. In contrast, in dog mesenteric vein, amplified UK-14304-induced contractions by TPA, KCl, and ET-1 were entirely dependent on alpha 2-adrenoceptors. Calphostin C (10(-6)M), which in control experiments had no effect on KCl-induced contraction and antagonized responses to TPA by 60.1%, inhibited UK-14304-induced contraction by 18.3%. Amplified UK-14304-induced contraction was antagonized by 10(-6)M calphostin C by 21.8% in KCl-precontracted tissues, 58.1% in ET-1-precontracted tissues, and 66.3% in TPA-precontracted tissues. In the ET-1- and TPA-pretreated dog mesenteric veins, 10(-6)M calphostin C decreased maximal tensions of enhanced UK-14304-induced contractions to the same level as the UK-14304-induced maximal tension inhibited by 10(-6)M calphostin C in untreated dog mesenteric vein. Therefore, TPA can be a precontracting agent that amplifies UK-14304-induced contractions through PKC activation in both dog mesenteric artery and vein. PKC predominantly mediates the contraction amplification mechanisms after exposure to ET-1 in dog mesenteric vein and does not play a major role in

  12. Intrathecal injection of adenosine 2A receptor agonists reversed neuropathic allodynia through protein kinase (PK)A/PKC signaling.

    PubMed

    Loram, Lisa C; Taylor, Frederick R; Strand, Keith A; Harrison, Jacqueline A; Rzasalynn, Rachael; Sholar, Paige; Rieger, Jayson; Maier, Steven F; Watkins, Linda R

    2013-10-01

    A single intrathecal dose of adenosine 2A receptor (A2AR) agonist was previously reported to produce a multi-week reversal of allodynia in a chronic constriction injury (CCI) model of neuropathic pain. We aimed to determine if this long-term reversal was induced by A2AR agonism versus more generalized across adenosine receptor subtypes, and begin to explore the intracellular signaling cascades involved. In addition, we sought to identify whether the enduring effect could be extended to other models of neuropathic pain. We tested an A1R and A2BR agonist in CCI and found the same long duration effect with A2BR but not A1R agonism. An A2AR agonist (ATL313) produced a significant long-duration reversal of mechanical allodynia induced by long established CCI (administered 6 weeks after surgery), spinal nerve ligation and sciatic inflammatory neuropathy. To determine if ATL313 had a direct effect on glia, ATL313 was coadministered with lipopolysaccharide to neonatal microglia and astrocytes in vitro. ATL313 significantly attenuated TNFα production in both microglia and astrocytes but had no effect on LPS induced IL-10. Protein kinase C significantly reversed the ATL313 effects on TNFα in vitro in microglia and astrocytes, while a protein kinase A inhibitor only effected microglia. Both intrathecal PKA and PKC inhibitors significantly reversed the effect of the A2AR agonist on neuropathic allodynia. Therefore, A2AR agonists administered IT remain an exciting novel target for the treatment of neuropathic pain.

  13. Contribution of PKC to the maintenance of 5-HT-induced short-term facilitation at sensorimotor synapses of Aplysia.

    PubMed

    Zhou, Lian; Baxter, Douglas A; Byrne, John H

    2014-10-15

    Aplysia sensorimotor synapses provide a useful model system for analyzing molecular processes that contribute to heterosynaptic plasticity. For example, previous studies demonstrated that multiple kinase cascades contribute to serotonin (5-HT)-induced short-term synaptic facilitation (STF), including protein kinase A (PKA) and protein kinase C (PKC). Moreover, the contribution of each kinase is believed to depend on the state of the synapse (e.g., depressed or nondepressed) and the time after application of 5-HT. Here, a previously unappreciated role for PKC-dependent processes was revealed to underlie the maintenance of STF at relatively nondepressed synapses. This PKC dependence was revealed when the synapse was stimulated repeatedly after application of 5-HT. The contributions of the PKA and PKC pathways were examined by blocking adenylyl cyclase-coupled 5-HT receptors with methiothepin and by blocking PKC with chelerythrine. STF was assessed 20 s after 5-HT application. The effects of PKC were consistent with enhanced mobilization of transmitter, as assessed by application of hypertonic sucrose solutions to measure the readily releasable pool of vesicles and recovery of the readily releasable pool after depletion. A computational model of transmitter release demonstrated that a PKC-dependent mobilization process was sufficient to explain the maintenance of STF at nondepressed synapses and the facilitation of depressed synapses.

  14. Orexin-A Protects Human Neuroblastoma SH-SY5Y Cells Against 6-Hydroxydopamine-Induced Neurotoxicity: Involvement of PKC and PI3K Signaling Pathways.

    PubMed

    Pasban-Aliabadi, Hamzeh; Esmaeili-Mahani, Saeed; Abbasnejad, Mehdi

    2017-04-01

    Parkinson's disease (PD) is a common neurodegenerative disorder that is characterized by progressive and selective death of dopaminergic neurons. Multifunctional neuropeptide orexin-A is involved in many biological events of the body. It has been shown that orexin-A has protective effects in neurodegenerative disease such as PD. However, its cellular mechanisms have not yet been fully clarified. Here, we investigated the intracellular signaling pathway of orexin-A neuroprotection in 6-hydroxydopamine (6-OHDA)-induced SH-SY5H cells damage as an in vitro model of PD. The cells were incubated with 150 μM 6-OHDA, and the viability was examined by 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazolium bromide (MTT) assay. Mitochondrial membrane potential and intracellular calcium were measured by fluorescent probes. Western blotting was also used to determine cyclooxygenase type 2 (COX-2), nuclear factor erythroid 2 related factor 2 (Nrf2), and HSP70 protein levels. The data showed that 6-OHDA has decreasing effects on cell viability, Nrf2, and HSP70 protein expression and increases the level of mitochondrial membrane potential, intracellular calcium, and COX-2 protein. Orexin-A (500 pM) significantly attenuated the 6-OHDA-induced cell damage. Furthermore, Orexin-A significantly prevented the mentioned effects of 6-OHDA on SH-SY5Y cells. Orexin 1 receptor antagonist (SB3344867), PKC, and PI3-kinase (PI3K) inhibitors (chelerythrin and LY294002, respectively) could suppress the orexin-A neuroprotective effect. In contrast, blockage of PKA by a selective inhibitor (KT5720) had no effects on the orexin protection. The results suggest that orexin-A protective effects against 6-OHDA-induced neurotoxicity are performed via its receptors, PKC and PI3K signaling pathways.

  15. Increased PDE5 activity and decreased Rho kinase and PKC activities in colonic muscle from caveolin-1-/- mice impair the peristaltic reflex and propulsion.

    PubMed

    Mahavadi, Sunila; Bhattacharya, Sayak; Kumar, Divya P; Clay, Chereena; Ross, Gracious; Akbarali, Hamid I; Grider, John R; Murthy, Karnam S

    2013-12-01

    Caveolae are specialized regions of the plasma membrane that concentrate receptors and associated signaling molecules critical in regulation of cellular response to transmitters and hormones. We have determined the effects of caveolin-1 (Cav-1) deletion, caveolin-1 siRNA, and caveolar disruption in mice on the signaling pathways that mediate contraction and relaxation in colonic smooth muscle and on the components of the peristaltic reflex in isolated tissue and propulsion in intact colonic segments. In Cav-1-/- mice, both relaxation and contraction were decreased in smooth muscle cells and muscle strips, as well as during both phases of the peristaltic reflex and colonic propulsion. The decrease in relaxation in response to the nitric oxide (NO) donor was accompanied by a decrease in cGMP levels and an increase in phosphodiesterase 5 (PDE5) activity. Relaxation by a PDE5-resistant cGMP analog was not affected in smooth muscle of Cav-1-/- mice, suggesting that inhibition of relaxation was due to augmentation of PDE5 activity. Similar effects on relaxation, PDE5 and cGMP were obtained in muscle cells upon disruption of caveolae by methyl-β-cyclodextrin or suppression of Cav-1. Sustained contraction mediated via inhibition of myosin light chain phosphatase (MLCP) activity is regulated by Rho kinase and PKC via phosphorylation of two endogenous inhibitors of MLCP: myosin phosphatase-targeting subunit (MYPT1) and 17-kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17), respectively. The activity of both enzymes and phosphorylation of MYPT1 and CPI-17 were decreased in smooth muscle from Cav-1-/- mice. We conclude that the integrity of caveolae is essential for contractile and relaxant activity in colonic smooth muscle and the maintenance of neuromuscular function at organ level.

  16. Altered expression of atypical PKC and Ryk in the spinal cord of a mouse model of amyotrophic lateral sclerosis

    PubMed Central

    Tury, Anna; Tolentino, Kristine; Zou, Yimin

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive paralysis due to the selective death of motor neurons of unknown causes. Increasing evidence indicates that Wnt signaling is altered in ALS. In this study, we focused on two non-canonical Wnt signaling components, atypical PKC (aPKC) and a Wnt receptor, Ryk, in a mouse model of ALS, SOD1 (G93A). aPKC mediates Wnt signaling to regulate growth cone guidance, axon differentiation and cell survival. Ryk is a Wnt repulsive receptor that regulates axon guidance and inhibits regeneration after spinal cord injury. aPKC expression was increased in motor neurons of the lumbar spinal cord in SOD1 (G93A) mice at different stages. Interestingly, aPKC was colocalized with SOD1 in motor neuron cell bodies and extracellular aggregates, and aPKC-containing extracellular aggregates increased with disease progression. Biochemical fractionation showed that aPKC protein level was increased in the detergent-insoluble protein fraction in SOD1 (G93A) mice at late stage but decreased in the detergent-soluble fraction at symptomatic stage. These results suggest that aPKC may be sequestered in SOD1 aggregates, impairing its ability to protect motor neurons from death. Ryk expression was also increased in the motor neurons and the white matter in the ventral lumbar spinal cord of mutant SOD1 mice with a peak at early stage. These observations indicate that Wnt/aPKC and Wnt/Ryk signaling are altered in SOD1 (G93A) mice, suggesting that changed Wnt signaling may contribute to neurodegeneration in ALS. PMID:24123880

  17. The synthetic genetic network around PKC1 identifies novel modulators and components of protein kinase C signaling in Saccharomyces cerevisiae.

    PubMed

    Krause, Sue A; Xu, Hong; Gray, Joseph V

    2008-11-01

    Budding yeast Saccharomyces cerevisiae contains one protein kinase C (PKC) isozyme encoded by the essential gene PKC1. Pkc1 is activated by the small GTPase Rho1 and plays a central role in the cell wall integrity (CWI) signaling pathway. This pathway acts primarily to remodel the cell surface throughout the normal life cycle and upon various environmental stresses. The pathway is heavily branched, with multiple nonessential branches feeding into and out of the central essential Rho1-Pkc1 module. In an attempt to identify novel components and modifiers of CWI signaling, we determined the synthetic lethal genetic network around PKC1 by using dominant-negative synthetic genetic array analysis. The resulting mutants are hypersensitive to lowered Pkc1 activity. The corresponding 21 nonessential genes are closely related to CWI function: 14 behave in a chemical-genetic epistasis test as acting in the pathway, and 6 of these genes encode known components. Twelve of the 21 null mutants display elevated CWI reporter activity, consistent with the idea that the pathway is activated by and compensates for loss of the gene products. Four of the 21 mutants display low CWI reporter activity, consistent with the idea that the pathway is compromised in these mutants. One of the latter group of mutants lacks Ack1(Ydl203c), an uncharacterized SEL-1 domain-containing protein that we find modulates pathway activity. Epistasis analysis places Ack1 upstream of Pkc1 in the CWI pathway and dependent on the upstream Rho1 GTP exchange factors Rom2 and Tus1. Overall, the synthetic genetic network around PKC1 directly and efficiently identifies known and novel components of PKC signaling in yeast.

  18. PKC and AMPK regulation of Kv1.5 potassium channels

    PubMed Central

    Andersen, Martin Nybo; Skibsbye, Lasse; Tang, Chuyi; Petersen, Frederic; MacAulay, Nanna; Rasmussen, Hanne Borger; Jespersen, Thomas

    2015-01-01

    The voltage-gated Kv1.5 potassium channel, conducting the ultra-rapid rectifier K+ current (IKur), is regulated through several pathways. Here we investigate if Kv1.5 surface expression is controlled by the 2 kinases PKC and AMPK, using Xenopus oocytes, MDCK cells and atrial derived HL-1 cells. By confocal microscopy combined with electrophysiology we demonstrate that PKC activation reduces Kv1.5 current, through a decrease in membrane expressed channels. AMPK activation was found to decrease the membrane expression in MDCK cells, but not in HL-1 cells and was furthermore shown to be dependent on co-expression of Nedd4–2 in Xenopus oocytes. These results indicate that Kv1.5 channels are regulated by both kinases, although through different molecular mechanisms in different cell systems. PMID:26043299

  19. Rise and Fall of Kir2.2 Current by TLR4 Signaling in Human Monocytes: PKC-Dependent Trafficking and PI3K-Mediated PIP2 Decrease.

    PubMed

    Kim, Kyung Soo; Jang, Ji Hyun; Lin, Haiyue; Choi, Seong Woo; Kim, Hang Rae; Shin, Dong Hoon; Nam, Joo Hyun; Zhang, Yin Hua; Kim, Sung Joon

    2015-10-01

    LPSs are widely used to stimulate TLR4, but their effects on ion channels in immune cells are poorly known. In THP-1 cells and human blood monocytes treated with LPS, inwardly rectifying K(+) channel current (IKir,LPS) newly emerged at 1 h, peaked at 4 h (-119 ± 8.6 pA/pF), and decayed afterward (-32 ± 6.7 pA/pF at 24 h). Whereas both the Kir2.1 and Kir2.2 mRNAs and proteins were observed, single-channel conductance (38 pS) of IKir,LPS and small interfering RNA-induced knockdown commonly indicated Kir2.2 than Kir2.1. LPS-induced cytokine release and store-operated Ca(2+) entry were commonly decreased by ML-133, a Kir2 inhibitor. Immunoblot, confocal microscopy, and the effects of vesicular trafficking inhibitors commonly suggested plasma membrane translocation of Kir2.2 by LPS. Both IKir,LPS and membrane translocation of Kir2.2 were inhibited by GF109203X (protein kinase C [PKC] inhibitor) or by transfection with small interfering RNA-specific PKCε. Interestingly, pharmacological activation of PKC by PMA induced both Kir2.1 and Kir2.2 currents. The spontaneously decayed IKir,LPS at 24 h was recovered by PI3K inhibitors but further suppressed by an inhibitor of phosphatidylinositol(3,4,5)-trisphosphate (PIP3) phosphatase (phosphatase and tensin homolog). However, IKir,LPS at 24 h was not affected by Akt inhibitors, suggesting that the decreased phosphatidylinositol(4,5)-bisphosphate availability, that is, conversion into PIP3 by PI3K, per se accounts for the decay of IKir,LPS. Taken together, to our knowledge these data are the first demonstrations that IKir is newly induced by TLR4 stimulation via PKC-dependent membrane trafficking of Kir2.2, and that conversion of phosphatidylinositol(4,5)-bisphosphate to PIP3 modulates Kir2.2. The augmentation of Ca(2+) influx and cytokine release suggests a physiological role for Kir2.2 in TLR4-stimulated monocytes.

  20. Stimulation of cleavage of membrane proteins by calmodulin inhibitors.

    PubMed Central

    Díaz-Rodríguez, E; Esparís-Ogando, A; Montero, J C; Yuste, L; Pandiella, A

    2000-01-01

    The ectodomain of several membrane-bound proteins can be shed by proteolytic cleavage. The activity of the proteases involved in shedding is highly regulated by several intracellular second messenger pathways, such as protein kinase C (PKC) and intracellular Ca(2+). Recently, the shedding of the adhesion molecule L-selectin has been shown to be regulated by the interaction of calmodulin (CaM) with the cytosolic tail of L-selectin. Prevention of CaM-L-selectin interaction by CaM inhibitors or mutation of a CaM binding site in L-selectin induced L-selectin ectodomain shedding. Whether this action of CaM inhibitors also affects other membrane-bound proteins is not known. In the present paper we show that CaM inhibitors also stimulate the cleavage of several other transmembrane proteins, such as the membrane-bound growth factor precursors pro-transforming growth factor-alpha and pro-neuregulin-alpha2c, the receptor tyrosine kinase, TrkA, and the beta-amyloid precursor protein. Cleavage induced by CaM inhibitors was a rapid event, and resulted from the activation of a mechanism that was independent of PKC or intracellular Ca(2+) increases, but was highly sensitive to hydroxamic acid-based metalloprotease inhibitors. Mutational analysis of the intracellular domain of the TrkA receptor indicated that CaM inhibitors may stimulate membrane-protein ectodomain cleavage by mechanisms independent of CaM-substrate interaction. PMID:10677354

  1. Munc18-1 redistributes in nerve terminals in an activity- and PKC-dependent manner

    PubMed Central

    Cijsouw, Tony; Weber, Jens P.; Broeke, Jurjen H.; Broek, Jantine A.C.; Schut, Desiree; Kroon, Tim; Saarloos, Ingrid

    2014-01-01

    Munc18-1 is a soluble protein essential for synaptic transmission. To investigate the dynamics of endogenous Munc18-1 in neurons, we created a mouse model expressing fluorescently tagged Munc18-1 from the endogenous munc18-1 locus. We show using fluorescence recovery after photobleaching in hippocampal neurons that the majority of Munc18-1 trafficked through axons and targeted to synapses via lateral diffusion together with syntaxin-1. Munc18-1 was strongly expressed at presynaptic terminals, with individual synapses showing a large variation in expression. Axon–synapse exchange rates of Munc18-1 were high: during stimulation, Munc18-1 rapidly dispersed from synapses and reclustered within minutes. Munc18-1 reclustering was independent of syntaxin-1, but required calcium influx and protein kinase C (PKC) activity. Importantly, a PKC-insensitive Munc18-1 mutant did not recluster. We show that synaptic Munc18-1 levels correlate with synaptic strength, and that synapses that recruit more Munc18-1 after stimulation have a larger releasable vesicle pool. Hence, PKC-dependent dynamic control of Munc18-1 levels enables individual synapses to tune their output during periods of activity. PMID:24590174

  2. Munc18-1 redistributes in nerve terminals in an activity- and PKC-dependent manner.

    PubMed

    Cijsouw, Tony; Weber, Jens P; Broeke, Jurjen H; Broek, Jantine A C; Schut, Desiree; Kroon, Tim; Saarloos, Ingrid; Verhage, Matthijs; Toonen, Ruud F

    2014-03-03

    Munc18-1 is a soluble protein essential for synaptic transmission. To investigate the dynamics of endogenous Munc18-1 in neurons, we created a mouse model expressing fluorescently tagged Munc18-1 from the endogenous munc18-1 locus. We show using fluorescence recovery after photobleaching in hippocampal neurons that the majority of Munc18-1 trafficked through axons and targeted to synapses via lateral diffusion together with syntaxin-1. Munc18-1 was strongly expressed at presynaptic terminals, with individual synapses showing a large variation in expression. Axon-synapse exchange rates of Munc18-1 were high: during stimulation, Munc18-1 rapidly dispersed from synapses and reclustered within minutes. Munc18-1 reclustering was independent of syntaxin-1, but required calcium influx and protein kinase C (PKC) activity. Importantly, a PKC-insensitive Munc18-1 mutant did not recluster. We show that synaptic Munc18-1 levels correlate with synaptic strength, and that synapses that recruit more Munc18-1 after stimulation have a larger releasable vesicle pool. Hence, PKC-dependent dynamic control of Munc18-1 levels enables individual synapses to tune their output during periods of activity.

  3. Structure-based lead discovery for protein kinase C zeta inhibitor design by exploiting kinase-inhibitor complex crystal structure data and potential therapeutics for preterm labour.

    PubMed

    Shao, Qing-Chun; Zhang, Cui-Juan; Li, Jie

    2014-10-14

    The protein kinase C (PKC) is a family of serine/threonine kinases with a broad range of cellular targets. Members of the PKC family participate at the diverse biological events involved in cellular proliferation, differentiation and survival. The PKC isoform zeta (PKCζ) is an atypical member that has recently been found to play an essential role in promoting human uterine contractility and thus been raised as a new target for treating preterm labour and other tocolytic diseases. In this study, an integrative protocol was described to graft hundreds of inhibitor ligands from their complex crystal structures with cognate kinases into the active pocket of PKCζ and, based on the modeled structures, to evaluate the binding strength of these inhibitors to the non-cognate PKCζ receptor by using a consensus scoring strategy. A total of 32 inhibitors with top score were compiled, and eight out of them were tested for inhibitory potency against PKCζ. Consequently, five compounds, i.e. CDK6 inhibitor fisetin, PIM1 inhibitor myricetin, CDK9 inhibitor flavopiridol and PknB inhibitor mitoxantrone as well as the promiscuous kinase inhibitor staurosporine showed high or moderate inhibitory activity on PKCζ, with IC50 values of 58 ± 9, 1.7 ± 0.4, 108 ± 17, 280 ± 47 and 0.019 ± 0.004 μM, respectively, while other three compounds, including two marketed drugs dasatinib and sunitinib as well as the Rho inhibitor fasudil, have not been detected to possess observable activity. Next, based on the modeled structure data we modified three flavonoid kinase inhibitors, i.e. fisetin, myricetin and flavopiridol, to generate a number of more potential molecular entities, two of which were found to have a moderately improved activity as compared to their parent compounds.

  4. Two amino acid sequences direct Aspergillus nidulans protein kinase C (PkcA) localization to hyphal apices and septation sites.

    PubMed

    Jackson-Hayes, Loretta; Hill, Terry W; Loprete, Darlene M; DelBove, Claire E; Shapiro, Justin A; Henley, Jordan L; Dawodu, Omolola O

    2015-01-01

    The Aspergillus nidulans ortholog of protein kinase C (pkcA) is involved in the organism's putative cell wall integrity (CWI) pathway, and PkcA also is highly localized at growing tips and forming septa. In the present work we identify the regions within PkcA that are responsible for its localization to hyphal tips and septation sites. To this end, we used serially truncated pkcA constructs and expressed them as green fluorescent protein (GFP) chimeras and identified two regions that direct PkcA localization. The first region is a 10 amino-acid sequence near the carboxyl end of the C2 domain that is required for localization to hyphal tips. Proteins containing this sequence also localize to septation sites. A second region between C2 and C1B (encompassing C1A) is sufficient for localization to septation sites but not to hyphal tips. We also report that localization to hyphal tips and septation sites alone is not sufficient for truncated constructs to complement hypersensitivity to the cell wall compromising agent calcofluor white in a strain bearing a mutation in the pkcA gene. Taken together, these results suggest that localization and stress response might be independent.

  5. aPKC phosphorylates JAM-A at Ser285 to promote cell contact maturation and tight junction formation.

    PubMed

    Iden, Sandra; Misselwitz, Steve; Peddibhotla, Swetha S D; Tuncay, Hüseyin; Rehder, Daniela; Gerke, Volker; Robenek, Horst; Suzuki, Atsushi; Ebnet, Klaus

    2012-03-05

    The PAR-3-atypical protein kinase C (aPKC)-PAR-6 complex has been implicated in the development of apicobasal polarity and the formation of tight junctions (TJs) in vertebrate epithelial cells. It is recruited by junctional adhesion molecule A (JAM-A) to primordial junctions where aPKC is activated by Rho family small guanosine triphosphatases. In this paper, we show that aPKC can interact directly with JAM-A in a PAR-3-independent manner. Upon recruitment to primordial junctions, aPKC phosphorylates JAM-A at S285 to promote the maturation of immature cell-cell contacts. In fully polarized cells, S285-phosphorylated JAM-A is localized exclusively at the TJs, and S285 phosphorylation of JAM-A is required for the development of a functional epithelial barrier. Protein phosphatase 2A dephosphorylates JAM-A at S285, suggesting that it antagonizes the activity of aPKC. Expression of nonphosphorylatable JAM-A/S285A interferes with single lumen specification during cyst development in three-dimensional culture. Our data suggest that aPKC phosphorylates JAM-A at S285 to regulate cell-cell contact maturation, TJ formation, and single lumen specification.

  6. Munc18-1 is a dynamically regulated PKC target during short-term enhancement of transmitter release

    PubMed Central

    Genç, Özgür; Kochubey, Olexiy; Toonen, Ruud F; Verhage, Matthijs; Schneggenburger, Ralf

    2014-01-01

    Transmitter release at synapses is regulated by preceding neuronal activity, which can give rise to short-term enhancement of release like post-tetanic potentiation (PTP). Diacylglycerol (DAG) and Protein-kinase C (PKC) signaling in the nerve terminal have been widely implicated in the short-term modulation of transmitter release, but the target protein of PKC phosphorylation during short-term enhancement has remained unknown. Here, we use a gene-replacement strategy at the calyx of Held, a large CNS model synapse that expresses robust PTP, to study the molecular mechanisms of PTP. We find that two PKC phosphorylation sites of Munc18-1 are critically important for PTP, which identifies the presynaptic target protein for the action of PKC during PTP. Pharmacological experiments show that a phosphatase normally limits the duration of PTP, and that PTP is initiated by the action of a ‘conventional’ PKC isoform. Thus, a dynamic PKC phosphorylation/de-phosphorylation cycle of Munc18-1 drives short-term enhancement of transmitter release during PTP. DOI: http://dx.doi.org/10.7554/eLife.01715.001 PMID:24520164

  7. Munc18-1 is a dynamically regulated PKC target during short-term enhancement of transmitter release.

    PubMed

    Genc, Ozgür; Kochubey, Olexiy; Toonen, Ruud F; Verhage, Matthijs; Schneggenburger, Ralf

    2014-02-11

    Transmitter release at synapses is regulated by preceding neuronal activity, which can give rise to short-term enhancement of release like post-tetanic potentiation (PTP). Diacylglycerol (DAG) and Protein-kinase C (PKC) signaling in the nerve terminal have been widely implicated in the short-term modulation of transmitter release, but the target protein of PKC phosphorylation during short-term enhancement has remained unknown. Here, we use a gene-replacement strategy at the calyx of Held, a large CNS model synapse that expresses robust PTP, to study the molecular mechanisms of PTP. We find that two PKC phosphorylation sites of Munc18-1 are critically important for PTP, which identifies the presynaptic target protein for the action of PKC during PTP. Pharmacological experiments show that a phosphatase normally limits the duration of PTP, and that PTP is initiated by the action of a 'conventional' PKC isoform. Thus, a dynamic PKC phosphorylation/de-phosphorylation cycle of Munc18-1 drives short-term enhancement of transmitter release during PTP. DOI: http://dx.doi.org/10.7554/eLife.01715.001.

  8. Autophosphorylation of the C2 domain inhibits translocation of the novel protein kinase C (nPKC) Apl II.

    PubMed

    Farah, Carole A; Lindeman, Amanda A; Siu, Vincent; Gupta, Micaela Das; Sossin, Wayne S

    2012-11-01

    Protein kinase Cs (PKCs) are critical signaling molecules controlled by complex regulatory pathways. Herein, we describe an important regulatory role for C2 domain phosphorylation. Novel PKCs (nPKCs) contain an N-terminal C2 domain that cannot bind to calcium. Previously, we described an autophosphorylation site in the Aplysia novel PKC Apl II that increased the binding of the C2 domain to lipids. In this study, we show that the function of this phosphorylation is to inhibit PKC translocation. Indeed, a phosphomimetic serine-glutamic acid mutation reduced translocation of PKC Apl II while blocking phosphorylation with a serine-alanine mutation enhanced translocation and led to the persistence of the kinase at the membrane longer after the end of the stimulation. Consistent with a role for autophosphorylation in regulating kinase translocation, inhibiting PKC activity using bisindolymaleimide 1 increased physiological translocation of PKC Apl II, whereas inhibiting phosphatase activity using calyculin A inhibited physiological translocation of PKC Apl II in neurons. Our results suggest a major role for autophosphorylation-dependent regulation of translocation.

  9. Sialic acid rescues repurified lipopolysaccharide-induced acute renal failure via inhibiting TLR4/PKC/gp91-mediated endoplasmic reticulum stress, apoptosis, autophagy, and pyroptosis signaling.

    PubMed

    Yang, Chih-Ching; Yao, Chien-An; Yang, Jyh-Chin; Chien, Chiang-Ting

    2014-09-01

    Lipopolysaccharides (LPS) through Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) activation induce systemic inflammation where oxidative damage plays a key role in multiple organ failure. Because of the neutralization of LPS toxicity by sialic acid (SA), we determined its effect and mechanisms on repurified LPS (rLPS)-evoked acute renal failure. We assessed the effect of intravenous SA (10 mg/kg body weight) on rLPS-induced renal injury in female Wistar rats by evaluating blood and kidney reactive oxygen species (ROS) responses, renal and systemic hemodynamics, renal function, histopathology, and molecular mechanisms. SA can interact with rLPS through a high binding affinity. rLPS dose- and time-dependently reduced arterial blood pressure, renal microcirculation and blood flow, and increased vascular resistance in the rats. rLPS enhanced monocyte/macrophage (ED-1) infiltration and ROS production and impaired kidneys by triggering p-IRE1α/p-JNK/CHOP/GRP78/ATF4-mediated endoplasmic reticulum (ER) stress, Bax/PARP-mediated apoptosis, Beclin-1/Atg5-Atg12/LC3-II-mediated autophagy, and caspase 1/IL-1β-mediated pyroptosis in the kidneys. SA treatment at 30 min, but not 60 min after rLPS stimulation, gp91 siRNA and protein kinase C-α (PKC) inhibitor efficiently rescued rLPS-induced acute renal failure via inhibition of TLR4/PKC/NADPH oxidase gp91-mediated ER stress, apoptosis, autophagy and pyroptosis in renal proximal tubular cells, and rat kidneys. In response to rLPS or IFNγ, the enhanced Atg5, FADD, LC3-II, and PARP expression can be inhibited by Atg5 siRNA. Albumin (10 mg/kg body weight) did not rescue rLPS-induced injury. In conclusion, early treatment (within 30 min) of SA attenuates rLPS-induced renal failure via the reduction in LPS toxicity and subsequently inhibiting rLPS-activated TLR4/PKC/gp91/ER stress/apoptosis/autophagy/pyroptosis signaling.

  10. CIN85 Modulates the Down-regulation of FcγRIIa Expression and Function by c-Cbl in a PKC-dependent Manner in Human Neutrophils*

    PubMed Central

    Marois, Louis; Vaillancourt, Myriam; Paré, Guillaume; Gagné, Valérie; Fernandes, Maria J. G.; Rollet-Labelle, Emmanuelle; Naccache, Paul H.

    2011-01-01

    We previously described a non-classical mechanism that arrests FcγRIIa signaling in human neutrophils once engaged by immune complexes or opsonized pathogens. The engagement of FcγRIIa leads to its ubiquitination by the ubiquitin ligase c-Cbl and degradation by the proteasome. Herein, we further examined some of the events regulating this novel pathway. The adaptor protein CIN85 was described in other systems to be involved in the regulation of the c-Cbl-dependent pathway. We found that CIN85 is expressed in human neutrophils and that it translocates like c-Cbl from the cytosol to the plasma membrane following receptor cross-linking. CIN85 was also recruited to the same subset of high density detergent-resistant membrane fractions in which stimulated FcγRIIa partitioned with c-Cbl. The integrity of these microdomains is essential to the FcγRIIa degradation process because the cholesterol-depleting agent methyl-β-cyclodextrin inhibits this event. Silencing the expression of CIN85 by siRNA in dibutyryl cyclic AMP-differentiated PLB 985 cells prevented FcγRIIa degradation and increased IgG-mediated phagocytosis. Confocal microscopy revealed that the presence of CIN85 is essential to the proper sorting of FcγRIIa during endocytosis. We also provide direct evidence that CIN85 is a substrate of serine/threonine kinase PKCs. Classical PKCs positively regulate FcγRIIa ubiquitination and degradation because these events were inhibited by Gö6976, a classical PKC inhibitor. We conclude that the ubiquitination and degradation of stimulated FcγRIIa mediated by c-Cbl are positively regulated by the adaptor protein CIN85 in a PKC-dependent manner and that these events contribute to the termination of FcγRIIa signaling. PMID:21372129

  11. p300/β-Catenin Interactions Regulate Adult Progenitor Cell Differentiation Downstream of WNT5a/Protein Kinase C (PKC)*

    PubMed Central

    Rieger, Megan E.; Zhou, Beiyun; Solomon, Nicola; Sunohara, Mitsuhiro; Li, Changgong; Nguyen, Cu; Liu, Yixin; Pan, Jie-hong; Minoo, Parviz; Crandall, Edward D.; Brody, Steven L.; Kahn, Michael; Borok, Zea

    2016-01-01

    Maintenance of stem/progenitor cell-progeny relationships is required for tissue homeostasis during normal turnover and repair. Wnt signaling is implicated in both maintenance and differentiation of adult stem/progenitor cells, yet how this pathway serves these dichotomous roles remains enigmatic. We previously proposed a model suggesting that specific interaction of β-catenin with either of the homologous Kat3 co-activators, p300 or CREB-binding protein, differentially regulates maintenance versus differentiation of embryonic stem cells. Limited knowledge of endogenous mechanisms driving differential β-catenin/co-activator interactions and their role in adult somatic stem/progenitor cell maintenance versus differentiation led us to explore this process in defined models of adult progenitor cell differentiation. We focused primarily on alveolar epithelial type II (AT2) cells, progenitors of distal lung epithelium, and identified a novel axis whereby WNT5a/protein kinase C (PKC) signaling regulates specific β-catenin/co-activator interactions to promote adult progenitor cell differentiation. p300/β-catenin but not CBP/β-catenin interaction increases as AT2 cells differentiate to a type I (AT1) cell-like phenotype. Additionally, p300 transcriptionally activates AT1 cell-specific gene Aqp-5. IQ-1, a specific inhibitor of p300/β-catenin interaction, prevents differentiation of not only primary AT2 cells, but also tracheal epithelial cells, and C2C12 myoblasts. p300 phosphorylation at Ser-89 enhances p300/β-catenin interaction, concurrent with alveolar epithelial cell differentiation. WNT5a, a traditionally non-canonical WNT ligand regulates Ser-89 phosphorylation and p300/β-catenin interactions in a PKC-dependent manner, likely involving PKCζ. These studies identify a novel intersection of canonical and non-canonical Wnt signaling in adult progenitor cell differentiation that has important implications for targeting β-catenin to modulate adult progenitor cell

  12. Inosine strongly enhances proliferation of human C32 melanoma cells through PLC-PKC-MEK1/2-ERK1/2 and PI3K pathways.

    PubMed

    Soares, Ana Sofia; Costa, Vera Marisa; Diniz, Carmen; Fresco, Paula

    2015-01-01

    Malignant melanoma is the most deadly type of skin cancer. The lack of effective pharmacological approaches for this tumour can be related to the incomplete understanding of the pathophysiological mechanisms involved in melanoma cell proliferation. Adenosine has growth-promoting and growth inhibitory effects on tumour cells. We aimed to investigate effects of adenosine and its metabolic product, inosine, on human C32 melanoma cells and the signalling pathways involved. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and bromodeoxyuridine (BrdU) proliferation assays were used to evaluate adenosine, adenosine deaminase and inosine effects, in the absence or presence of adenosine receptor (AR), A3 AR and P2Y1 R antagonists and PLC, PKC, MEK1/2 and PI3K inhibitors. ERK1/2 levels were determined using an ELISA kit. Adenosine and inosine levels were quantified using an enzyme-coupled assay. Adenosine caused cell proliferation through AR activation. Adenosine deaminase increased inosine levels (nanomolar concentrations) on the extracellular space, in a time-dependent manner, inducing proliferation through A3 AR activation. Micromolar concentrations of inosine enhanced proliferation through A3 AR activation, causing an increase in ERK1/2 levels, and P2Y1 R activation via ENT-dependent mechanisms. We propose the simultaneous activation of PLC-PKC-MEK1/2-ERK1/2 and PI3K pathways as the main mechanism responsible for the proliferative effect elicited by inosine and its significant role in melanoma cancer progression.

  13. Shikonin inhibits TNF-α production through suppressing PKC-NF-κB-dependent decrease of IL-10 in rheumatoid arthritis-like cell model.

    PubMed

    Sun, Wen-Xiao; Liu, Yan; Zhou, Wei; Li, He-Wei; Yang, Jian; Chen, Zhen-Bing

    2017-04-01

    Shikonin, a major effective component in the Chinese herbal medicine Lithospermum erythrorhizon Sieb., exhibits an anti-inflammatory property towards rheumatoid arthritis (RA), but the potential mechanism is unclear. Our aim was to investigate the mechanism of shikonin on the lipopolysaccharide (LPS)-induced fibroblast-like synoviocyte (LiFLS) inflammation model. Fibroblast-like synoviocytes (FLSs) were treated with 200 μg/ml of LPS for 24 h to establish the RA-like model, LiFLS. FLSs were pretreated with shikonin (0.1-1 μM) for 30 min in the treatment groups. Quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assays were used to detect mRNA and protein levels of interleukin (IL)-10 and tumor necrosis factor (TNF)-α. Signal proteins involved in IL-10 production were analyzed by Western blotting. Shikonin significantly reversed the inhibitory effects of LPS on IL-10 expression in FLSs by inactivating the PKC-NF-κB pathway. In addition, shikonin inhibited LPS-induced TNF-α expression in FLSs, and this effect was markedly diminished by IL-10-neutralizing antibody. The IL-10-mediated suppression of TNF-α transcription was demonstrated by no response to the protein synthesis inhibitor cyclohexamide and no mRNA decay. Shikonin inhibits LPS-induced TNF-α production in FLSs through suppressing the PKC-NF-κB-dependent decrease in IL-10, and this study also highlights the potential application of shikonin in the treatment of RA.

  14. Steady-state concentrations of mRNA encoding two inhibitors of protein kinase C in ovine luteal tissue.

    PubMed

    Juengel, J L; Melner, M H; Clapper, J A; Turzillo, A M; Moss, G E; Nett, T M; Niswender, G D

    1998-07-01

    Prostaglandin F2 alpha (PGF2 alpha) decreases secretion of progesterone from the corpus luteum in domestic ruminants. However, it is less effective during the early part of the oestrous cycle (Louis et al., 1973) and at the time of maternal recognition of pregnancy (Silvia and Niswender, 1984; Lacroix and Kann, 1986). Decreased luteal responsiveness may be due to failure of PGF2 alpha to activate fully its normal second messenger system, protein kinase C (PKC). Alternatively, increased resistance of the corpus luteum to PGF2 alpha might be attributable to greater concentrations of recently identified biological inhibitors of PKC. These possibilities were addressed by measuring steady-state concentrations of mRNA encoding PGF2 alpha receptor and two inhibitors of PKC, protein kinase C inhibitor-1 (PKCI-1) and kinase C inhibitor protein-1 (KCIP-1, brain 14-3-3 protein), in corpora lutea collected from ewes on days 4, 10 and 15 of the oestrous cycle (n = 5 per day) and day 15 of pregnancy (n = 7). There were no differences in mean concentrations of mRNA encoding PGF2 alpha receptor among the groups. However, concentrations of mRNA encoding both inhibitors of PKC were higher (P < 0.01) on day 4 of the oestrous cycle compared with the other groups. Treatment of ewes with a luteolytic dose of PGF2 alpha, which activates PKC, did not change concentrations of mRNA encoding either PKCI-1 or KCIP-I up to 24 h later. Luteal expression of mRNA encoding the PKC inhibitors and PGF2 alpha receptor was also examined in ewes treated with oestradiol in vivo for 16 h in the midluteal phase. High concentrations of oestradiol in serum (20 and 70 pg ml-1) did not influence quantities of any of the mRNAs examined. Therefore, an increase in PKC inhibitors may be involved in resistance of the corpus luteum to PGF2 alpha during the early part of the oestrous cycle but does not appear to mediate the increased resistance of the corpus luteum to PGF2 alpha during maternal recognition of

  15. Interaction between protein kinase C and protein kinase A can modulate transmitter release at the rat neuromuscular synapse.

    PubMed

    Santafé, M M; Garcia, N; Lanuza, M A; Tomàs, M; Tomàs, J

    2009-02-15

    We used intracellular recording to investigate the functional interaction between protein kinase C (PKC) and protein kinase A (PKA) signal transduction cascades in the control of transmitter release in the neuromuscular synapses from adult rats. Our results indicate that: 1) PKA and PKC are independently involved in asynchronous release. 2) Evoked acetylcholine (ACh) release is enhanced with the PKA agonist Sp-8-BrcAMP and the PKC agonist phorbol ester (PMA). 3) PKA has a constitutive role in promoting a component of normal evoked transmitter release because, when the kinase is inhibited with H-89, the release diminishes. However, the PKC inhibitor calphostin C (CaC) does not affect ACh release. 4) PKA regulates neurotransmission without PKC involvement because, after PMA or CaC modulation of the PKC activity, coupling to the ACh release of PKA can normally be stimulated with Sp-8-BrcAMP or inhibited with H-89. 5) After PKA inhibition with H-89, PKC stimulation with PMA (or inhibition with CaC) does not lead to any change in evoked ACh release. However, in PKA-stimulated preparations with Sp-8-BrcAMP, PKC becomes tonically active, thus potentiating a component of release that can now be blocked with CaC. In normal conditions, therefore, PKA was able to modulate ACh release independently of PKC activity, whereas PKA stimulation caused the PKC coupling to evoked release. In contrast, PKA inhibition prevent PKC stimulation (with the phorbol ester) and coupling to ACh output. There was therefore some dependence of PKC on PKA activity in the fine control of the neuromuscular synaptic functionalism and ACh release.

  16. PKC phosphorylates residues in the N-terminal of the DA transporter to regulate amphetamine-induced DA efflux.

    PubMed

    Wang, Qiang; Bubula, Nancy; Brown, Jason; Wang, Yunliang; Kondev, Veronika; Vezina, Paul

    2016-05-27

    The DA transporter (DAT), a phosphoprotein, controls extracellular dopamine (DA) levels in the central nervous system through transport or reverse transport (efflux). Multiple lines of evidence support the claim that PKC significantly contributes to amphetamine-induced DA efflux. Other signaling pathways, involving CaMKII and ERK, have also been shown to regulate DAT mediated efflux. Here we assessed the contribution of putative PKC residues (S4, S7, S13) in the N-terminal of the DAT to amphetamine-induced DA efflux by transfecting DATs containing different serine to alanine (S-A) point mutations into DA pre-loaded HEK-293 cells and incubating these cells in amphetamine (2μM). The effects of a S-A mutation at the non-PKC residue S12 and a threonine to alanine (T-A) mutation at the ERK T53 residue were also assessed for comparison. WT-DATs were used as controls. In an initial experiment, we confirmed that inhibiting PKC with Go6976 (130nM) significantly reduced amphetamine-induced DA efflux. In subsequent experiments, cells transfected with the S4A, S12A, S13A, T53A and S4,7,13A mutants showed a reduction in amphetamine-induced DA efflux similar to that observed with Go6976. Interestingly, cells transfected with the S7A mutant, identified by some as a PKC-PKA residue, showed unperturbed WT-DAT levels of amphetamine-induced DA efflux. These results indicate that phosphorylation by PKC of select residues in the DAT N-terminal can regulate amphetamine-induced efflux. PKC can act either independently or in concert with other kinases such as ERK to produce this effect.

  17. Inhibitory responses in Aplysia pleural sensory neurons act to block excitability, transmitter release, and PKC Apl II activation.

    PubMed

    Dunn, Tyler W; Farah, Carole A; Sossin, Wayne S

    2012-01-01

    Expression of the 5-HT(1Apl(a)) receptor in Aplysia pleural sensory neurons inhibited 5-HT-mediated translocation of the novel PKC Apl II in sensory neurons and prevented PKC-dependent synaptic facilitation at sensory to motoneuron synapses (Nagakura et al. 2010). We now demonstrate that the ability of inhibitory receptors to block PKC activation is a general feature of inhibitory receptors and is found after expression of the 5-HT(1Apl(b)) receptor and with activation of endogenous dopamine and FMRFamide receptors in sensory neurons. Pleural sensory neurons are heterogeneous for their inhibitory response to endogenous transmitters, with dopamine being the most prevalent, followed by FMRFamide, and only a small number of neurons with inhibitory responses to 5-HT. The inhibitory response is dominant, reduces membrane excitability and synaptic efficacy, and can reverse 5-HT facilitation at both naive and depressed synapses. Indeed, dopamine can reverse PKC translocation during the continued application of 5-HT. Reversal of translocation can also be seen after translocation mediated by an analog of diacylglycerol, suggesting inhibition is not through blockade of diacylglycerol production. The effects of inhibition on PKC translocation can be rescued by phosphatidic acid, consistent with the inhibitory response involving a reduction or block of production of this lipid. However, phosphatidic acid could not recover PKC-dependent synaptic facilitation due to an additional inhibitory effect on the non-L-type calcium flux linked to synaptic transmission. In summary, we find a novel mechanism downstream of inhibitory receptors linked to inhibition of PKC activation in Aplysia sensory neurons.

  18. Thrombin/Matrix Metalloproteinase-9-Dependent SK-N-SH Cell Migration is Mediated Through a PLC/PKC/MAPKs/NF-κB Cascade.

    PubMed

    Yang, Chien-Chung; Lin, Chih-Chung; Chien, Peter Tzu-Yu; Hsiao, Li-Der; Yang, Chuen-Mao

    2016-11-01

    Thrombin has been known to activate inflammatory genes including matrix metalloproteinases (MMPs). The elevated expression of MMP-9 has been observed in patients with neuroinflammatory diseases and may contribute to the pathology of brain diseases. However, the mechanisms underlying thrombin-induced MMP-9 expression in SK-N-SH cells remain unknown. The effects of thrombin on MMP-9 expression were examined in SK-N-SH cells by gelatin zymography, Western blot, real-time PCR, promoter activity assay, and cell migration assay. The detailed mechanisms were analyzed by using pharmacological inhibitors and small intefering RNA (siRNA) transfection. Here, we demonstrated that thrombin induced the expression of proform MMP-9 and migration of SK-N-SH cells, which were attenuated by pretreatment with the inhibitor of thrombin (PPACK), Gq (GPA2A), PC-PLC (D609), PI-PLC (ET-18-OCH3), nonselective protien kinase C (PKC, GF109203X), PKCα/βII (Gö6983), PKCδ (Rottlerin), p38 mitogen-activated protein kinases (MAPK) (SB202190), JNK1/2 (SP600125), or NF-κB (Bay11-7082 or Helenalin) and transfection with siRNA of Gq, PKCα, PKCβ, PKCδ, p38, JNK1/2, IKKα, IKKβ, or p65. Moreover, thrombin-stimulated PKCα/βII, PKCδ, p38 MAPK, JNK1/2, or p65 phosphorylation was abrogated by their respective inhibitor of PPACK, GPA2A, D609, ET-18-OCH3, Gö6983, Rottlerin, SB202190, SP600125, Bay11-7082, or Helenalin. Pretreatment with these inhibitors or transfection with MMP-9 siRNA also blocked thrombin-induced SK-N-SH cell migration. Our results show that thrombin stimulates a Gq/PLC/PKCs/p38 MAPK and JNK1/2 cascade, which in turn triggers NF-κB activation and ultimately induces MMP-9 expression and cell migration in SK-N-SH cells.

  19. Serotonin receptor antagonists discriminate between PKA- and PKC-mediated plasticity in aplysia sensory neurons.

    PubMed

    Dumitriu, Bogdan; Cohen, Jonathan E; Wan, Qin; Negroiu, Andreea M; Abrams, Thomas W

    2006-04-01

    Highly selective serotonin (5-hydroxytryptamine, 5-HT) receptor antagonists developed for mammals are ineffective in Aplysia due to the evolutionary divergence of neurotransmitter receptors and because the higher ionic strength of physiological saline for marine invertebrates reduces antagonist affinity. It has therefore been difficult to identify antagonists that specifically block individual signaling cascades initiated by 5-HT. We studied two broad-spectrum 5-HT receptor antagonists that have been characterized biochemically in Aplysia CNS: methiothepin and spiperone. Methiothepin is highly effective in inhibiting adenylyl cyclase (AC)-coupled 5-HT receptors in Aplysia. Spiperone, which blocks phospholipase C (PLC)-coupled 5-HT receptors in mammals, does not block AC-coupled 5-HT receptors in Aplysia. In electrophysiological studies, we explored whether methiothepin and spiperone can be used in parallel to distinguish between the AC-cAMP and PLC-protein kinase C (PKC) modulatory cascades that are initiated by 5-HT. 5-HT-induced broadening of the sensory neuron action potential in the presence of tetraethylammonium/nifedipine, which is mediated by modulation of the S-K+ currents, was used an assay for the AC-cAMP cascade. Spike broadening initiated by 5 microM 5-HT was unaffected by 100 microM spiperone, whereas it was effectively blocked by 100 microM methiothepin. Facilitation of highly depressed sensory neuron-to-motor neuron synapses by 5-HT was used as an assay for the PLC-PKC cascade. Spiperone completely blocked facilitation of highly depressed synapses by 5 microM 5-HT. In contrast, methiothepin produced a modest, nonsignificant, reduction in the facilitation of depressed synapses. Interestingly, these experiments revealed that the PLC-PKC cascade undergoes desensitization during exposure to 5-HT.

  20. Downregulation of transient K+ channels in dendrites of hippocampal CA1 pyramidal neurons by activation of PKA and PKC.

    PubMed

    Hoffman, D A; Johnston, D

    1998-05-15

    We have reported recently a high density of transient A-type K+ channels located in the distal dendrites of CA1 hippocampal pyramidal neurons and shown that these channels shape EPSPs, limit the back-propagation of action potentials, and prevent dendritic action potential initiation (). Because of the importance of these channels in dendritic signal propagation, their modulation by protein kinases would be of significant interest. We investigated the effects of activators of cAMP-dependent protein kinase (PKA) and the Ca2+-dependent phospholipid-sensitive protein kinase (PKC) on K+ channels in cell-attached patches from the distal dendrites of hippocampal CA1 pyramidal neurons. Inclusion of the membrane-permeant PKA activators 8-bromo-cAMP (8-br-cAMP) or forskolin in the dendritic patch pipette resulted in a depolarizing shift in the activation curve for the transient channels of approximately 15 mV. Activation of PKC by either of two phorbol esters also resulted in a 15 mV depolarizing shift of the activation curve. Neither PKA nor PKC activation affected the sustained or slowly inactivating component of the total outward current. This downregulation of transient K+ channels in the distal dendrites may be responsible for some of the frequently reported increases in cell excitability found after PKA and PKC activation. In support of this hypothesis, we found that activation of either PKA or PKC significantly increased the amplitude of back-propagating action potentials in distal dendrites.

  1. PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination

    PubMed Central

    Barrera, Susana P.; Castrejon-Tellez, Vicente; Trinidad, Margarita; Robles-Escajeda, Elisa; Vargas-Medrano, Javier; Varela-Ramirez, Armando; Miranda, Manuel

    2015-01-01

    Neurotransmitter transporter ubiquitination is emerging as the main mechanism for endocytosis and sorting of cargo into lysosomes. In this study, we demonstrate PKC-dependent ubiquitination of three different isoforms of the glycine transporter 1 (GlyT1). Incubation of cells expressing transporter with the PKC activator phorbol ester induced a dramatic, time-dependent increase in GlyT1 ubiquitination, followed by accumulation of GlyT1 in EEA1 positive early endosomes. This occurred via a mechanism that was abolished by inhibition of PKC. GlyT1 endocytosis was confirmed in both retinal sections and primary cultures of mouse amacrine neurons. Replacement of only all lysines in the N-and C-termini to arginines prevented ubiquitination and endocytosis, displaying redundancy in the mechanism of ubiquitination. Interestingly, a 40–50% reduction in glycine uptake was detected in phorbol-ester stimulated cells expressing the WT-GlyT1, whereas no significant change was for the mutant protein, demonstrating that endocytosis participates in the reduction of uptake. Consistent with previous findings for the dopamine transporter DAT, ubiquitination of GlyT1 tails functions as sorting signal to deliver transporter into the lysosome and removal of ubiquitination sites dramatically attenuated the rate of GlyT1 degradation. Finally, we showed for the first time that PKC-dependent GlyT1 phosphorylation was not affected by removal of ubiquitination sites, suggesting separate PKC-dependent signaling events for these posttranslational modifications. PMID:26418248

  2. Importance of PKC and PI3Ks in ethanol-induced contraction of cerebral arterial smooth muscle.

    PubMed

    Yang, Z W; Wang, J; Zheng, T; Altura, B T; Altura, B M

    2001-05-01

    We investigated the relationships of two potential intracellular signaling pathways, protein kinase C (PKC) and phosphatidylinositol 3-kinases (PI3Ks), to ethanol-induced contractions in cerebral arteries. Ethanol (20-200 mM) induces concentration-dependent constriction in isolated canine basilar arteries that is inhibited in a concentration-dependent manner by pretreatment of these vessels with 10(-9)-10(-3) M Gö-6976 (an antagonist selective for PKC-alpha and PKC-betaI), 10(-10)-10(-4) M bisindolylmaleimide I (a specific antagonist of PKC), and 10(-10)-10(-4) M wortmannin or 10(-8)-10(-2) M LY-294002 (selective antagonists of PI3Ks). Ethanol-induced increases in intracellular Ca(2+) concentration (from approximately 100 to approximately 500 nM) in canine basilar smooth muscle cells are also suppressed markedly (approximately 20-70%) in the presence of a similar concentration range of Gö-6976, bisindolymaleimide I, wortmannin, or LY-294002. This study suggests that activation of PKC isoforms and PI3Ks appears to be an important signaling pathway in ethanol-induced vasoconstriction of cerebral blood vessels.

  3. Aloe-emodin modulates PKC isozymes, inhibits proliferation, and induces apoptosis in U-373MG glioma cells.

    PubMed

    Acevedo-Duncan, Mildred; Russell, Christopher; Patel, Sapna; Patel, Rekha

    2004-12-20

    Aloe-emodin (1,8-dihydroy-3-[hydroxymethyl]-anthraquione) purified from Aloe vera leaves has been reported to have antitumor activity. The objectives of our research were to determine how aloe-emodin regulates the cell cycle, cell proliferation and protein kinase C (PKC) during glioma growth and development. To establish the cell cycle effects of aloe-emodin on brain cells [transformed glia cell line (SVG) and human glioma U-373MG cell line (U-373MG)], cells were treated with either dimethylsulfoxide (DMSO; control) or aloe-emodin (40 microM). Results from flow cytometry demonstrated that aloe-emodin delayed the number of cells entering and exiting DNA synthesis (S) phase in both SVG and U-373MG cells indicating that aloe-emodin may inhibit S phase progression. Assessment of cell viability demonstrated that SVG and U-373MG glioma cell were highly sensitive to aloe-emodin. The aloe-emodin-induced decreased proliferation was sustained at 48-96 h. A PKC activity assay was quantified to establish the role of PKC in aloe-emodin's mode of action. Exposure of SVG and U-373MG glioma cells to aloe-emodin suppressed PKC activity and reduced the protein content of most of the PKC isozymes. We determined that cancer growth inhibition by aloe-emodin was due to apoptosis (i.e., programmed cell death). Taken together, these results support the hypothesis that aloe-emodin represents a novel antitumor chemotherapeutic drug.

  4. Clueless regulates aPKC activity and promotes self-renewal cell fate in Drosophila lgl mutant larval brains.

    PubMed

    Goh, Li Hui; Zhou, Xiu; Lee, Mei Chin; Lin, Shuping; Wang, Huashan; Luo, Yan; Yang, Xiaohang

    2013-09-15

    Asymmetric cell division of Drosophila neural stem cells or neuroblasts is an important process which gives rise to two different daughter cells, one of which is the stem cell itself and the other, a committed or differentiated daughter cell. During neuroblast asymmetric division, atypical Protein Kinase C (aPKC) activity is tightly regulated; aberrant levels of activity could result in tumorigenesis in third instar larval brain. We identified clueless (clu), a genetic interactor of parkin (park), as a novel regulator of aPKC activity. It preferentially binds to the aPKC/Bazooka/Partition Defective 6 complex and stabilizes aPKC levels. In clu mutants, Miranda (Mira) and Numb are mislocalized in small percentages of dividing neuroblasts. Adult mutants are short-lived with severe locomotion defects. Clu promotes tumorigenesis caused by loss of function of lethal(2) giant larvae (lgl) in the larval brain. Removal of clu in lgl mutants rescues Mira and Numb mislocalization and restores the enlarged brain size. Western blot analyses indicate that the rescue is due to the down-regulation of aPKC levels in the lgl clu double mutant. Interestingly, the phenotype of the park mutant, which causes Parkinson's Disease-like symptoms in adult flies, is reminiscent of that of clu in neuroblast asymmetric division. Our study provides the first clue for the potential missing pathological link between temporally separated neurogenesis and neurodegeneration events; the minor defects during early neurogenesis could be a susceptible factor contributing to neurodegenerative diseases at later stages of life.

  5. Enhancement of insulin-induced PI3K/Akt/GSK-3beta and ERK signaling by neuronal nicotinic receptor/PKC-alpha/ERK pathway: up-regulation of IRS-1/-2 mRNA and protein in adrenal chromaffin cells.

    PubMed

    Sugano, Takashi; Yanagita, Toshihiko; Yokoo, Hiroki; Satoh, Shinya; Kobayashi, Hideyuki; Wada, Akihiko

    2006-07-01

    In cultured bovine adrenal chromaffin cells treated with nicotine (10 microm for 24 h), phosphorylation of Akt, glycogen synthase kinase-3beta (GSK-3beta) and extracellular signal-regulated kinase (ERK)1/2 induced by insulin (100 nm for 10 min) was enhanced by approximately 62%, without altering levels of these protein kinases. Nicotine produced time (> 12 h)- and concentration (EC(50) 3.6 and 13 microm)-dependent increases in insulin receptor substrate (IRS)-1 and IRS-2 levels by approximately 125 and 105%, without altering cell surface density of insulin receptors. In these cells, insulin-induced tyrosine phosphorylation of IRS-1/IRS-2 and recruitment of phosphoinositide 3-kinase (PI3K) to IRS-1/IRS-2 were augmented by approximately 63%. The increase in IRS-1/IRS-2 levels induced by nicotine was prevented by nicotinic acetylcholine receptor (nAChR) antagonists, the Ca(2+) chelator 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid tetrakis-acetoxymethyl ester, cycloheximide or actinomycin D. Nicotine increased IRS-1 and IRS-2 mRNA levels by approximately 57 and approximately 50%, and this was prevented by conventional protein kinase C (cPKC) inhibitor Gö6976, or ERK kinase inhibitors PD98059 and U0126. Nicotine phosphorylated cPKC-alpha, thereby increasing phosphorylation of ERK1/ERK2, as demonstrated by using Gö6976, PD98059 or U0126. Selective activation of cPKC-alpha by thymeleatoxin mimicked these effects of nicotine. Thus, stimulation of nAChRs up-regulated expression of IRS-1/IRS-2 via Ca(2+)-dependent sequential activation of cPKC-alpha and ERK, and enhanced insulin-induced PI3K/Akt/GSK-3beta and ERK signaling pathways.

  6. Induction of TRIM22 by IFN-γ Involves JAK and PC-PLC/PKC, but Not MAPKs and pI3K/Akt/mTOR Pathways.

    PubMed

    Gao, Bo; Xu, Wei; Wang, Yaxin; Zhong, Linmao; Xiong, Sidong

    2013-10-01

    Tripartite motif (TRIM) 22 plays an important role in interferons (IFNs)-mediated antiviral activity. We previously demonstrated that interferon regulatory factor-1 (IRF-1) played a central role in IFN-γ-induced TRIM22 expression via binding to a special cis-element named 5' extended IFN-stimulating response element (5'eISRE). In this study, we sought to identify the signaling pathways involved in TRIM22 induction by IFN-γ. By using various pharmacological inhibitors, it was found that the activity of tyrosine kinase and phosphatidylcholine-phospholipase C (PC-PLC), but not phosphatidylinositol-phospholipase C (PI-PLC) and phospholipase D (PLD), was required for IFN-γ-induced TRIM22 expression in HepG2 cells. Tyrosine kinase Janus kinase (JAK), not SRC and PYK2, played an indispensable role in TRIM22 induction. Inhibition of protein kinase C (PKC) activity also significantly attenuated IFN-γ induction of TRIM22. Although treatment with IFN-γ resulted in the stimulation of mitogen-activated protein kinases (MAPKs) (p38, ERK, and JNK) and pI3K/Akt/mTOR pathways in HepG2 cells, the inhibition of their activity did not affect IFN-γ-stimulated TRIM22 expression. Further studies showed that overexpression of JAK1 and PKCα activated TRIM22 promoter activity in a 5'eISRE-dependent manner, and inhibition of not only JAK but also PC-PLC/PKC pathways significantly attenuated IFN-γ-induced IRF-1 expression in HepG2 cells. Taken together, these data indicated that IFN-γ induced TRIM22 expression via activation of JAK and PC-PLC/PKC signaling pathways, which involved the cis-element 5'eISRE and the transactivator IRF-1.

  7. Cholesterol Inhibits M-type K+ Channels via Protein Kinase C-dependent Phosphorylation in Sympathetic Neurons*

    PubMed Central

    Lee, Seul-Yi; Choi, Hyun-Kyung; Kim, Seong-Tae; Chung, Sungkwon; Park, Myoung Kyu; Cho, Jung-Hwa; Ho, Won-Kyung; Cho, Hana

    2010-01-01

    M-type (KCNQ) potassium channels play an important role in regulating the action potential firing in neurons. Here, we investigated the effect of cholesterol on M current in superior cervical ganglion (SCG) sympathetic neurons, using the patch clamp technique. M current was inhibited in a dose-dependent manner by cholesterol loading with a methyl-β-cyclodextrin-cholesterol complex. This effect was prevented when membrane cholesterol level was restored by including empty methyl-β-cyclodextrin in the pipette solution. Dialysis of cells with AMP-PNP instead of ATP prevented cholesterol action on M currents. Protein kinase C (PKC) inhibitor, calphostin C, abolished cholesterol-induced inhibition whereas the PKC activator, PDBu, mimicked the inhibition of M currents by cholesterol. The in vitro kinase assay showed that KCNQ2 subunits of M channel can be phosphorylated by PKC. A KCNQ2 mutant that is defective in phosphorylation by PKC failed to show current inhibition not only by PDBu but also by cholesterol. These results indicate that cholesterol-induced inhibition of M currents is mediated by PKC phosphorylation. The inhibition of M currents by PDBu and cholesterol was completely blocked by PIP2 loading, indicating that the decrease in PIP2-channel interaction underlies M channel inhibition by PKC-mediated phosphorylation. We conclude that cholesterol specifically regulates M currents in SCG neurons via PKC activation. PMID:20123983

  8. Involvement of PKA, CaMKII, PKC, MAPK/ERK and PI3K in the acute antidepressant-like effect of ferulic acid in the tail suspension test.

    PubMed

    Zeni, Ana Lúcia Bertarello; Zomkowski, Andréa Dias Elpo; Maraschin, Marcelo; Rodrigues, Ana Lúcia Severo; Tasca, Carla Inês

    2012-12-01

    Ferulic acid (FA, 4-hydroxy-3-methoxycinnamic acid) is a phytochemical compound naturally present in several plants and foods that is approved as an antioxidant additive and food preservative. It exerts a beneficial action in chronic mild stress-induced depressive-like behavior and produces an acute antidepressant-like effect in the tail suspension test (TST) through the activation of the serotonergic system. This study was aimed at investigating the possible involvement of signaling pathways in the antidepressant-like effect of acute and oral administration of FA, in the TST in mice. The anti-immobility effect of orally administered FA (0.01mg/kg, p.o.) was prevented by pretreatment of mice with H-89 (1μg/site, i.c.v., an inhibitor of PKA), KN-62 (1μg/site, i.c.v., an inhibitor of CaMKII), GF109203X (5ng/site, i.c.v., an inhibitor of PKC), U0126 (5μg/site, i.c.v., an inhibitor of MAPK/ERK) or LY294002 (10nmol/site, i.c.v., an inhibitor of PI3K), all involved with neurotrophic signaling pathways. The results demonstrated that FA exerts antidepressant-like effect in the TST in mice, through the activation of signaling pathways related to neuroplasticity, neurogenesis and cell survival.

  9. Agonist-induced activation of histamine H3 receptor signals to extracellular signal-regulated kinases 1 and 2 through PKC-, PLD-, and EGFR-dependent mechanisms.

    PubMed

    Lai, Xiangru; Ye, Lingyan; Liao, Yuan; Jin, Lili; Ma, Qiang; Lu, Bing; Sun, Yi; Shi, Ying; Zhou, Naiming

    2016-04-01

    The histamine H3 receptor (H3R), abundantly expressed in the central and the peripheral nervous system, has been recognized as a promising target for the treatment of various important CNS diseases including narcolepsy, Alzheimer's disease, and attention deficit hyperactivity disorder. The H3R acts via Gi/o -proteins to inhibit adenylate cyclase activity and modulate MAPK activity. However, the underlying molecular mechanisms for H3R mediation of the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) remain to be elucidated. In this study, using HEK293 cells stably expressing human H3R and mouse primary cortical neurons endogenously expressing mouse H3R, we found that the H3R-mediated activation of ERK1/2 was significantly blocked by both the pertussis toxin and the MEK1/2 inhibitor U0126. Upon stimulation by H3R agonist histamine or imetit, H3R was shown to rapidly induce ERK1/2 phosphorylation via PLC/PKC-, PLDs-, and epidermal growth factor receptor (EGFR) transactivation-dependent pathways. Furthermore, it was also indicated that while the βγ-subunits play a key role in H3R-activated ERK1/2 phosphorylation, β-arrestins were not required for ERK1/2 activation. In addition, when the cultured mouse cortical neurons were exposed to oxygen and glucose deprivation conditions (OGD), imetit exhibited neuroprotective properties through the H3R. Treatment of cells with the inhibitor UO126 abolished these protective effects. This suggests a possible neuroprotective role of the H3R-mediated ERK1/2 pathway under hypoxia conditions. These observations may provide new insights into the pharmacological effects and the physiological functions modulated by the H3R-mediated activation of ERK1/2. Histamine H3 receptors are abundantly expressed in the brain and play important roles in various CNS physiological functions. However, the underlying mechanisms for H3R-induced activation of extracellular signal-regulated kinase (ERK)1/2 remain largely unknown. Here

  10. PKC in motorneurons underlies self-learning, a form of motor learning in Drosophila

    PubMed Central

    Colomb, Julien

    2016-01-01

    Tethering a fly for stationary flight allows for exquisite control of its sensory input, such as visual or olfactory stimuli or a punishing infrared laser beam. A torque meter measures the turning attempts of the tethered fly around its vertical body axis. By punishing, say, left turning attempts (in a homogeneous environment), one can train a fly to restrict its behaviour to right turning attempts. It was recently discovered that this form of operant conditioning (called operant self-learning), may constitute a form of motor learning in Drosophila. Previous work had shown that Protein Kinase C (PKC) and the transcription factor dFoxP were specifically involved in self-learning, but not in other forms of learning. These molecules are specifically involved in various forms of motor learning in other animals, such as compulsive biting in Aplysia, song-learning in birds, procedural learning in mice or language acquisition in humans. Here we describe our efforts to decipher which PKC gene is involved in self-learning in Drosophila. We also provide evidence that motorneurons may be one part of the neuronal network modified during self-learning experiments. The collected evidence is reminiscent of one of the simplest, clinically relevant forms of motor learning in humans, operant reflex conditioning, which also relies on motorneuron plasticity. PMID:27168980

  11. Activation of transient receptor potential vanilloid 1 by lipoxygenase metabolites depends on PKC phosphorylation.

    PubMed

    Kumar, Rakesh; Hazan, Adina; Geron, Matan; Steinberg, Rebbeca; Livni, Lital; Matzner, Henry; Priel, Avi

    2017-03-01

    Peripheral neuronal activation by inflammatory mediators is a multifaceted physiological response that involves a multitude of regulated cellular functions. One key pathway that has been shown to be involved in inflammatory pain is Gq/GPCR, whose activation by inflammatory mediators is followed by the regulated response of the cation channel transient receptor potential vanilloid 1 (TRPV1). However, the mechanism that underlies TRPV1 activation downstream of the Gq/GPCR pathway has yet to be fully defined. In this study, we employ pharmacological and molecular biology tools to dissect this activation mechanism via perforated-patch recordings and calcium imaging of both neurons and a heterologous system. We showed that TRPV1 activity downstream of Gq/GPCR activation only produced a subdued current, which was noticeably different from the robust current that is typical of TRPV1 activation by exogenous stimuli. Moreover, we specifically demonstrated that 2 pathways downstream of Gq/GPCR signaling, namely endovanilloid production by lipoxygenases and channel phosphorylation by PKC, converge on TRPV1 to evoke a tightly regulated response. Of importance, we show that only when both pathways are acting on TRPV1 is the inflammatory-mediated response achieved. We propose that the requirement of multiple signaling events allows subdued TRPV1 activation to evoke regulated neuronal response during inflammation.-Kumar R., Hazan, A., Geron, M., Steinberg, R., Livni, L., Matzner, H., Priel, A. Activation of transient receptor potential vanilloid 1 by lipoxygenase metabolites depends on PKC phosphorylation.

  12. Protein synthesis required for long-term memory is induced by PKC activation on days before associative learning.

    PubMed

    Alkon, Daniel L; Epstein, Herman; Kuzirian, Alan; Bennett, M Catherine; Nelson, Thomas J

    2005-11-08

    Protein synthesis has long been known to be required for associative learning to consolidate into long-term memory. Here we demonstrate that PKC isozyme activation on days before training can induce the synthesis of proteins necessary and sufficient for subsequent long-term memory consolidation. Bryostatin (Bryo), a macrolide lactone with efficacy in subnanomolar concentrations and a potential therapeutic for Alzheimer's disease, is a potent activator of PKC, some of whose isozymes undergo prolonged activation after associative learning. Under normal conditions, two training events with paired visual and vestibular stimuli cause short-term memory of the mollusc Hermissenda that lasts approximately 7 min. However, after 4-h exposures to Bryo (0.25 ng/ml) on two preceding days, the same two training events produced long-term conditioning that lasted >1 week and that was not blocked by anisomycin (1 mug/ml). Anisomycin, however, eliminated long-term memory lasting at least 1 week after nine training events. Both the nine training events alone and two Bryo exposures plus two training event regimens caused comparably increased levels of the PKC alpha-isozyme substrate calexcitin in identified type B neurons and enhanced PKC activity in the membrane fractions. Furthermore, Bryo increased overall protein synthesis in cultured mammalian neurons by up to 60% for >3 days. The specific PKC antagonist Ro-32-0432 blocked much of this Bryo-induced protein synthesis as well as the Bryo-induced enhancement of the behavioral conditioning. Thus, Bryo-induced PKC activation produces those proteins necessary and sufficient for long-term memory on days in advance of the training events themselves.

  13. A screen for Fli-1 transcriptional modulators identifies PKC agonists that induce erythroid to megakaryocytic differentiation and suppress leukemogenesis.

    PubMed

    Liu, Tangjingjun; Yao, Yao; Zhang, Gang; Wang, Ye; Deng, Bin; Song, Jialei; Li, Xiaogang; Han, Fei; Xiao, Xiao; Yang, Jue; Xia, Lei; Li, You-Jun; Plachynta, Maksym; Zhang, Mu; Yan, Chen; Mu, Shuzhen; Luo, Heng; Zacksenhaus, Eldad; Hao, Xiaojiang; Ben-David, Yaacov

    2016-12-30

    The ETS-related transcription factor Fli-1 affects many developmental programs including erythroid and megakaryocytic differentiation, and is frequently de-regulated in cancer. Fli-1 was initially isolated following retrovirus insertional mutagenesis screens for leukemic initiator genes, and accordingly, inhibition of this transcription factor can suppress leukemia through induction of erythroid differentiation. To search for modulators of Fli-1, we hereby performed repurposing drug screens with compounds isolated from Chinese medicinal plants. We identified agents that can transcriptionally activate or inhibit a Fli-1 reporter. Remarkably, agents that increased Fli-1 transcriptional activity conferred a strong anti-cancer activity upon Fli-1-expressing leukemic cells in culture. As opposed to drugs that suppress Fli1 activity and lead to erythroid differentiation, growth suppression by these new Fli-1 transactivating compounds involved erythroid to megakaryocytic conversion (EMC). The identified compounds are structurally related to diterpene family of small molecules, which are known agonists of protein kinase C (PKC). In accordance, these PKC agonists (PKCAs) induced PKC phosphorylation leading to activation of the mitogen-activated protein kinase (MAPK) pathway, increased cell attachment and EMC, whereas pharmacological inhibition of PKC or MAPK diminished the effect of our PKCAs. Moreover, in a mouse model of leukemia initiated by Fli-1 activation, the PKCA compounds exhibited strong anti-cancer activity, which was accompanied by increased presence of CD41/CD61 positive megakaryocytic cells in leukemic spleens. Thus, PKC agonists offer a novel approach to combat Fli-1-induced leukemia, and possibly other cancers,by inducing EMC in part through over-activation of the PKC-MAPK-Fli-1 pathway.

  14. Effect of trehalose on the properties of mutant {gamma}PKC, which causes spinocerebellar ataxia type 14, in neuronal cell lines and cultured Purkinje cells.

    PubMed

    Seki, Takahiro; Abe-Seki, Nana; Kikawada, Takahiro; Takahashi, Hideyuki; Yamamoto, Kazuhiro; Adachi, Naoko; Tanaka, Shigeru; Hide, Izumi; Saito, Naoaki; Sakai, Norio

    2010-10-22

    Several missense mutations in the protein kinase Cγ (γPKC) gene have been found to cause spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that the mutant γPKC found in SCA14 is susceptible to aggregation, which induces apoptotic cell death. The disaccharide trehalose has been reported to inhibit aggregate formation and to alleviate symptoms in cellular and animal models of Huntington disease, Alzheimer disease, and prion disease. Here, we show that trehalose can be incorporated into SH-SY5Y cells and reduces the aggregation of mutant γPKC-GFP, thereby inhibiting apoptotic cell death in SH-SY5Y cells and primary cultured Purkinje cells (PCs). Trehalose acts by directly stabilizing the conformation of mutant γPKC without affecting protein turnover. Trehalose was also found to alleviate the improper development of dendrites in PCs expressing mutant γPKC-GFP without aggregates but not in PCs with aggregates. In PCs without aggregates, trehalose improves the mobility and translocation of mutant γPKC-GFP, probably by inhibiting oligomerization and thereby alleviating the improper development of dendrites. These results suggest that trehalose counteracts various cellular dysfunctions that are triggered by mutant γPKC in both neuronal cell lines and primary cultured PCs by inhibiting oligomerization and aggregation of mutant γPKC.

  15. High-content screening identifies small molecules that remove nuclear foci, affect MBNL distribution and CELF1 protein levels via a PKC-independent pathway in myotonic dystrophy cell lines.

    PubMed

    Ketley, Ami; Chen, Catherine Z; Li, Xin; Arya, Sukrat; Robinson, Thelma E; Granados-Riveron, Javier; Udosen, Inyang; Morris, Glenn E; Holt, Ian; Furling, Denis; Chaouch, Soraya; Haworth, Ben; Southall, Noel; Shinn, Paul; Zheng, Wei; Austin, Christopher P; Hayes, Christopher J; Brook, J David

    2014-03-15

    Myotonic dystrophy (DM) is a multi-system neuromuscular disorder for which there is no treatment. We have developed a medium throughput phenotypic assay, based on the identification of nuclear foci in DM patient cell lines using in situ hybridization and high-content imaging to screen for potentially useful therapeutic compounds. A series of further assays based on molecular features of DM have also been employed. Two compounds that reduce and/or remove nuclear foci have been identified, Ro 31-8220 and chromomycin A3. Ro 31-8220 is a PKC inhibitor, previously shown to affect the hyperphosphorylation of CELF1 and ameliorate the cardiac phenotype in a DM1 mouse model. We show that the same compound eliminates nuclear foci, reduces MBNL1 protein in the nucleus, affects ATP2A1 alternative splicing and reduces steady-state levels of CELF1 protein. We demonstrate that this effect is independent of PKC activity and conclude that this compound may be acting on alternative kinase targets within DM pathophysiology. Understanding the activity profile for this compound is key for the development of targeted therapeutics in the treatment of DM.

  16. Early memory formation disrupted by atypical PKC inhibitor ZIP in the medial prefrontal cortex but not hippocampus.

    PubMed

    Evuarherhe, Obaro; Barker, Gareth R I; Savalli, Giorgia; Warburton, Elizabeth C; Brown, Malcolm W

    2014-08-01

    Atypical isoforms of protein kinase C (aPKCs; particularly protein kinase M zeta: PKMζ) have been hypothesized to be necessary and sufficient for the maintenance of long-term potentiation (LTP) and long term memory by maintaining postsynaptic AMPA receptors via the GluA2 subunit. A myristoylated PKMζ pseudosubstrate peptide (ZIP) blocks PKMζ activity. We examined the actions of ZIP in medial prefrontal cortex (mPFC) and hippocampus in associative recognition memory in rats during early memory formation and memory maintenance. ZIP infusion in either hippocampus or mPFC impaired memory maintenance. However, early memory formation was impaired by ZIP in mPFC but not hippocampus; and blocking GluA2-dependent removal of AMPA receptors did not affect this impairment caused by ZIP in the mPFC. The findings indicate: (i) a difference in the actions of ZIP in hippocampus and medial prefrontal cortex, and (ii) a GluA2-independent target of ZIP (possibly PKCλ) in the mPFC during early memory formation.

  17. Simultaneous determination of multi-mycotoxins in palm kernel cake (PKC) using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

    PubMed

    Yibadatihan, S; Jinap, S; Mahyudin, N A

    2014-01-01

    Palm kernel cake (PKC) is a useful source of protein and energy for livestock. Recently, it has been used as an ingredient in poultry feed. Mycotoxin contamination of PKC due to inappropriate handling during production and storage has increased public concern about economic losses and health risks for poultry and humans. This concern has accentuated the need for the evaluation of mycotoxins in PKC. Furthermore, a method for quantifying mycotoxins in PKC has so far not been established. The aims of this study were therefore (1) to develop a method for the simultaneous determination of mycotoxins in PKC and (2) to validate and verify the method. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using an electrospray ionisation interface (ESI) in both positive- and negative-ion modes was developed for the simultaneous determination of aflatoxins (AFB₁, AFB₂, AFG₁ and AFG₂), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisins (FB₁ and FB₂), T-2 and HT-2 toxin in PKC. An optimum method using a 0.2 ml min⁻¹ flow rate, 0.2% formic acid in aqueous phase, 10% organic phase at the beginning and 90% organic phase at the end of the gradient was achieved. The extraction of mycotoxins was performed using a solvent mixture of acetonitrile-water-formic acid (79:20:1, v/v) without further clean-up. The mean recoveries of mycotoxins in spiked PKC samples ranged from 81% to 112%. Limits of detection (LODs) and limits of quantification (LOQs) for mycotoxin standards and PKC samples ranged from 0.02 to 17.5 μg kg⁻¹ and from 0.06 to 58.0 μg kg⁻¹, respectively. Finally, the newly developed method was successfully applied to PKC samples. The results illustrated the fact that the method is efficient and accurate for the simultaneous multi-mycotoxin determination in PKC, which can be ideal for routine analysis.

  18. Rho1- and Pkc1-dependent phosphorylation of the F-BAR protein Syp1 contributes to septin ring assembly

    PubMed Central

    Merlini, Laura; Bolognesi, Alessio; Juanes, Maria Angeles; Vandermoere, Franck; Courtellemont, Thibault; Pascolutti, Roberta; Séveno, Martial; Barral, Yves; Piatti, Simonetta

    2015-01-01

    In many cell types, septins assemble into filaments and rings at the neck of cellular appendages and/or at the cleavage furrow to help compartmentalize the plasma membrane and support cytokinesis. How septin ring assembly is coordinated with membrane remodeling and controlled by mechanical stress at these sites is unclear. Through a genetic screen, we uncovered an unanticipated link between the conserved Rho1 GTPase and its effector protein kinase C (Pkc1) with septin ring stability in yeast. Both Rho1 and Pkc1 stabilize the septin ring, at least partly through phosphorylation of the membrane-associated F-BAR protein Syp1, which colocalizes asymmetrically with the septin ring at the bud neck. Syp1 is displaced from the bud neck upon Pkc1-dependent phosphorylation at two serines, thereby affecting the rigidity of the new-forming septin ring. We propose that Rho1 and Pkc1 coordinate septin ring assembly with membrane and cell wall remodeling partly by controlling Syp1 residence at the bud neck. PMID:26179915

  19. Role of the Chemokine MCP-1 in Sensitization of PKC-Mediated Apoptosis in Prostate Cancer Cells

    DTIC Science & Technology

    2009-02-01

    proteolytic cleavage of PKCδ. J. Biol. Chem. 275: 7574-7582 (2000). 4. Garcia -Bermejo, M.L., Leskow, F.C., Fujii, T., Wang, Q., Blumberg, P.M., Ohba...M., Kuroki, T., Han, K.C., Lee, J., Marquez , V.E., and Kazanietz, M.G. Diacylglycerol (DAG)- lactones, a new class of protein kinase C (PKC) agonists

  20. Decline in the Recovery from Synaptic Depression in Heavier Aplysia Results from Decreased Serotonin-Induced Novel PKC Activation.

    PubMed

    Dunn, Tyler William; Sossin, Wayne S

    2015-01-01

    The defensive withdrawal reflexes of Aplysia are important behaviors for protecting the animal from predation. Habituation and dishabituation allow for experience-dependent tuning of these reflexes and the mechanisms underlying these forms of behavioral plasticity involve changes in transmitter release from the sensory to motor neuron synapses through homosynaptic depression and the serotonin-mediated recovery from depression, respectively. Interestingly, dishabituation is reduced in older animals with no corresponding change in habituation. Here we show that the cultured sensory neurons of heavier animals (greater than 120 g) that form synaptic connections with motor neurons have both reduced recovery from depression and reduced novel PKC Apl II activation with 5HT. The decrease in the recovery from depression correlated better with the size of the animal than the age of the animal. Much of this change in PKC activation and synaptic facilitation following depression can be rescued by direct activation of PKC Apl II with phorbol dibutyrate, suggesting a change in the signal transduction pathway upstream of PKC Apl II activation in the sensory neurons of larger animals.

  1. Hair cell BK channels interact with RACK1, and PKC increases its expression on the cell surface by indirect phosphorylation.

    PubMed

    Surguchev, Alexei; Bai, Jun-Ping; Joshi, Powrnima; Navaratnam, Dhasakumar

    2012-07-15

    Large conductance (BK) calcium activated potassium channels (Slo) are ubiquitous and implicated in a number of human diseases including hypertension and epilepsy. BK channels consist of a pore forming α-subunit (Slo) and a number of accessory subunits. In hair cells of nonmammalian vertebrates these channels play a critical role in electrical resonance, a mechanism of frequency selectivity. Hair cell BK channel clusters on the surface and currents increase along the tonotopic axis and contribute significantly to the responsiveness of these hair cells to sounds of high frequency. In contrast, messenger RNA levels encoding the Slo gene show an opposite decrease in high frequency hair cells. To understand the molecular events underlying this paradox, we used a yeast two-hybrid screen to isolate binding partners of Slo. We identified Rack1 as a Slo binding partner and demonstrate that PKC activation increases Slo surface expression. We also establish that increased Slo recycling of endocytosed Slo is at least partially responsible for the increased surface expression of Slo. Moreover, analysis of several PKC phosphorylation site mutants confirms that the effects of PKC on Slo surface expression are likely indirect. Finally, we show that Slo clusters on the surface of hair cells are also increased by increased PKC activity and may contribute to the increasing amounts of channel clusters on the surface of high-frequency hair cells.

  2. The Par3/aPKC interaction is essential for end bud remodeling and progenitor differentiation during mammary gland morphogenesis

    PubMed Central

    McCaffrey, Luke Martin; Macara, Ian G.

    2009-01-01

    Mammalian polarity proteins have been studied predominantly in cell culture systems, and little is known about their functions in vivo. To address this issue, we used a shRNA lentiviral system to manipulate gene expression in mouse mammary stem/progenitor cells. Transplantation of Par3-depleted stem/progenitor cells into the mammary fat pad severely disrupted mammary development, and glands were characterized by ductal hyperplasia, luminal filling, and highly disorganized end bud structures that were unable to remodel into normal ductal structures. Unexpectedly, Par3-depleted mammary glands also had an expanded progenitor population. We identified a novel function for the atypical protein kinase C (aPKC)-binding domain of Par3 in restricting Par3 and aPKC to the apical region in mammary epithelia in vivo, and found that mammary morphogenesis is dependent on the ability of Par3 to directly bind aPKC. These results reveal a new function for Par3 in the regulation of progenitor differentiation and epithelial morphogenesis in vivo and demonstrate for the first time an essential requirement for the Par3–aPKC interaction. PMID:19528321

  3. PKC/NADPH oxidase are involved in the protective effect of pioglitazone in high homocysteine-induced paracrine dyfunction in endothelial progenitor cells

    PubMed Central

    Xu, Shengjie; Zhao, Yanbo; Jin, Chongying; Yu, Lu; Ding, Fang; Fu, Guosheng; Zhu, Junhui

    2017-01-01

    Increasing evidence suggests that EPCs improve neovascularization and endothelial regeneration via the production of paracrine factors. VEGF and IL-8 are major cytokines involved in EPC-mediated angiogenesis and re-endothelialization. In our previous studies, Hcy impaired EPC migratory and adhesive activities. We devised this study to determine whether Hcy could affect the expression and secretion of VEGF and IL-8 from EPCs. We found that high levels of Hcy (100-500 μM) decreased the EPC-mediated protein secretion and mRNA expression of VEGF and IL-8. Moreover, PIO, a PPARγ agonist, has been suggested to regulate EPC adhesion, migration, survival. In this study, PIO normalized the production of these cytokines by EPCs stimulated with Hcy. These effects of Hcy and PIO were primarily mediated by PKC and ROS via NADPH oxidase. We further confirmed this mechanism via knockdown of the NADPH oxidase subunits p67phox and Nox2. Furthermore, the PPARγ inhibitor GW9662 was not observed to abrogate the beneficial effect of PIO, indicating that PIO protected EPC paracrine function against Hcy in a PPARγ-independent manner. PMID:28386331

  4. Signaling through Lrg1, Rho1 and Pkc1 Governs Candida albicans Morphogenesis in Response to Diverse Cues

    PubMed Central

    Leach, Michelle D.; Hogan, Deborah A.; Robbins, Nicole; Cowen, Leah E.

    2016-01-01

    The capacity to transition between distinct morphological forms is a key virulence trait for diverse fungal pathogens. A poignant example of a leading opportunistic fungal pathogen of humans for which an environmentally responsive developmental program underpins virulence is Candida albicans. C. albicans mutants that are defective in the transition between yeast and filamentous forms typically have reduced virulence. Although many positive regulators of C. albicans filamentation have been defined, there are fewer negative regulators that have been implicated in repression of filamentation in the absence of inducing cues. To discover novel negative regulators of filamentation, we screened a collection of 1,248 C. albicans homozygous transposon insertion mutants to identify those that were filamentous in the absence of inducing cues. We identified the Rho1 GAP Lrg1, which represses filamentous growth by stimulating Rho1 GTPase activity and converting Rho1 to its inactive, GDP-bound form. Deletion of LRG1 or introduction of a RHO1 mutation that locks Rho1 in constitutively active, GTP-bound state, leads to filamentation in the absence of inducing cues. Deletion of the Rho1 downstream effector PKC1 results in defective filamentation in response to diverse host-relevant inducing cues, including serum. We further established that Pkc1 is not required to sense filament-inducing cues, but its kinase activity is critical for the initiation of filamentous growth. Our genetic analyses revealed that Pkc1 regulates filamentation independent of the canonical MAP kinase cascade. Further, although Ras1 activation is not impaired in a pkc1Δ/pkc1Δ mutant, adenylyl cyclase activity is reduced, consistent with a model in which Pkc1 functions in parallel with Ras1 in regulating Cyr1 activation. Thus, our findings delineate a signaling pathway comprised of Lrg1, Rho1 and Pkc1 with a core role in C. albicans morphogenesis, and illuminate functional relationships that govern activation

  5. Absence of PKC-alpha attenuates lithium-induced nephrogenic diabetes insipidus.

    PubMed

    Sim, Jae H; Himmel, Nathaniel J; Redd, Sara K; Pulous, Fadi E; Rogers, Richard T; Black, Lauren N; Hong, Seongun M; von Bergen, Tobias N; Blount, Mitsi A

    2014-01-01

    Lithium, an effective antipsychotic, induces nephrogenic diabetes insipidus (NDI) in ∼40% of patients. The decreased capacity to concentrate urine is likely due to lithium acutely disrupting the cAMP pathway and chronically reducing urea transporter (UT-A1) and water channel (AQP2) expression in the inner medulla. Targeting an alternative signaling pathway, such as PKC-mediated signaling, may be an effective method of treating lithium-induced polyuria. PKC-alpha null mice (PKCα KO) and strain-matched wild type (WT) controls were treated with lithium for 0, 3 or 5 days. WT mice had increased urine output and lowered urine osmolality after 3 and 5 days of treatment whereas PKCα KO mice had no change in urine output or concentration. Western blot analysis revealed that AQP2 expression in medullary tissues was lowered after 3 and 5 days in WT mice; however, AQP2 was unchanged in PKCα KO. Similar results were observed with UT-A1 expression. Animals were also treated with lithium for 6 weeks. Lithium-treated WT mice had 19-fold increased urine output whereas treated PKCα KO animals had a 4-fold increase in output. AQP2 and UT-A1 expression was lowered in 6 week lithium-treated WT animals whereas in treated PKCα KO mice, AQP2 was only reduced by 2-fold and UT-A1 expression was unaffected. Urinary sodium, potassium and calcium were elevated in lithium-fed WT but not in lithium-fed PKCα KO mice. Our data show that ablation of PKCα preserves AQP2 and UT-A1 protein expression and localization in lithium-induced NDI, and prevents the development of the severe polyuria associated with lithium therapy.

  6. Evidence of PKC Binding and Translocation to explain the anticancer mechanism of chlorogenic acid in breast cancer cells.

    PubMed

    Deka, S J; Gorai, S; Manna, D; Trivedi, V

    2017-02-09

    Chlorogenic acid (CGA) exhibits potentials towards liver, breast and skin cancer. Cancer cells stimulated with CGA exhibits differential expression of transcriptional factors and regulatory molecules but the molecular target of the molecule is not known. Superposition of biophoric elements of CGA with Curcumin gives maximum common substructure score of 0.90. Molecular modeling studies further suggest that CGA fits into the C1b domain of PKC with extensive interaction with residues lining binding site. It binds PKC in a concentration dependent manner with dissociation constant KD, 28.84±3.95 μM. PKC-CGA complex is stable with minimal distortion to the 3-D structure and maintains the hydrogen bonding between ligand and receptor during simulation period. Cells stimulated with CGA causes 12.1 ± 0.56% PKC translocation from the cytosol to the plasma membrane. It disturbs the cell cycle and arrest the cancer cell at the G1 phase with a reduction in S-phase. Chlorogenic acid exhibits killing of cancer cells in a dose-dependent manner with an IC50 of 75.88 ± 4.54µg/ml and 52.5 ± 4.72µg/ml towards MDAMB-231 and MCF-7 cells respectively. It induces apoptosis in cancer cells as evident by AO/EtBr staining and degradation of genomic DNA to give a laddering pattern. Apoptosis in cancer cells involves mitochondrial pathway as supported by a reduction in mitochondrial potentials and release of cyt-C into the cytosol. Hence, the current study has established PKC as an important signaling molecule to the observed anti-cancer effects of CGA and provides the impetus to design better CGA analogs for improved anti-cancer potential against the malignant tumor.

  7. A pathway in the yeast cell division cycle linking protein kinase C (Pkc1) to activation of Cdc28 at START.

    PubMed Central

    Marini, N J; Meldrum, E; Buehrer, B; Hubberstey, A V; Stone, D E; Traynor-Kaplan, A; Reed, S I

    1996-01-01

    In an effort to study further the mechanism of Cdc28 function and cell cycle commitment, we describe here a genetic approach to identify components of pathways downstream of the Cdc28 kinase at START by screening for mutations that decrease the effectiveness of signaling by Cdc28. The first locus to be characterized in detail using this approach was PKC1 which encodes a homolog of the Ca(2+)-dependent isozymes of the mammalian protein kinase C (PKC) superfamily (Levin et al., 1990). By several genetic criteria, we show a functional interaction between CDC28 and PKC1 with PKC1 apparently functioning with respect to bud emergence downstream of START. Consistent with this, activity of the MAP kinase homolog Mpk1 (a putative Pkc1 effector) is stimulated by activation of Cdc28. Furthermore, we demonstrate a cell cycle-dependent hydrolysis of phosphatidylcholine to diacylglycerol (a PKC activator) and choline phosphate at START. Diacylglycerol production is stimulated by Cdc28 in cycling cells and is closely associated with Cdc28 activation at START. These results imply that the activation of Pkc1, which is known to be necessary during bud morphogenesis, is mediated via the CDC28-dependent stimulation of PC-PLC activity in a novel cell cycle-regulated signaling pathway. Images PMID:8670805

  8. Assessment of roles for the Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI) Ly-GDI in platelet function: a spatial systems approach.

    PubMed

    Ngo, Anh T P; Thierheimer, Marisa L D; Babur, Özgün; Rocheleau, Anne D; Huang, Tao; Pang, Jiaqing; Rigg, Rachel A; Mitrugno, Annachiara; Theodorescu, Dan; Burchard, Julja; Nan, Xiaolin; Demir, Emek; McCarty, Owen J T; Aslan, Joseph E

    2017-02-01

    Upon activation at sites of vascular injury, platelets undergo morphological alterations essential to hemostasis via cytoskeletal reorganizations driven by the Rho GTPases Rac1, Cdc42 and RhoA. Here we investigate roles for Rho-specific guanine nucleotide dissociation inhibitor proteins (RhoGDIs) in platelet function. We find that platelets express two RhoGDI family members, RhoGDI and Ly-GDI. While RhoGDI localizes throughout platelets in a granule-like manner, Ly-GDI shows an asymmetric, polarized localization that largely overlaps with Rac1 and Cdc42 as well as microtubules and protein kinase C (PKC) in platelets adherent to fibrinogen. Antibody interference and platelet spreading experiments suggest a specific role for Ly-GDI in platelet function. Intracellular signaling studies based on interactome and pathways analyses also support a regulatory role for Ly-GDI, which is phosphorylated at PKC substrate motifs in a PKC-dependent manner in response to the platelet collagen receptor glycoprotein (GP)VI-specific agonist collagen-related peptide. Additionally, PKC inhibition diffuses the polarized organization of Ly-GDI in spread platelets relative to its colocalization with Rac1 and Cdc42. Together our results suggest a role for Ly-GDI in the localized regulation of Rho GTPases in platelets and hypothesize a link between the PKC and Rho GTPase signaling systems in platelet function.

  9. Protein Kinase C Inhibitors Sensitize GNAQ Mutant Uveal Melanoma Cells to Ionizing Radiation

    PubMed Central

    Cerne, Jasmina Ziva; Hartig, Sean Michael; Hamilton, Mark Patrick; Chew, Sue Anne; Mitsiades, Nicholas; Poulaki, Vassiliki; McGuire, Sean Eric

    2014-01-01

    Purpose. Uveal melanoma (UM) tumors require large doses of radiation therapy (RT) to achieve tumor ablation, which frequently results in damage to adjacent normal tissues, leading to vision-threatening complications. Approximately 50% of UM patients present with activating somatic mutations in the gene encoding for G protein αq-subunit (GNAQ), which lead to constitutive activation of downstream pathways, including protein kinase C (PKC). In this study, we investigated the impact of small-molecule PKC inhibitors bisindolylmaleimide I (BIM) and sotrastaurin (AEB071), combined with ionizing radiation (IR), on survival in melanoma cell lines. Methods. Cellular radiosensitivity was determined by using a combination of proliferation, viability, and clonogenic assays. Cell-cycle effects were measured by flow cytometry. Transcriptomic and proteomic profiling were performed by quantitative real-time PCR, reverse-phase protein array analysis, and immunofluorescence. Results. We found that the PKC inhibitors combined with IR significantly decreased the viability, proliferation, and clonogenic potential of GNAQmt, but not GNAQwt/BRAFmt cells, compared with IR alone. Combined treatment increased the antiproliferative and proapoptotic effects of IR in GNAQmt cells through delayed DNA-damage resolution and enhanced induction of proteins involved in cell-cycle arrest, cell-growth arrest, and apoptosis. Conclusions. Our preclinical results suggest that combined modality treatment may allow for reductions in the total RT dose and/or fraction size, which may lead to better functional organ preservation in the treatment of primary GNAQmt UM. These findings suggest future clinical trials combining PKC inhibitors with RT in GNAQmt UM warrant consideration. PMID:24595385

  10. Involvement of protein kinase C in the response of Neurospora crassa to blue light.

    PubMed

    Arpaia, G; Cerri, F; Baima, S; Macino, G

    1999-09-01

    As a first step towards understanding the process of blue light perception, and the signal transduction mechanisms involved, in Neurospora crassa we have used a pharmacological approach to screen a wide range of second messengers and chemical compounds known to interfere with the activity of well-known signal transducing molecules in vivo. We tested the influence of these compounds on the induction of the al-3 gene, a key step in light-induced carotenoid biosynthesis. This approach has implicated protein kinase C (PKC) as a component of the light transduction machinery. The conclusion is based on the effects of specific inhibitors (calphostin C and chelerythrine chloride) and activators of PKC (1,2-dihexanoyl-sn-glycerol). During vegetative growth PKC may be responsible for desensitization to light because inhibitors of the enzyme cause an increase in the total amount of mRNA transcribed after illumination. PKC is therefore proposed here to be an important regulator of transduction of the blue light signal, and may act through modification of the protein White Collar-1, which we show to be a substrate for PKC in N. crassa.

  11. Activating PKC-β1 at the blood-brain barrier reverses induction of P-glycoprotein activity by dioxin and restores drug delivery to the CNS.

    PubMed

    Wang, Xueqian; Hawkins, Brian T; Miller, David S

    2011-06-01

    Upregulation of blood-brain barrier (BBB) P-glycoprotein expression causes central nervous system (CNS) pharmacoresistance. However, activation of BBB protein kinase C-β1 (PKC-β1) rapidly reduces basal P-glycoprotein transport activity. We tested whether PKC-β1 activation would reverse CNS drug resistance caused by dioxin acting through aryl hydrocarbon receptor. A selective PKC-β1 agonist abolished the increase in P-glycoprotein activity induced by dioxin in isolated rat brain capillaries and reversed the effect of dioxin on brain uptake of verapamil in dioxin-dosed rats. Thus, targeting BBB PKC-β1 may be an effective strategy to improve drug delivery to the brain, even in drug-resistant individuals.

  12. aPKC regulates apical localization of Lgl to restrict elongation of microridges in developing zebrafish epidermis

    PubMed Central

    Raman, Renuka; Damle, Indraneel; Rote, Rahul; Banerjee, Shamik; Dingare, Chaitanya; Sonawane, Mahendra

    2016-01-01

    Epithelial cells exhibit apical membrane protrusions, which confer specific functions to epithelial tissues. Microridges are short actin protrusions that are laterally long and form a maze-like pattern in the apical domain. They are widely found on vertebrate squamous epithelia including epidermis and have functions in mucous retention, membrane storage and abrasion resistance. It is largely unknown how the formation of these laterally long actin projections is regulated. Here, we show that antagonistic interactions between aPKC and Lgl–regulators of apical and basolateral domain identity, respectively,–control the length of microridges in the zebrafish periderm, the outermost layer of the epidermis. aPKC regulates the levels of Lgl and the active form of non-muscle myosinII at the apical cortex to prevent actin polymerization-dependent precocious fusion and elongation of microridges. Our data unravels the functional significance of exclusion of Lgl from the apical domain in epithelial cells. PMID:27249668

  13. Transcriptional upregulation of the human MRP2 gene expression by serine/threonine protein kinase inhibitors.

    PubMed

    Pułaski, L; Szemraj, J; Uchiumi, T; Kuwano, M; Bartosz, G

    2005-01-01

    Transcriptional regulation by cellular signalling pathways of multidrug resistance proteins that pump anticancer drugs out of cells is one of key issues in the development of the multidrug resistance phenotype. In our study, we have used the reporter gene approach as well as determination of mRNA levels in two cancer cell lines of human origin, MCF-7 and A549, to study the regulation of multidrug resistance proteins 2 and 3 (MRP2 AND MRP3) by serine/threonine protein kinases. Since a prototypic PKC inducer, PMA, caused a marked upregulation of transcription from both human MRP2 and MRP3 promoters, a role for PKC isoforms in positive control of expression of these proteins could be postulated. Interestingly, broad-spectrum serine-threonine protein kinase inhibitors which also inhibit PKC, staurosporine and H-7, stimulated expression from the MRP2 promoter instead of inhibiting it. This effect was not seen for MRP3. MRP2 induction by staurosporine and H-7 was shown to have phenotypic consequences in whole cells, rendering them more resistant to etoposide and increasing their ability to export calcein through the plasma membrane. These results point to the involvement of serine/threonine protein kinases in negative regulation of the human MRP2 gene and to the necessity of testing novel anti-cancer drugs acting as protein kinase inhibitors with regard to their potential ability to induce multidrug resistance.

  14. F-actin links Epac-PKC signaling to purinergic P2X3 receptor sensitization in dorsal root ganglia following inflammation

    PubMed Central

    Gu, Yanping; Wang, Congying; Li, GuangWen

    2016-01-01

    Sensitization of purinergic P2X3 receptors (P2X3Rs) contributes to the production of exaggerated nociceptive responses following inflammatory injury. We showed previously that prostaglandin E2 (PGE2) potentiates P2X3R-mediated ATP currents in dorsal root ganglion neurons isolated from both control and complete Freund’s adjuvant-induced inflamed rats. PGE2 potentiation of ATP currents depends only on PKA signaling in control neurons, but it depends on both PKA and PKC signaling in inflamed neurons. We further found that inflammation evokes an increase in exchange proteins directly activated by cAMP (Epacs) in dorsal root ganglions. This increase promotes the activation of PKC to produce a much enhanced PGE2 effect on ATP currents and to elicit Epac-dependent flinch nocifensive behavioral responses in complete Freund’s adjuvant rats. The link between Epac-PKC signaling and P2X3R sensitization remains unexplored. Here, we show that the activation of Epacs promotes the expression of phosphorylated PKC and leads to an increase in the cytoskeleton, F-actin, expression at the cell perimeter. Depolymerization of F-actin blocks PGE2-enhanced ATP currents and inhibits P2X3R-mediated nocifensive responses after inflammation. Thus, F-actin is dynamically involved in the Epac-PKC-dependent P2X3R sensitization. Furthermore, Epacs induce a PKC-dependent increase in the membrane expression of P2X3Rs. This increase is abolished by F-actin depolymerization, suggesting that F-actin mediates Epac-PKC signaling of P2X3R membrane expression. Thus, after inflammation, an Epac-PKC dependent increase in F-actin in dorsal root ganglion neurons enhances the membrane expression of P2X3Rs to bring about sensitization of P2X3Rs and abnormal pain behaviors. PMID:27385722

  15. EXPRESS: F-actin links Epac-PKC signaling to purinergic P2X3 receptors sensitization in dorsal root ganglia following inflammation.

    PubMed

    Gu, Yanping; Wang, Congying; Li, Guangwen; Huang, Li-Yen Mae

    2016-01-01

    Sensitization of purinergic P2X3 receptors (P2X3Rs) contributes to the production of exaggerated nociceptive responses following inflammatory injury. We showed previously that prostaglandin E2 (PGE2) potentiates P2X3R-mediated ATP currents in dorsal root ganglion neurons isolated from both control and complete Freund’s adjuvant-induced inflamed rats. PGE2 potentiation of ATP currents depends only on PKA signaling in control neurons, but it depends on both PKA and PKC signaling in inflamed neurons. We further found that inflammation evokes an increase in exchange proteins directly activated by cAMP (Epacs) in dorsal root ganglions. This increase promotes the activation of PKC to produce a much enhanced PGE2 effect on ATP currents and to elicit Epac-dependent flinch nocifensive behavioral responses in complete Freund’s adjuvant rats. The link between Epac-PKC signaling and P2X3R sensitization remains unexplored. Here, we show that the activation of Epacs promotes the expression of phosphorylated PKC and leads to an increase in the cytoskeleton, F-actin, expression at the cell perimeter. Depolymerization of F-actin blocks PGE2-enhanced ATP currents and inhibits P2X3R-mediated nocifensive responses after inflammation. Thus, F-actin is dynamically involved in the Epac-PKC-dependent P2X3R sensitization. Furthermore, Epacs induce a PKC-dependent increase in the membrane expression of P2X3Rs. This increase is abolished by F-actin depolymerization, suggesting that F-actin mediates Epac-PKC signaling of P2X3R membrane expression. Thus, after inflammation, an Epac-PKC dependent increase in F-actin in dorsal root ganglion neurons enhances the membrane expression of P2X3Rs to bring about sensitization of P2X3Rs and abnormal pain behaviors.

  16. The Novel Functions of the PLC/PKC/PKD Signaling Axis in G Protein-Coupled Receptor-Mediated Chemotaxis of Neutrophils

    PubMed Central

    Xu, Xuehua; Jin, Tian

    2015-01-01

    Chemotaxis, a directional cell migration guided by extracellular chemoattractant gradients, plays an essential role in the recruitment of neutrophils to sites of inflammation. Chemotaxis is mediated by the G protein-coupled receptor (GPCR) signaling pathway. Extracellular stimuli trigger activation of the PLC/PKC/PKD signaling axis, which controls several signaling pathways. Here, we concentrate on the novel functions of PLC/PKC/PKD signaling in GPCR-mediated chemotaxis of neutrophils. PMID:26605346

  17. Notch and PKC are involved in formation of the lateral region of the dorso-ventral axis in Drosophila embryos.

    PubMed

    Tremmel, Daniel M; Resad, Sedat; Little, Christopher J; Wesley, Cedric S

    2013-01-01

    The Notch gene encodes an evolutionarily conserved cell surface receptor that generates regulatory signals based on interactions between neighboring cells. In Drosophila embryos it is normally expressed at a low level due to strong negative regulation. When this negative regulation is abrogated neurogenesis in the ventral region is suppressed, the development of lateral epidermis is severely disrupted, and the dorsal aminoserosa is expanded. Of these phenotypes only the anti-neurogenic phenotype could be linked to excess canonical Notch signaling. The other phenotypes were linked to high levels of Notch protein expression at the surface of cells in the lateral regions indicating that a non-canonical Notch signaling activity normally functions in these regions. Results of our studies reported here provide evidence. They show that Notch activities are inextricably linked to that of Pkc98E, the homolog of mammalian PKCδ. Notch and Pkc98E up-regulate the levels of the phosphorylated form of IκBCactus, a negative regulator of Toll signaling, and Mothers against dpp (MAD), an effector of Dpp signaling. Our data suggest that in the lateral regions of the Drosophila embryos Notch activity, in conjunction with Pkc98E activity, is used to form the slopes of the opposing gradients of Toll and Dpp signaling that specify cell fates along the dorso-ventral axis.

  18. The participation of NMDA receptors, PKC, and MAPK in the formation of memory following operant conditioning in Lymnaea

    PubMed Central

    2010-01-01

    Background Memory is the ability to store, retain, and later retrieve information that has been learned. Intermediate term memory (ITM) that persists for up to 3 h requires new protein synthesis. Long term memory (LTM) that persists for at least 24 h requires: DNA transcription, RNA translation, and the trafficking of newly synthesized proteins. It has been shown in a number of different model systems that NMDA receptors, protein kinase C (PKC) and mitogen activated protein kinase (MAPK) are all involved in the memory formation process. Results Here we show that snails trained in control conditions are capable of forming, depending on the training procedure used, either ITM or LTM. However, blockage of NMDA receptors (MK 801), inhibition of PKC (GF109203X hydrochloride) and MAPK activity (UO126) prevent the formation of both ITM and LTM. Conclusions The injection of either U0126 or GF109203X, which inhibit MAPK and PKC activity respectively, 1 hour prior to training results in the inhibition of both ITM and LTM formation. We further found that NMDA receptor activity was necessary in order for both ITM and LTM formation. PMID:20807415

  19. Huntingtin Is Required for Epithelial Polarity through RAB11A-Mediated Apical Trafficking of PAR3-aPKC

    PubMed Central

    Elias, Salah; McGuire, John Russel; Yu, Hua; Humbert, Sandrine

    2015-01-01

    The establishment of apical-basolateral polarity is important for both normal development and disease, for example, during tumorigenesis and metastasis. During this process, polarity complexes are targeted to the apical surface by a RAB11A-dependent mechanism. Huntingtin (HTT), the protein that is mutated in Huntington disease, acts as a scaffold for molecular motors and promotes microtubule-based dynamics. Here, we investigated the role of HTT in apical polarity during the morphogenesis of the mouse mammary epithelium. We found that the depletion of HTT from luminal cells in vivo alters mouse ductal morphogenesis and lumen formation. HTT is required for the apical localization of PAR3-aPKC during epithelial morphogenesis in virgin, pregnant, and lactating mice. We show that HTT forms a complex with PAR3, aPKC, and RAB11A and ensures the microtubule-dependent apical vesicular translocation of PAR3-aPKC through RAB11A. We thus propose that HTT regulates polarized vesicular transport, lumen formation and mammary epithelial morphogenesis. PMID:25942483

  20. Ellagic acid induces novel and atypical PKC isoforms and promotes caspase-3 dependent apoptosis by blocking energy metabolism.

    PubMed

    Mishra, Sudha; Vinayak, Manjula

    2014-01-01

    Antioxidant ellagic acid is a herbal polyphenolic compound shown to possess growth-inhibiting and apoptotic activities in cancer. Protein kinase C (PKC) plays an important role in cell proliferation, apoptosis, and differentiation. Apoptosis of tumor cells is induced by inactivation of glycolytic enzyme of anaerobic metabolism, lactate dehydrogenase (LDH)-A, and by activating apoptotic protein caspase-3 via PKCδ. The present study aims to analyze the role of ellagic acid on regulation of novel and atypical isozymes of PKC to modulate apoptosis and anaerobic metabolism to prevent lymphoma growth as its role on classical PKCs is reported earlier. Expression of novel and atypical isozymes of PKC, activity of PKCδ, expression and activity of caspase-3, and LDH-A have been analyzed. Expression is measured by RT-PCR, activities of PKCδ as level of its catalytic fragment, caspase-3 as level of its p17 fragment, and LDH-A by specific staining. Lymphoma bearing mice were treated with 3 different doses of ellagic acid. The treatment enhanced expression of all novel and atypical PKCs, activity and expression of caspase-3, and activity of PKCδ but decreased activity and expression of LDH-A. Our results suggest that ellagic acid induces apoptosis via novel and atypical PKCs in association with caspase-3 and induces cancer cell death by blocking the energy metabolism.

  1. Curcumin supplementation could improve diabetes-induced endothelial dysfunction associated with decreased vascular superoxide production and PKC inhibition

    PubMed Central

    2010-01-01

    Background Curcumin, an Asian spice and food-coloring agent, is known for its anti-oxidant properties. We propose that curcumin can improve diabetes-induced endothelial dysfunction through superoxide reduction. Methods Diabetes (DM) was induced in rats by streptozotocin (STZ). Daily curcumin oral feeding was started six weeks after the STZ injection. Twelve weeks after STZ injection, mesenteric arteriolar responses were recorded in real time using intravital fluorescence videomicroscopy. Superoxide and vascular protein kinase C (PKC-βII) were examined by hydroethidine and immunofluorescence, respectively. Results The dilatory response to acetylcholine (ACh) significantly decreased in DM arterioles as compared to control arterioles. There was no difference among groups when sodium nitroprusside (SNP) was used. ACh responses were significantly improved by both low and high doses (30 and 300 mg/kg, respectively) of curcumin supplementation. An oxygen radical-sensitive fluorescent probe, hydroethidine, was used to detect intracellular superoxide anion (O2●-) production. O2●- production was markedly increased in DM arterioles, but it was significantly reduced by supplementation of either low or high doses of curcumin. In addition, with a high dose of curcumin, diabetes-induced vascular PKC-βII expression was diminished. Conclusion Therefore, it is suggested that curcumin supplementation could improve diabetes-induced endothelial dysfunction significantly in relation to its potential to decrease superoxide production and PKC inhibition. PMID:20946622

  2. Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

    SciTech Connect

    Vorhagen, Susanne; Niessen, Carien M.

    2014-11-01

    Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.

  3. Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate.

    PubMed

    Vorhagen, Susanne; Niessen, Carien M

    2014-11-01

    Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.

  4. Polycystin-1 binds Par3/aPKC and controls convergent extension during renal tubular morphogenesis

    NASA Astrophysics Data System (ADS)

    Castelli, Maddalena; Boca, Manila; Chiaravalli, Marco; Ramalingam, Harini; Rowe, Isaline; Distefano, Gianfranco; Carroll, Thomas; Boletta, Alessandra

    2013-10-01

    Several organs, including the lungs and kidneys, are formed by epithelial tubes whose proper morphogenesis ensures correct function. This is best exemplified by the kidney, where defective establishment or maintenance of tubular diameter results in polycystic kidney disease, a common genetic disorder. Most polycystic kidney disease cases result from loss-of-function mutations in the PKD1 gene, encoding Polycystin-1, a large receptor of unknown function. Here we demonstrate that PC-1 has an essential role in the establishment of correct tubular diameter during nephron development. Polycystin-1 associates with Par3 favouring the assembly of a pro-polarizing Par3/aPKC complex and it regulates a programme of cell polarity important for oriented cell migration and for a convergent extension-like process during tubular morphogenesis. Par3 inactivation in the developing kidney results in defective convergent extension and tubular morphogenesis, and in renal cyst formation. Our data define Polycystin-1 as central to cell polarization and to epithelial tube morphogenesis and homeostasis.

  5. The 5-HT7 receptor triggers cerebellar long-term synaptic depression via PKC-MAPK.

    PubMed

    Lippiello, Pellegrino; Hoxha, Eriola; Speranza, Luisa; Volpicelli, Floriana; Ferraro, Angela; Leopoldo, Marcello; Lacivita, Enza; Perrone-Capano, Carla; Tempia, Filippo; Miniaci, Maria Concetta

    2016-02-01

    The 5-HT7 receptor (5-HT7R) mediates important physiological effects of serotonin, such as memory and emotion, and is emerging as a therapeutic target for the treatment of cognitive disorders and depression. Although previous studies have revealed an expression of 5-HT7R in cerebellum, particularly at Purkinje cells, its functional role and signaling mechanisms have never been described. Using patch-clamp recordings in cerebellar slices of adult mice, we investigated the effects of a selective 5-HT7R agonist, LP-211, on the main plastic site of the cerebellar cortex, the parallel fiber-Purkinje cell synapse. Here we show that 5-HT7R activation induces long-term depression of parallel fiber-Purkinje cell synapse via a postsynaptic mechanism that involves the PKC-MAPK signaling pathway. Moreover, a 5-HT7R antagonist abolished the expression of PF-LTD, produced by pairing parallel fiber stimulation with Purkinje cell depolarization; whereas, application of a 5-HT7R agonist impaired LTP induced by 1 Hz parallel fiber stimulation. Our results indicate for the first time that 5-HT7R exerts a fine regulation of cerebellar bidirectional synaptic plasticity that might be involved in cognitive processes and neuropsychiatric disorders involving the cerebellum.

  6. Protein kinase Cμ mediates adenosine-stimulated steroidogenesis in primary rat adrenal cells.

    PubMed

    Chen, Yung-Chia; Chen, Ying; Huang, Shih-Horng; Wang, Seu-Mei

    2010-11-05

    Adenosine (Ado), an endogenous nucleoside, can stimulate corticosterone synthesis in adrenal cells via the A(2A)/A(2B) adenosine receptors (ARs). This study evaluated the contribution of protein kinase C (PKC) isoforms in Ado-induced steroidogenesis. The PKC inhibitor calphostin c blocked Ado-induced steroidogenesis, the mitogen-activated protein kinase (MEK)-extracellular signal-related regulated kinase (ERK)-cyclic AMP responsive element-binding protein cascade, and the mRNA expression of steroidogenic acute regulatory protein and CYP11B1. Further analyses revealed that PKCμ was indeed activated by Ado. Moreover, downregulation of PKCμ by small interfering RNA (siRNA) inhibited Ado-stimulated steroidogenesis and ERK phosphorylation. Finally, inhibition of either A(2A)AR or A(2B)AR led to the suppression of PKCμ phosphorylation. Together, these findings suggest that A(2)AR-PKCμ-MEK signaling mediates Ado-stimulated adrenal steroidogenesis.

  7. Modulation of human c-mpl gene expression by thrombopoietin through protein kinase C.

    PubMed

    Sunohara, M; Morikawa, S; Sato, T; Sato, I; Sato, T; Fuse, A

    2003-01-01

    The c-Mpl, thrombopoietin (TPO) receptor specificially controls megakaryocytic growth and differentiation. TPO increased the c-mpl promoter activity determined by a transient expression system using a vector containing the luciferase gene as a reporter in the human megakaryoblastic cell line CMK. The maximal promoter activity of c-mpl was obtained 24 hr after pretreatment with TPO for 3 hr and then declined with time. This increase was completely abolished by protein kinase C (PKC) inhibitors (GF109203, calphostin C and H7). Phorbol 12-myristate 13-acetate (PMA) treatment led to an increase in c-mpl promoter activity. These results demonstrate that the promoter activity of c-mpl is modulated by transcription through a PKC-dependent pathway.

  8. Enzastaurin (LY317615), a Protein Kinase C Beta Selective Inhibitor, Enhances Antiangiogenic Effect of Radiation

    SciTech Connect

    Willey, Christopher D.; Xiao Dakai; Tu Tianxiang; Kim, Kwang Woon; Moretti, Luigi; Niermann, Kenneth J.; Tawtawy, Mohammed N.; Quarles, Chad C. Ph.D.; Lu Bo

    2010-08-01

    Purpose: Angiogenesis has generated interest in oncology because of its important role in cancer growth and progression, particularly when combined with cytotoxic therapies, such as radiotherapy. Among the numerous pathways influencing vascular growth and stability, inhibition of protein kinase B(Akt) or protein kinase C(PKC) can influence tumor blood vessels within tumor microvasculature. Therefore, we wanted to determine whether PKC inhibition could sensitize lung tumors to radiation. Methods and Materials: The combination of the selective PKC{beta} inhibitor Enzastaurin (ENZ, LY317615) and ionizing radiation were used in cell culture and a mouse model of lung cancer. Lung cancer cell lines and human umbilical vascular endothelial cells (HUVEC) were examined using immunoblotting, cytotoxic assays including cell proliferation and clonogenic assays, and Matrigel endothelial tubule formation. In vivo, H460 lung cancer xenografts were examined for tumor vasculature and proliferation using immunohistochemistry. Results: ENZ effectively radiosensitizes HUVEC within in vitro models. Furthermore, concurrent ENZ treatment of lung cancer xenografts enhanced radiation-induced destruction of tumor vasculature and proliferation by IHC. However, tumor growth delay was not enhanced with combination treatment compared with either treatment alone. Analysis of downstream effectors revealed that HUVEC and the lung cancer cell lines differed in their response to ENZ and radiation such that only HUVEC demonstrate phosphorylated S6 suppression, which is downstream of mTOR. When ENZ was combined with the mTOR inhibitor, rapamycin, in H460 lung cancer cells, radiosensitization was observed. Conclusion: PKC appears to be crucial for angiogenesis, and its inhibition by ENZ has potential to enhance radiotherapy in vivo.

  9. Activated niacin receptor HCA2 inhibits chemoattractant-mediated macrophage migration via Gβγ/PKC/ERK1/2 pathway and heterologous receptor desensitization

    PubMed Central

    Shi, Ying; Lai, Xiangru; Ye, Lingyan; Chen, Keqiang; Cao, Zheng; Gong, Wanghua; Jin, Lili; Wang, Chunyan; Liu, Mingyong; Liao, Yuan; Wang, Ji Ming; Zhou, Naiming

    2017-01-01

    The niacin receptor HCA2 is implicated in controlling inflammatory host responses with yet poorly understood mechanistic basis. We previously reported that HCA2 in A431 epithelial cells transduced Gβγ-protein kinase C- and Gβγ-metalloproteinase/EGFR-dependent MAPK/ERK signaling cascades. Here, we investigated the role of HCA2 in macrophage-mediated inflammation and the underlying mechanisms. We found that proinflammatory stimulants LPS, IL-6 and IL-1β up-regulated the expression of HCA2 on macrophages. Niacin significantly inhibited macrophage chemotaxis in response to chemoattractants fMLF and CCL2 by disrupting polarized distribution of F-actin and Gβ protein. Niacin showed a selected additive effect on chemoattractant-induced activation of ERK1/2, JNK and PI3K pathways, but only the MEK inhibitor UO126 reduced niacin-mediated inhibition of macrophage chemotaxis, while activation of ERK1/2 by EGF alone did not inhibit fMLF-mediated migration of HEK293T cells co-expressing HCA2 and fMLF receptor FPR1. In addition, niacin induced heterologous desensitization and internalization of FPR1. Furthermore, niacin rescued mice from septic shock by diminishing inflammatory symptoms and the effect was abrogated in HCA2−/− mice. These results suggest that Gβγ/PKC-dependent ERK1/2 activation and heterologous desensitization of chemoattractant receptors are involved in the inhibition of chemoattractant-induced migration of macrophages by niacin. Thus, HCA2 plays a critical role in host protection against pro-inflammatory insults. PMID:28186140

  10. Antidepressant phenotype by inhibiting the phospholipase Cβ(1)--protein kinase Cγ pathway in the forced swim test.

    PubMed

    Galeotti, Nicoletta; Ghelardini, Carla

    2011-05-01

    Although great advances have recently been made in the study of signal transduction, the pathogenesis of affective disorders is still unknown. There is mounting evidence suggesting that elevated phosphoinositide-protein kinase C (PI-PKC) signal transduction pathway may be a pathophysiological feature of bipolar and major depressive disorders. The aim of the present study was to further investigated the phospholipase C-protein kinase C (PLC-PKC) cascade by evaluating the effect produced by an acute blockade of this intracellular pathway at PLC and PKC level. Adult male mice were administered with pharmacological inhibitors of PLC or PKC and then subjected to the forced swim test (FST), an animal model which emulates the behavioural despair paradigm of depression. In this study we also tested the hypothesis that it might be possible to selectively modulate depressive behaviour by inhibiting the expression of specific PLC and PKC isoforms by means of specific antisense oligonucleotides (aODNs). Administration of the PLC inhibitors neomycin and U73122 as well as of the PKC inhibitors calphostin C and chelerytrine dose-dependently reduced the immobility time in the FST producing an antidepressant-like behaviour. Selective knockdown of the PLCβ(1) and PKCγ isoforms also induced an antidepressant phenotype. Conversely, the inhibition of the expression of PLCβ(3) was unable to modify the immobility time values. The PLC and PKC modulators used, at the highest effective doses, altered neither locomotor activity nor motor coordination. We demonstrate that selective blockade of PLCβ(1)-PKCγ signalling pathway produces an antidepressant-like phenotype in mice.

  11. Nicotine decreases the activity of glutamate transporter type 3.

    PubMed

    Yoon, Hea-Jo; Lim, Young-Jin; Zuo, Zhiyi; Hur, Wonseok; Do, Sang-Hwan

    2014-02-10

    Nicotine, the main ingredient of tobacco, elicits seizures in animal models and cigarette smoking is regarded as a behavioral risk factor associated with epilepsy or seizures. In the hippocampus, the origin of nicotine-induced seizures, most glutamate uptake could be performed primarily by excitatory amino acid transporter type 3 (EAAT3). An association between temporal lobe epilepsy and EAAT3 downregulation has been reported. Therefore, we hypothesized that nicotine may elicit seizures through the attenuation of EAAT3 activity. We investigated chronic nicotine exposure (72 h) cause reduction of the activity of EAAT3 in a Xenopus oocyte expression system using a two-electrode voltage clamp. The roles of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) were also determined. Nicotine (0.001-1 μM) resulted in a time- and dose-dependent decrease in EAAT3 activity with maximal inhibition at nicotine concentrations of 0.03 μM or higher and at an exposure time of 72 h. Vmax on the glutamate response was significantly reduced in the nicotine group (0.03 μM for 72 h), but the Km value of EAAT3 for glutamate was not altered. When nicotine-exposed oocytes (0.03 μM for 72 h) were pretreated with phorbol-12-myristate-13-acetate (PMA, a PKC activator), the nicotine-induced reduction in EAAT3 activity was abolished. PKC inhibitors (staurosporine, chelerythrine, and calphostin C) significantly reduced basal EAAT3 activity, but there were no significant differences among the PKC inhibitors, nicotine, and PKC inhibitors+nicotine groups. Similar response patterns were observed among PI3K inhibitors (wortmannin and LY294002), nicotine, and PI3K inhibitors+nicotine. In conclusion, this study suggests that nicotine decreases EAAT3 activity, and that this inhibition seems to be dependent on PKC and PI3K. Our results may provide an additional mechanism for nicotine-induced seizure.

  12. [Apoptosis of human leukemic cells induced by topoisomerase I and II inhibitors].

    PubMed

    Solary, E; Dubrez, L; Eymin, B; Bertrand, R; Pommier, Y

    1996-03-01

    Comparison between five human leukemic lines (BV173, HL60, U937, K562, KCL22) suggest that the main determinant of their sensitivity to topoisomerase I (camptothecin) and II (VP-16) inhibitors is their ability to regulate cell cycle progression in response to specific DNA damage, then to die through apoptosis: the more the cells inhibit cell cycle progression, the less sensitive they are. The final pathway of apoptosis induction involves a cytoplasmic signal, active at neutral pH, needing magnesium, sensitive to various protease inhibitors and activated directly by staurosporine. Modulators of intracellular signaling (calcium chelators, calmodulin inhibitors, PKC modulators, kinase and phosphatase inhibitors) have no significant influence upon apoptosis induction. Conversely, apoptosis induction pathway is modified during monocytic differentiation of HL60 cells induced by phorbol esters. Lastly, poly(ADP-ribosyl)ation and chromatine structure should regulate apoptotic DNA fragmentation that is prevented by 3-aminobenzamide and spermine, respectively.

  13. Angiogenesis Inhibitors

    MedlinePlus

    ... inhibitors: current strategies and future prospects. CA: A Cancer Journal for Clinicians 2010; 60(4):222–243. [PubMed Abstract] Chen HX, Cleck JN. Adverse effects of anticancer agents that target the VEGF pathway. Nature Reviews Clinical Oncology 2009; 6(8):465– ...

  14. Carboxylesterase inhibitors

    PubMed Central

    Hatfield, M. Jason; Potter, Philip M.

    2011-01-01

    Introduction Carboxylesterases play major roles in the hydrolysis of numerous therapeutically active compounds. This is, in part, due to the prevalence of the ester moiety in these small molecules. However, the impact these enzymes may play on drug stability and pharmacokinetics is rarely considered prior to molecule development. Therefore, the application of selective inhibitors of this class of proteins may have utility in modulating the metabolism, distribution and toxicity of agents that are subjected to enzyme hydrolysis. Areas covered This review details the development of all such compounds dating back to 1986, but principally focuses on the very recent identification of selective human carboxylesterases inhibitors. Expert opinion The implementation of carboxylesterase inhibitors may significantly revolutionize drug discovery. Such molecules may allow for improved efficacy of compounds inactivated by this class of enzymes and/or reduce the toxicity of agents that are activated by these proteins. Furthermore, since lack of carboxylesterase activity appears to have no obvious biological consequence, these compounds could be applied in combination with virtually any esterified drug. Therefore, inhibitors of these proteins may have utility in altering drug hydrolysis and distribution in vivo. The characteristics, chemical and biological properties, and potential uses of such agents, are discussed here. PMID:21609191

  15. 14-3-3ζ and aPKC-ι synergistically facilitate epithelial-mesenchymal transition of cholangiocarcinoma via GSK-3β/snail signaling pathway

    PubMed Central

    He, Jun-chuang; Wang, Jian-ming; Schemmer, Peter; Ma, Chao-qun; Qian, Ya-wei; Yao, Wei; Zhang, Jian; Qi, Wei-peng; Fu, Yang; Feng, Wei; Yang, Tao

    2016-01-01

    Cholangiocarcinoma (CCA) invasion and metastasis are the primary causes of poor survival rates in patients. The epithelial-mesenchymal transition (EMT) is a crucial step in cancer invasion and metastasis. However, it is still unclear of the molecular mechanism. In this study, the expression of 14-3-3ζ and atypical protein kinase C-ι (aPKC-ι) was further detected in CCA tissues and cell lines. Meanwhile, we established the EMT model of CCA cells and investigated 14-3-3ζ and aPKC-ι co-regulatory effect on the EMT in vitro and in vivo. Further, we identified the downstream molecular glycogen synthase kinase 3 beta (GSK-3β)/Snail signalling pathway that contribute to regulating the EMT. Our data showed that the expression of 14-3-3ζ and aPKC-ι was synergistically increased in CCA tissues compared with adjacent noncancerous tissues and was intimately associated with differentiation and the tumour-node-metastasis (TNM) stage. Multivariate Cox regression analysis indicated that high 14-3-3ζ and aPKC-ι expression separately predicted a poor prognosis and were independent prognostic indicators in patients with CCA. The CO-IP experiment confirmed that the mutual binding relationship between 14-3-3ζ and aPKC-ι. Small interfering RNAs and siRNA rescue experiment demonstrated that 14-3-3ζ and aPKC-ι regulated each other. In addition, 14-3-3ζ and aPKC-ι pretreatment by si-RNA inhibit the phosphorylated GSK-3β and Snail expression during EMT. Meanwhile, silence of 14-3-3ζ or aPKC-ι suppressed CCA cells migration, metastasis and proliferation in vitro and in vivo. Our study demonstrates that 14-3-3ζ and aPKC-ι synergistically facilitate EMT of CCA via GSK-3β/Snail signalling pathway, and may be potential therapeutic target for CCA. PMID:27409422

  16. The stromal cell-derived factor-1alpha dependent migration of human cord blood CD34 haematopoietic stem and progenitor cells switches from protein kinase C (PKC)-alpha dependence to PKC-alpha independence upon prolonged culture in the presence of Flt3-ligand and interleukin-6.

    PubMed

    Kasenda, Benjamin; Kassmer, Susannah H; Niggemann, Bernd; Schiermeier, Sven; Hatzmann, Wolfgang; Zänker, Kurt S; Dittmar, Thomas

    2008-09-01

    Addition of the inflammatory cytokine interleukin (IL)-6 to the culture medium of human cord blood haematopoietic stem and progenitor cells (HSPCs) has been shown to lead to an altered stromal cell-derived factor-1alpha-dependent migratory phenotype. This study investigated whether this effect was attributed to a differential engagement of protein kinase C (PKC) isotypes. The migratory activity of both Flt3-ligand and Flt3-ligand/IL-6 cultured cord blood HSPCs was PKC-alpha dependent on day 1, but PKC-alpha independent after 5 d of cultivation. PKC-alpha expression was not down-regulated in cells cultured for 5 d indicating a switch of signalling molecules directing cell migration.

  17. PKC-dependent stimulation of EAAT3 glutamate transporter does not require the integrity of actin cytoskeleton.

    PubMed

    Bianchi, Massimiliano G; Rotoli, Bianca Maria; Dall'Asta, Valeria; Gazzola, Gian C; Gatti, Rita; Bussolati, Ovidio

    2006-04-01

    The activity and the membrane expression of EAAT3 glutamate transporter are stimulated upon PKC activation by phorbol esters in C6 rat glioma cells. To investigate the role of cytoskeleton in these effects, we have employed actin-perturbing toxins and found that the perturbation of actin cytoskeleton inhibits basal but not phorbol-stimulated EAAT3 activity and membrane trafficking. In the absence of phorbols, latrunculin A, a toxin that disassembles actin cytoskeleton, produced a rapid inhibition of EAAT3 activity, due to a decrease in transport V(max). The inhibitory effect was fully reversible and was not detected for other sodium dependent transport systems for amino acids. However, latrunculin did not prevent the increase in transport caused by phorbol esters and, moreover, cells pre-treated with phorbols were resistant to the inhibitory effect of the toxin on EAAT3 activity. Biotinylation experiments indicated that the inhibitory effect of latrunculin was attributable to a decreased expression of the carrier on the membrane, while the toxin did not suppress the PKC-dependent increase in EAAT3 membrane abundance. Latrunculin A effects on EAAT3 were shared by cytochalasin D, a toxin that disorganizes actin filaments with a distinct mechanism of action. On the contrary, a small, but significant, increase of EAAT3 activity was observed upon incubation with jasplakinolide, a drug that stabilizes actin microfilaments. Also jasplakinolide, however, did not hinder phorbol-dependent stimulation of aspartate transport. Colchicine, a toxin that disrupts microtubules, also lowered EAAT3 activity without preventing transport stimulation by phorbols, while microtubule stabilization by paclitaxel led to an increase in aspartate transport. It is concluded that, in C6 cells, the PKC-mediated stimulatory effects on EAAT3 are cytoskeleton-independent, while in the absence of phorbols, the transporter is partially inhibited by the disorganization of either actin microfilaments or

  18. ROS, MAPK/ERK and PKC play distinct roles in EGF-stimulated human corneal cell proliferation and migration.

    PubMed

    Huo, Y-N; Chen, W; Zheng, X-X

    2015-11-08

    Cornea is at the outermost surface of eye globe, and it easily receives damage from ultraviolet light exposure, physiology wounding, and infections. It is essential to understand the mechanisms controlling human corneal epithelial (HCE) cell proliferation and wound healing. Epidermal growth factor (EGF) could stimulate cell proliferation and migration in various cell types. Therefore, we investigated the roles and mechanisms of EGF on HCE cell proliferation and migration. CCK-8 kit and wound healing experiment were used to investigate HCE cell proliferation and cell migration, respectively. ROS activity was quantified by DCFDA and flow cytometry. Western blot and Q-PCR were performed to examine protein and RNA levels. EGF could promote HCE cell proliferation and migration in both physiology status and UV irradiation conditions, which is used to mimic the disease condition in human corneal epithelial cells. Interestingly, the promotion effect of EGF on HCE cell proliferation is mainly mediated by activated ROS signaling under disease condition. However, the EGF function is mediated by ROS and MAPK/ERK pathway in EGF-treated corneal epithelial cells in physiology status, in which ROS and MAPK/ERK pathway have no mutual influence on the other signaling pathway in EGF-stimulated corneal epithelial cells. We also revealed that MAPK/ERK pathway instead of ROS mediates EGF-stimulated HCE cell migration. Interestingly, we found that PKC proteins were downregulated by EGF in HCE cells that is partially mediated by ROS signaling, while PKC pathway was not involved in EGF-stimulated corneal cell proliferation and migration. EGF promotes human corneal cell proliferation and migration both in physiology and disease conditions, and ROS, MAPK/ERK and PKC pathways play different roles in these processes.

  19. Mouse Sphingosine Kinase 1a Is Negatively Regulated through Conventional PKC-Dependent Phosphorylation at S373 Residue

    PubMed Central

    Oh, Yong-Seok; Bae, Sun Sik; Park, Jong Bae; Ha, Sang Hoon; Ryu, Sung Ho; Suh, Pann-Ghill

    2015-01-01

    Sphingosine kinase is a lipid kinase that converts sphingosine into sphingosine-1-phosphate, an important signaling molecule with intracellular and extracellular functions. Although diverse extracellular stimuli influence cellular sphingosine kinase activity, the molecular mechanisms underlying its regulation remain to be clarified. In this study, we investigated the phosphorylation-dependent regulation of mouse sphingosine kinase (mSK) isoforms 1 and 2. mSK1a was robustly phosphorylated in response to extracellular stimuli such as phorbol ester, whereas mSK2 exhibited a high basal level of phosphorylation in quiescent cells regardless of agonist stimulation. Interestingly, phorbol ester-induced phosphorylation of mSK1a correlated with suppression of its activity. Chemical inhibition of conventional PKCs (cPKCs) abolished mSK1a phosphorylation, while overexpression of PKCα, a cPKC isoform, potentiated the phosphorylation, in response to phorbol ester. Furthermore, an in vitro kinase assay showed that PKCα directly phosphorylated mSK1a. In addition, phosphopeptide mapping analysis determined that the S373 residue of mSK1a was the only site phosphorylated by cPKC. Interestingly, alanine substitution of S373 made mSK1a refractory to the inhibitory effect of phorbol esters, whereas glutamate substitution of the same residue resulted in a significant reduction in mSK1a activity, suggesting the significant role of this phosphorylation event. Taken together, we propose that mSK1a is negatively regulated through cPKC-dependent phosphorylation at S373 residue. PMID:26642194

  20. Mouse Sphingosine Kinase 1a Is Negatively Regulated through Conventional PKC-Dependent Phosphorylation at S373 Residue.

    PubMed

    Oh, Yong-Seok; Bae, Sun Sik; Park, Jong Bae; Ha, Sang Hoon; Ryu, Sung Ho; Suh, Pann-Ghill

    2015-01-01

    Sphingosine kinase is a lipid kinase that converts sphingosine into sphingosine-1-phosphate, an important signaling molecule with intracellular and extracellular functions. Although diverse extracellular stimuli influence cellular sphingosine kinase activity, the molecular mechanisms underlying its regulation remain to be clarified. In this study, we investigated the phosphorylation-dependent regulation of mouse sphingosine kinase (mSK) isoforms 1 and 2. mSK1a was robustly phosphorylated in response to extracellular stimuli such as phorbol ester, whereas mSK2 exhibited a high basal level of phosphorylation in quiescent cells regardless of agonist stimulation. Interestingly, phorbol ester-induced phosphorylation of mSK1a correlated with suppression of its activity. Chemical inhibition of conventional PKCs (cPKCs) abolished mSK1a phosphorylation, while overexpression of PKCα, a cPKC isoform, potentiated the phosphorylation, in response to phorbol ester. Furthermore, an in vitro kinase assay showed that PKCα directly phosphorylated mSK1a. In addition, phosphopeptide mapping analysis determined that the S373 residue of mSK1a was the only site phosphorylated by cPKC. Interestingly, alanine substitution of S373 made mSK1a refractory to the inhibitory effect of phorbol esters, whereas glutamate substitution of the same residue resulted in a significant reduction in mSK1a activity, suggesting the significant role of this phosphorylation event. Taken together, we propose that mSK1a is negatively regulated through cPKC-dependent phosphorylation at S373 residue.

  1. JAM-A and aPKC: A close pair during cell-cell contact maturation and tight junction formation in epithelial cells.

    PubMed

    Ebnet, Klaus

    2013-01-01

    Cell-cell adhesion plays a critical role in the formation of barrier-forming epithelia. The molecules which mediate cell-cell adhesion frequently act as signaling molecules by recruiting and/or assembling cytoplasmic protein complexes. Junctional Adhesion Molecule (JAM)-A interacts with the cell polarity protein PAR-3, a member of the PAR-3-aPKC-PAR-6 complex, which regulates the formation of cell-cell contacts and the development of tight junctions (TJs). In our recent study we found that JAM-A is localized at primordial, spot-like cell-cell junctions (pAJs) in a non-phosphorylated form. After the recruitment of the PAR-aPKC complex and its activation at pAJs, aPKC phosphorylates JAM-A at Ser285 to promote the maturation of immature junctions. In polarized epithelial cells, aPKC phosphorylates JAM-A selectively at the TJs to maintain the barrier function of TJs. Thus, through mutual regulation, JAM-A and aPKC form a functional unit that regulates the establishment of barrier-forming junctions in vertebrate epithelial cells.

  2. Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation

    PubMed Central

    Li, Jasmine; Hardy, Kristine; Phetsouphanh, Chan; Tu, Wen Juan; Sutcliffe, Elissa L.; McCuaig, Robert; Sutton, Christopher R.; Zafar, Anjum; Munier, C. Mee Ling; Zaunders, John J.; Xu, Yin; Theodoratos, Angelo; Tan, Abel; Lim, Pek Siew; Knaute, Tobias; Masch, Antonia; Zerweck, Johannes; Brezar, Vedran; Milburn, Peter J.; Dunn, Jenny; Casarotto, Marco G.; Turner, Stephen J.; Seddiki, Nabila; Kelleher, Anthony D.

    2016-01-01

    ABSTRACT Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation. This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood. By genome-wide ChIP-sequencing ex vivo human CD4+ T cells, here, we show that the signaling enzyme, protein kinase C theta (PKC-θ) directly relays stimulatory signals to chromatin by binding to transcriptional-memory-responsive genes to induce transcriptional activation. Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4+ T cells. Within the nucleus, PKC-θ catalytic activity maintains the Ser536 phosphorylation on the p65 subunit of NF-κB (also known as RelA) and can directly influence chromatin accessibility at transcriptional memory genes by regulating H2B deposition through Ser32 phosphorylation. Furthermore, using a cytoplasm-restricted PKC-θ mutant, we highlight that chromatin-anchored PKC-θ integrates activating signals at the chromatin template to elicit transcriptional memory responses in human memory T cells. PMID:27149922

  3. Protein Kinase C (PKC)-promoted Endocytosis of Glutamate Transporter GLT-1 Requires Ubiquitin Ligase Nedd4-2-dependent Ubiquitination but Not Phosphorylation*

    PubMed Central

    García-Tardón, Noemí; González-González, Inmaculada M.; Martínez-Villarreal, Jaime; Fernández-Sánchez, Enrique; Giménez, Cecilio; Zafra, Francisco

    2012-01-01

    Glutamate transporter-1 (GLT-1) is the main glutamate transporter in the central nervous system, and its concentration severely decreases in neurodegenerative diseases. The number of transporters in the plasma membrane reflects the balance between their insertion and removal, and it has been reported that the regulated endocytosis of GLT-1 depends on its ubiquitination triggered by protein kinase C (PKC) activation. Here, we identified serine 520 of GLT-1 as the primary target for PKC-dependent phosphorylation, although elimination of this serine did not impair either GLT-1 ubiquitination or endocytosis in response to phorbol esters. In fact, we present evidence indicating that the ubiquitin ligase Nedd4-2 mediates the PKC-dependent ubiquitination and down-regulation of GLT-1. Overexpression of Nedd4-2 increased the ubiquitination of the transporter and promoted its degradation. Moreover, phorbol myristate acetate enhanced Nedd4-2 phosphorylation and the formation of GLT-1·Nedd4-2 complexes, whereas siRNA knockdown of Nedd4-2 prevented ubiquitination, endocytosis, and the concomitant decrease in GLT-1 activity triggered by PKC activation. These results indicate that GLT-1 endocytosis is independent of its phosphorylation and that Nedd4-2 mediates PKC-dependent down-regulation of the transporter. PMID:22505712

  4. eNOS S-nitrosylates β-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1.

    PubMed

    García-Ortiz, Almudena; Martín-Cofreces, Noa B; Ibiza, Sales; Ortega, Ángel; Izquierdo-Álvarez, Alicia; Trullo, Antonio; Victor, Víctor M; Calvo, Enrique; Sot, Begoña; Martínez-Ruiz, Antonio; Vázquez, Jesús; Sánchez-Madrid, Francisco; Serrador, Juan M

    2017-04-01

    The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-θ (PKC-θ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of β-actin and PKC-θ from the lamellipodium-like distal (d)-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated β-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding defective mutants of β-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-θ at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS.

  5. High D-glucose reduces SLC29A1 promoter activity and adenosine transport involving specific protein 1 in human umbilical vein endothelium.

    PubMed

    Puebla, Carlos; Farías, Marcelo; González, Marcelo; Vecchiola, Andrea; Aguayo, Claudio; Krause, Bernardo; Pastor-Anglada, Marçal; Casanello, Paola; Sobrevia, Luis

    2008-06-01

    High D-glucose reduces human equilibrative nucleoside transporter 1 (hENT1)-mediated adenosine uptake involving endothelial nitric oxide synthase (eNOS), mitogen-activated protein (MAP) kinase kinases 1 and 2/MAP kinases p42/44 (MEK/ERKs), and protein kinase C (PKC) activation in human umbilical vein endothelium (HUVEC). Since NO represses SLC29A1 gene (hENT1) promoter activity we studied whether D-glucose-reduced hENT1-adenosine transport results from lower SLC29A1 expression in HUVEC primary cultures. HUVEC incubation (24 h) with high D-glucose (25 mM) reduced hENT1-adenosine transport and pGL3-hENT1(-1114) construct SLC29A1 reporter activity compared with normal D-glucose (5 mM). High D-glucose also reduced pGL3-hENT1(-1114) reporter activity compared with cells transfected with pGL3-hENT1(-795) construct. N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor), PD-98059 (MEK1/2 inhibitor), and/or calphostin C (PKC inhibitor) blocked D-glucose effects. Insulin (1 nM) and phorbol 12-myristate 13-acetate (PMA, 100 nM, PKC activator), but not 4alpha-phorbol 12,13-didecanoate (4alphaPDD, 100 nM, PMA less active analogue) reduced hENT1-adenosine transport. L-NAME and PD-98059 blocked insulin effects. L-NAME, PD-98059, and calphostin C increased hENT1 expression without altering protein or mRNA stability. High D-glucose increased Sp1 transcription factor protein abundance and binding to SLC29A1 promoter, phenomena blocked by L-NAME, PD-98059, and calphostin C. Sp1 overexpression reduced SLC29A1 promoter activity in normal D-glucose, an effect reversed by L-NAME and further reduced by S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor) in high D-glucose. Thus, reduced hENT1-mediated adenosine transport in high D-glucose may result from increased Sp1 binding to SLC29A1 promoter down-regulating hENT1 expression. This phenomenon depends on eNOS, MEK/ERKs, and PKC activity, suggesting potential roles for these molecules in hyperglycemia-associated endothelial

  6. TCR-induced, PKC-θ-mediated NF-κB Activation Is Regulated by a Caspase-8-Caspase-9-Caspase-3 Cascade

    PubMed Central

    Zhao, Yixia; Lei, Minxiang; Wang, Zhaoyuan; Qiao, Guilin; Yang, Tianlun; Zhang, Jian

    2014-01-01

    It has been documented that caspase-8, a central player in apoptosis, is also crucial for TCR-mediated NF-κB activation. However, whether other caspases are also involved this process is unknown. In this report, we showed that in addition to caspase-8, caspase-9 is required for TCR-mediated NF-κB activation. Caspase-9 induces activation of PKC-θ, phosphorylation of Bcl10 and NF-κB activation in a caspase-3-dependent manner, but it appears that Bcl10 phosphorylation is uncoupled from NF-κB activation. Furthermore, caspase-8 lies upstream of caspase-9 during T cell activation. Therefore, TCR ligation elicits a caspase cascade involving caspase-8, caspase-9 and caspase-3 which initiates PKC-θ-dependent pathway leading to NF-κB activation and PKC-θ-independent Bcl10 phosphorylation which limits NF-kB activity. PMID:24924627

  7. Activation of muscarinic receptors in porcine airway smooth muscle elicits a transient increase in phospholipase D activity.

    PubMed

    Mamoon, A M; Smith, J; Baker, R C; Farley, J M

    1999-01-01

    Phospholipase D (PLD) is a phosphodiesterase that catalyses hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. In the presence of ethanol, PLD also catalyses the formation of phosphatidylethanol, which is a unique characteristic of this enzyme. Muscarinic receptor-induced changes in the activity of PLD were investigated in porcine tracheal smooth muscle by measuring the formation of [3H]phosphatidic acid ([3H]PA) and [3H]phosphatidylethanol ([3H]PEth) after labeling the muscle strips with [3H]palmitic acid. The cholinergic receptor agonist acetylcholine (Ach) significantly but transiently increased formation of both [3H]PA and [3H]PEth in a concentration-dependent manner (>105-400% vs. controls in the presence of 10(-6) to 10(-4) M Ach) when pretreated with 100 mM ethanol. The Ach receptor-mediated increase in PLD activity was inhibited by atropine (10(-6) M), indicating that activation of PLD occurred via muscarinic receptors. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) increased PLD activity that was effectively blocked by the PKC inhibitors calphostin C (10(-8) to 10(-6) M) and GFX (10(-8) to 10(-6) M). Ach-induced increases in PLD activity were also significantly, but incompletely, inhibited by both GFX and calphostin C. From the present data, we conclude that in tracheal smooth muscle, muscarinic acetylcholine receptor-induced PLD activation is transient in nature and coupled to these receptors via PKC. However, PKC activation is not solely responsible for Ach-induced activation of PLD in porcine tracheal smooth muscle.

  8. Clostridium perfringens phospholipase C induced ROS production and cytotoxicity require PKC, MEK1 and NFκB activation.

    PubMed

    Monturiol-Gross, Laura; Flores-Díaz, Marietta; Pineda-Padilla, Maria Jose; Castro-Castro, Ana Cristina; Alape-Giron, Alberto

    2014-01-01

    Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the most toxic extracellular enzyme produced by this bacteria and is essential for virulence in gas gangrene. At lytic concentrations, CpPLC causes membrane disruption, whereas at sublytic concentrations this toxin causes oxidative stress and activates the MEK/ERK pathway, which contributes to its cytotoxic and myotoxic effects. In the present work, the role of PKC, ERK 1/2 and NFκB signalling pathways in ROS generation induced by CpPLC and their contribution to CpPLC-induced cytotoxicity was evaluated. The results demonstrate that CpPLC induces ROS production through PKC, MEK/ERK and NFκB pathways, the latter being activated by the MEK/ERK signalling cascade. Inhibition of either of these signalling pathways prevents CpPLC's cytotoxic effect. In addition, it was demonstrated that NFκB inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of CpPLC in mice. Understanding the role of these signalling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridialmyonecrosis.

  9. Clostridium perfringens Phospholipase C Induced ROS Production and Cytotoxicity Require PKC, MEK1 and NFκB Activation

    PubMed Central

    Monturiol-Gross, Laura; Flores-Díaz, Marietta; Pineda-Padilla, Maria Jose; Castro-Castro, Ana Cristina; Alape-Giron, Alberto

    2014-01-01

    Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the most toxic extracellular enzyme produced by this bacteria and is essential for virulence in gas gangrene. At lytic concentrations, CpPLC causes membrane disruption, whereas at sublytic concentrations this toxin causes oxidative stress and activates the MEK/ERK pathway, which contributes to its cytotoxic and myotoxic effects. In the present work, the role of PKC, ERK 1/2 and NFκB signalling pathways in ROS generation induced by CpPLC and their contribution to CpPLC-induced cytotoxicity was evaluated. The results demonstrate that CpPLC induces ROS production through PKC, MEK/ERK and NFκB pathways, the latter being activated by the MEK/ERK signalling cascade. Inhibition of either of these signalling pathways prevents CpPLC's cytotoxic effect. In addition, it was demonstrated that NFκB inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of CpPLC in mice. Understanding the role of these signalling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridialmyonecrosis. PMID:24466113

  10. 1–42 β-Amyloid peptide requires PDK1/nPKC/Rac 1 pathway to induce neuronal death

    PubMed Central

    Manterola, L; Hernando-Rodríguez, M; Ruiz, A; Apraiz, A; Arrizabalaga, O; Vellón, L; Alberdi, E; Cavaliere, F; Lacerda, H M; Jimenez, S; Parada, L A; Matute, C; Zugaza, J L

    2013-01-01

    1–42 β-Amyloid (Aβ1–42) peptide is a key molecule involved in the development of Alzheimer's disease. Some of its effects are manifested at the neuronal morphological level. These morphological changes involve loss of neurites due to cytoskeleton alterations. However, the mechanism of Aβ1–42 peptide activation of the neurodegenerative program is still poorly understood. Here, Aβ1–42 peptide-induced transduction of cellular death signals through the phosphatidylinositol 3-kinase (PI3K)/phosphoinositol-dependent kinase (PDK)/novel protein kinase C (nPKC)/Rac 1 axis is described. Furthermore, pharmacological inhibition of PDK1 and nPKC activities blocks Rac 1 activation and neuronal cell death. Our results provide insights into an unsuspected connection between PDK1, nPKCs and Rac 1 in the same signal-transduction pathway and points out nPKCs and Rac 1 as potential therapeutic targets to block the toxic effects of Aβ1–42 peptide in neurons. PMID:23340502

  11. The interplay between intracellular progesterone receptor and PKC plays a key role in migration and invasion of human glioblastoma cells.

    PubMed

    Marquina-Sánchez, Brenda; González-Jorge, Jesús; Hansberg-Pastor, Valeria; Wegman-Ostrosky, Talia; Baranda-Ávila, Noemi; Mejía-Pérez, Sonia; Camacho-Arroyo, Ignacio; González-Arenas, Aliesha

    2016-10-04

    Intracellular progesterone receptors (PRs) and protein kinases C (PKCs) are known regulators of cancer cell proliferation and metastasis. Both PRs and PKCs are found overexpressed in grade IV human astrocytomas, also known as glioblastomas, which are the most frequent and aggressive brain tumors. In the present study, we investigated whether PR activation by PKC induces the migration and invasion of glioblastoma derived cell lines and if PKCα and δ isoforms are involved in PR activation. We observed that PKC activation with tetradecanoylphorbol acetate (TPA) increases the migration and invasion capacity of two human glioblastoma derived human cell lines (U251 MG and U87) and that the treatment with the PR receptor antagonist RU486 blocks these processes. Interestingly, the pharmacological inhibition of the isoenzymes PKCα and PKCδ also resulted in a blocked PR transcriptional activity. Also, TPA-dependent PR activation increases the expression of progesterone-induced blocking factor (PIBF), a known PR target gene. These results hint to an existing cross-talk between PKCs and PRs in regulating the infiltration process of human glioblastomas.

  12. Differential effects of protein kinase C inhibitors on chemokine production in human synovial fibroblasts.

    PubMed Central

    Jordan, N. J.; Watson, M. L.; Yoshimura, T.; Westwick, J.

    1996-01-01

    1. Rheumatoid arthritis is associated with the accumulation and activation of selected populations of inflammatory cells within the arthritic joint. One putative signal for this process is the production, by resident cells, of a group of inflammatory mediators known as the chemokines. 2. The chemokines interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1) and RANTES (regulated on activation normal T-cell expressed and presumably secreted) are target-cell specific chemoattractants produced by synovial fibroblasts in response to stimulation with interleukin-1 alpha (IL-1 alpha) or tumour necrosis factor alpha (TNF alpha). The signalling pathways involved in their production are not well defined. We therefore used four different protein kinase C inhibitors to investigate the role of this kinase in the regulation of chemokine mRNA and protein expression in human cultured synovial fibroblasts. 3. The non-selective PKC inhibitor, staurosporine (1-300 nM) significantly increased the production of IL-1 alpha-induced IL-8 mRNA and protein. A specific PKC inhibitor, chelerythrine chloride (0.1-3 microM), also caused a small concentration-dependent increase in IL-8 mRNA and protein production. In contrast, 3-[1-[3-(amidinothio)propyl]-3-indoly]-4-(1-methyl-3-indolyl )- 1H-pyrrole-2,5-dione methanesulphonate (Ro 31-8220) and 2[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3- yl)-maleimide (GF 109203X), two selective PKC inhibitors of the substituted bisindolylmaleimide family had a concentration-dependent biphasic effect on IL-1 alpha or TNF alpha-induced chemokine expression. At low concentrations they caused a stimulation in chemokine production, which was especially evident at the mRNA level. At higher concentrations both inhibited IL-1 alpha or TNF alpha-induced chemokine mRNA and protein production. Ro 31-8220 was 10 fold more potent than GF 109203X, with an IC50 of 1.6 +/- 0.08 microM (mean +/- s.e.mean, n = 4) for IL-1 alpha induced IL-8 production. Ro 31

  13. Autophagy inhibitors.

    PubMed

    Pasquier, Benoit

    2016-03-01

    Autophagy is a lysosome-dependent mechanism of intracellular degradation. The cellular and molecular mechanisms underlying this process are highly complex and involve multiple proteins, including the kinases ULK1 and Vps34. The main function of autophagy is the maintenance of cell survival when modifications occur in the cellular environment. During the past decade, extensive studies have greatly improved our knowledge and autophagy has exploded as a research field. This process is now widely implicated in pathophysiological processes such as cancer, metabolic, and neurodegenerative disorders, making it an attractive target for drug discovery. In this review, we will summarize the different types of inhibitors that affect the autophagy machinery and provide some potential therapeutic perspectives.

  14. Regulation of gap junctional intercellular communication by TCDD in HMEC and MCF-7 breast cancer cells

    SciTech Connect

    Gakhar, Gunjan Schrempp, Diane Nguyen, Thu Annelise

    2009-03-01

    Previous studies suggest that many neoplastic tissues exhibit a decrease in gap junctional intercellular communication (GJIC). Many hydrocarbons and organochlorine compounds are environmental pollutants known to be carcinogenic. The effect of an organochlorine compound, TCDD, on GJIC in human breast cell lines has not been established. In the present study, we showed that TCDD causes an inhibition in the gap junctional activity in MCF-7 (breast cancer cells). In MCF-7 cells, an increase in the phosphorylated form of gap junctional protein, connexin 43 (Cx43), and PKC {alpha} was seen in the presence of TCDD. Gap junctional plaque formation was significantly decreased in MCF-7 cells in the presence of TCDD. Immunoprecipitation studies of PKC {alpha} showed that TCDD caused a significant 40% increase in the phosphorylated Cx43 in MCF-7 cells. TCDD also modulated the translocation of PKC {alpha} from the cytosol to the membrane and caused a 2-fold increase in the PKC {alpha} activity at 50 nM TCDD in MCF-7 cells. Calphostin C, an inhibitor of PKC {alpha}, showed a significant inhibition of PKC {alpha} activity in the presence of TCDD. Furthermore, TCDD also caused a decrease in the gap junctional activity and Cx43 protein in human mammary epithelial cells (HMEC). However, we observed a shift in the Cx43 plaques towards the perinuclear membrane in the presence of TCDD by confocal microscopy and Western blot. Overall, these results conclude that TCDD decreases GJIC by phosphorylating Cx43 via PKC {alpha} signaling pathway in MCF-7 cells; however, TCDD decreases the GJIC by affecting the localization of Cx43 in HMEC. These new findings elucidate the differential mode of effect of TCDD in the downregulation of GJIC in HMEC and MCF-7 cells.

  15. Very low density lipoprotein-mediated signal transduction and plasminogen activator inhibitor type 1 in cultured HepG2 cells.

    PubMed

    Banfi, C; Mussoni, L; Risé, P; Cattaneo, M G; Vicentini, L; Battaini, F; Galli, C; Tremoli, E

    1999-07-23

    In normal subjects and in patients with cardiovascular disease, plasma triglycerides are positively correlated with plasminogen activator inhibitor type 1 (PAI-1) levels. Moreover, in vitro studies indicate that VLDLs induce PAI-1 synthesis in cultured cells, ie, endothelial and HepG2 cells. However, the signaling pathways involved in the effect of VLDL on PAI-1 synthesis have not yet been investigated. We report that VLDLs induce a signaling cascade that leads to an enhanced secretion of PAI-1 by HepG2 cells. In myo-[(3)H]inositol-labeled HepG2 cells, VLDL (100 microg/mL) caused a time-dependent increase in [(3)H]inositol phosphates, the temporal sequence being tris>bis>monophosphate. VLDL brought about a time-dependent stimulation of membrane-associated protein kinase C (PKC) activity and arachidonate release. Finally, VLDL stimulated mitogen-activated protein (MAP) kinase, and this effect was reduced by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), which suggests that PKC plays a pivotal role in MAP kinase phosphorylation. VLDL-induced PAI-1 secretion was completely prevented by U73122, a specific inhibitor of phosphatidylinositol-specific phospholipase C, by H7 or by PKC downregulation, and by mepacrine (all P<0.01 versus VLDL-treated cells). 3,4,5-Trimethoxybenzoic acid 8-(diethylamino)-octyl ester, which prevents Ca2+ release from intracellular stores, inhibited VLDL-induced PAI-1 secretion by 60% (P<0.05), and the MAP kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059 completely suppressed both basal and VLDL-induced PAI-1 secretion. These data demonstrate that VLDL-induced PAI-1 biosynthesis results from a principal signaling pathway involving PKC-mediated MAP kinase activation.

  16. Reduction of α1GABAA receptor mediated by tyrosine kinase C (PKC) phosphorylation in a mouse model of fragile X syndrome

    PubMed Central

    Zhao, Weidong; Wang, Jiaqin; Song, Shunyi; Li, Fang; Yuan, Fangfang

    2015-01-01

    Fragile X syndrome (FXS) caused by lack of fragile X mental retardation protein (Fmr1) is the most common cause of inherited intellectual disability and characterized by many cognitive disturbances like attention deficit, autistic behavior, and audiogenic seizure and have region-specific altered expression of some gamma-aminobutyric acid (GABAA) receptor subunits. Quantitative real-time polymerase chain reaction and western blot experiments were performed in the cultured cortical neurons and forebrain obtained from wild-type (WT) and Fmr1 KO mice demonstrate the reduction in the expression of α1 gamma-aminobutyric acid (α1GABAA) receptor, phospho-α1GABAA receptor, PKC and phosphor-PKC in Fmr1 KO mice comparing with WT mice, both in vivo and in vitro. Furthermore, we found that the phosphorylation of the α1GABAA receptor was mediated by PKC. Our results elucidate that the lower phosphorylation of the α1GABAA receptor mediated by PKC neutralizes the seizure-promoting effects in Fmr1 KO mice and point to the potential therapeutic targets of α1GABAA agonists for the treatment of fragile X syndrome. PMID:26550246

  17. Replacement of the Bryostatin A- and B-Pyran Rings With Phenyl Rings Leads to Loss of High Affinity Binding With PKC.

    PubMed

    Petersen, Mark E; Kedei, Noemi; Lewin, Nancy E; Blumberg, Peter M; Keck, Gary E

    2016-10-19

    We describe a convergent synthesis of a bryostatin analogue in which the natural A- and B-ring pyrans have been replaced by phenyl rings. The new analogue exhibited PMA like behavior in cell assays, but failed to maintain high affinity binding for PKC, despite retaining an unaltered C-ring 'binding domain'.

  18. Inositol hexaphosphate represses telomerase activity and translocates TERT from the nucleus in mouse and human prostate cancer cells via the deactivation of Akt and PKC{alpha}

    SciTech Connect

    Jagadeesh, Shankar; Banerjee, Partha P. . E-mail: ppb@georgetown.edu

    2006-11-03

    Inositol hexaphosphate (IP6) has anti-proliferative effects on a variety of cancer cells, including prostate cancer. However, the molecular mechanism of anti-proliferative effects of IP6 is not entirely understood. Since the activation of telomerase is crucial for cells to gain immortality and proliferation ability, we examined the role of IP6 in the regulation of telomerase activity in prostate cancer cells. Here, we show that IP6 represses telomerase activity in mouse and human prostate cancer cells dose-dependently. In addition, IP6 prevents the translocation of TERT to the nucleus. Since phosphorylation of TERT by Akt and/or PKC{alpha} is necessary for nuclear translocation, we examined phosphorylation of Akt and PKC{alpha} after IP6 treatments. Our results show that IP6 inhibits phosphorylation of Akt and PKC{alpha}. These results show for the first time that IP6 represses telomerase activity in prostate cancer cells by posttranslational modification of TERT via the deactivation of Akt and PKC{alpha}.

  19. Alterations in the immunoreactivity for muscarinic acetylcholine receptors and colocalized PKC gamma in mouse hippocampus induced by spatial discrimination learning.

    PubMed

    Van der Zee, E A; Compaan, J C; Bohus, B; Luiten, P G

    1995-01-01

    group over all other groups. In contrast to the CA1-CA2 region, both pseudotrained and trained mice revealed high mAChR staining in the CA3-CA4 region and the DG. These results indicate that prolonged exposure to the hole board is sufficient for an enhanced mAChR-ir in the CA3-CA4 and DG, whereas the increase in CA1-CA2 pyramidal cells is a training-specific feature related to spatial orientation. Nonpyramidal neurons within the CA1-CA2 region with enhanced mAChR-ir in the pyramidal cells, however, revealed a decreased level of mAChR-ir. The opposing effect of pyramidal and nonpyramidal cells suggests a shift in the excitability of the hippocampal microcircuitry. Previously we demonstrated an increase and redistribution of hippocampal protein kinase C gamma-immunoreactivity (PKC gamma-ir) induced by hole board learning in mice (Van der Zee et al., 1992, J Neurosci 12:4808-4815). Immunofluorescence double-labeling experiments conducted in the present study in naive and trained animals revealed that the principal cells and DG interneurons co-express mAChRs and PKC gamma, and that the immunoreactivity for both markers increased in relation to spatial orientation within these neurons. The mAChR-positive nonpyramidal cells of the CA1-CA2 region were devoid of PKC gamma and revealed an opposite training-induced effect. These results suggest that the postsynaptic changes in mAChR- and PKC gamma-ir reflect functional alterations of the hippocampal formation induced by spatial learning.

  20. Impaired muscarinic type 3 (M3) receptor/PKC and PKA pathways in islets from MSG-obese rats.

    PubMed

    Ribeiro, Rosane Aparecida; Balbo, Sandra Lucinei; Roma, Letícia Prates; Camargo, Rafael Ludemann; Barella, Luiz Felipe; Vanzela, Emerielle Cristine; de Freitas Mathias, Paulo Cesar; Carneiro, Everardo Magalhães; Boschero, Antonio Carlos; Bonfleur, Maria Lúcia

    2013-07-01

    Monosodium glutamate-obese rats are glucose intolerant and insulin resistant. Their pancreatic islets secrete more insulin at increasing glucose concentrations, despite the possible imbalance in the autonomic nervous system of these rats. Here, we investigate the involvement of the cholinergic/protein kinase (PK)-C and PKA pathways in MSG β-cell function. Male newborn Wistar rats received a subcutaneous injection of MSG (4 g/kg body weight (BW)) or hyperosmotic saline solution during the first 5 days of life. At 90 days of life, plasma parameters, islet static insulin secretion and protein expression were analyzed. Monosodium glutamate rats presented lower body weight and decreased nasoanal length, but had higher body fat depots, glucose intolerance, hyperinsulinemia and hypertrigliceridemia. Their pancreatic islets secreted more insulin in the presence of increasing glucose concentrations with no modifications in the islet-protein content of the glucose-sensing proteins: the glucose transporter (GLUT)-2 and glycokinase. However, MSG islets presented a lower secretory capacity at 40 mM K(+) (P < 0.05). The MSG group also released less insulin in response to 100 μM carbachol, 10 μM forskolin and 1 mM 3-isobutyl-1-methyl-xantine (P < 0.05, P < 0.0001 and P < 0.01). These effects may be associated with a the decrease of 46 % in the acetylcholine muscarinic type 3 (M3) receptor, and a reduction of 64 % in PKCα and 36 % in PKAα protein expressions in MSG islets. Our data suggest that MSG islets, whilst showing a compensatory increase in glucose-induced insulin release, demonstrate decreased islet M3/PKC and adenylate cyclase/PKA activation, possibly predisposing these prediabetic rodents to the early development of β-cell dysfunction.

  1. Effect of captopril and the bradykinin-PKC pathway on ROS production in type 1 diabetic rats.

    PubMed

    Rodrigues de Araujo, Glaucy; Granato de Faria, Karine; Lima, Wanderson Geraldo; Pádua, Bruno da Cruz; Rossoni, Joamyr Victor; Souza, Aline Arlindo; Chianca-Júnior, Deoclecio; Silva, Marcelo Eustáquio; Pedrosa, Maria Lucia; Chaves, Miriam Martins; Costa, Daniela Caldeira

    2011-12-01

    The aim of this study was to investigate the possible effects of captopril as a promoter in modulating the oxidant-antioxidant balance in rats with type 1 diabetes, and the influence of protein kinase C (PKC) pathways in the production of reactive oxygen species (ROS) induced by bradykinin in type 1 diabetic rats. This study evaluated the redox status in both the cardiac tissue and at the cellular level (neutrophils). Two concentrations of captopril were utilized: (i) 5 mg·(kg body mass)(-1), which was considered a therapeutic dose; and (ii) 10 mg·(kg body mass)(-1). Body mass, plasma glucose, and serum insulin were evaluated. To investigate the redox status of the cardiac tissue, we analyzed lipid peroxidation, concentration of carbonylated protein, catalase activity, and the concentration of glutathione. For a more accurate assessment of the possible antioxidant effect of captopril, we also analyzed ROS in neutrophils (in vivo), and ROS production induced by bradykinin and the influence of the PKC pathway in this production (in vitro). Our data show that the hearts of diabetic animals have increased oxidative damage, exemplified by the increased concentration of carbonylated protein and thiobarbituric acid reactive substances (TBARS). However, animals treated with captopril at both concentrations showed lower concentrations of carbonylated protein compared with untreated diabetic animals. We found an increase of catalase activity in the heart of diabetic rats, which was reversed by captopril treatment at both of the dosages tested. Our data showed that captopril was able to reduce ROS production in the neutrophils of diabetic rats at a dose of 10 mg captopril·(kg body mass)(-1). However, the antioxidant effect of captopril is independent of bradykinin. Diabetes induces oxidative stress, and these results suggest that captopril has an antioxidant effect and can modulate the production of ROS in circulating neutrophils.

  2. A novel specificity protein 1 (SP1)-like gene regulating protein kinase C-1 (Pkc1)-dependent cell wall integrity and virulence factors in Cryptococcus neoformans.

    PubMed

    Adler, Amos; Park, Yoon-Dong; Larsen, Peter; Nagarajan, Vijayaraj; Wollenberg, Kurt; Qiu, Jin; Myers, Timothy G; Williamson, Peter R

    2011-06-10

    Eukaryotic cells utilize complex signaling systems to detect their environments, responding and adapting as new conditions arise during evolution. The basidiomycete fungus Cryptococcus neoformans is a leading cause of AIDS-related death worldwide and utilizes the calcineurin and protein kinase C-1 (Pkc1) signaling pathways for host adaptation and expression of virulence. In the present studies, a C-terminal zinc finger transcription factor, homologous both to the calcineurin-responsive zinc fingers (Crz1) of ascomycetes and to the Pkc1-dependent specificity protein-1 (Sp1) transcription factors of metazoans, was identified and named SP1 because of its greater similarity to the metazoan factors. Structurally, the Cryptococcus neoformans Sp1 (Cn Sp1) protein was found to have acquired an additional zinc finger motif from that of Crz1 and showed Pkc1-dependent phosphorylation, nuclear localization, and whole genome epistatic associations under starvation conditions. Transcriptional targets of Cn Sp1 shared functional similarities with Crz1 factors, such as cell wall synthesis, but gained the regulation of processes involved in carbohydrate metabolism, including trehalose metabolism, and lost others, such as the induction of autophagy. In addition, overexpression of Cn Sp1 in a pkc1Δ mutant showed restoration of altered phenotypes involved in virulence, including cell wall stability, nitrosative stress, and extracellular capsule production. Cn Sp1 was also found to be important for virulence of the fungus using a mouse model. In summary, these data suggest an evolutionary shift in C-terminal zinc finger proteins during fungal evolution, transforming them from calcineurin-dependent to PKC1-dependent transcription factors, helping to shape the role of fungal pathogenesis of C. neoformans.

  3. Effects of propofol on the activity of rat glutamate transporter type 3 expressed in Xenopus oocytes: the role of protein kinase C.

    PubMed

    Do, Sang-Hwan; Ham, Byung-Moon; Zuo, Zhiyi

    2003-06-05

    We investigated the effects of propofol on one type of glutamate transporter, excitatory amino acid transporter 3 (EAAT3) and the role of protein kinase C (PKC) in mediating these effects. Rat EAAT3 was expressed in Xenopus oocytes. L-glutamate (30 microM)-induced membrane currents were measured. Propofol increased glutamate-induced inward currents significantly at two tested concentrations (30 and 100 microM) but not at other concentrations. Propofol (30 microM) significantly increased V(max), but not K(m) of EAAT3 for glutamate. The combination of phorbol-12-myrisate-13-acetate (PMA, a PKC activator) and propofol did not increase the responses further compared with PMA or propofol alone. Three PKC inhibitors (staurosporine, calphostin C, and chelerythrine) did not affect basal EAAT3 activity but significantly inhibited the propofol-enhanced EAAT3 activity. Our results suggest that propofol enhances EAAT3 activity at clinically relevant concentrations and PKC may mediate these effects.

  4. Activation of ERK1/2 by protein kinase C-alpha in response to hydrogen peroxide-induced cell death in human gingival fibroblasts.

    PubMed

    Gutiérrez-Venegas, Gloria; Arreguín-Cano, Juan Antonio; Arroyo-Cruz, Rita; Villeda-Navarro, Mónica; Méndez-Mejía, José Antonio

    2010-02-01

    Hydrogen peroxide (H(2)O(2)) increases protein tyrosine phosphorylation of numerous proteins in human gingival fibroblasts (HGFs). Two main proteins, with an apparent molecular weight of 44 and 42kDa, were phosphorylated after hydrogen peroxide stimulation of the human gingival fibroblasts. Further analysis identified these two proteins as ERK1/2. Maximum phosphorylation was detected at 10min post-H(2)O(2) treatment. Pretreatment with an MEK inhibitor, PD98059, inhibited H(2)O(2)-stimulated ERK1/2 phosphorylation in a dose-dependent manner. Treatment with H(2)O(2) also induced phosphorylation of protein kinase C-alpha (PKCalpha). Staurosporine, a PKC inhibitor, blocked ERK1/2 phosphorylation induced by H(2)O(2). In addition, H(2)O(2)-induced cell death was prevented by PD98059, SB203580, and calphostin C, which are MEK, p38 and PKC inhibitors, respectively. These results suggest that H(2)O(2) leads to the phosphorylation and activation of ERK1/2 in a PKC-dependent manner. These findings demonstrate that the MAPK signaling pathway plays an active role in mediating the H(2)O(2)-induced decrease in HGF cell viability and ATP depletion.

  5. The angiotensin receptor type 1-Gq protein-phosphatidyl inositol phospholipase Cbeta-protein kinase C pathway is involved in activation of proximal tubule Na+-ATPase activity by angiotensin(1-7) in pig kidneys.

    PubMed

    Lara, Lucienne S; Correa, Juliana S; Lavelle, Anouchka B; Lopes, Anibal G; Caruso-Neves, Celso

    2008-05-01

    In a previous study, we observed that angiotensin(1-7) (Ang(1-7)) stimulates proximal tubule Na+-ATPase activity through the angiotensin receptor type 1 (AT1R). Here we aimed to study the signalling pathways involved. Our results show that the stimulatory effect of Ang(1-7) on Na+-ATPase activity through AT1R involves a Gq protein-phosphatidyl inositol-phospholipase Cbeta (PI-PLCbeta) pathway because: (1) the effect was reversed by GDPbetaS, a non-hydrolysable GDP analogue, and by a monoclonal Gq protein antibody; (2) the effect was similar and not additive to that of GTPgammaS, a non-hydrolysable GTP analogue; (3) Ang(1-7) induced a rapid decrease (30 s) in phosphatidylinositol 4,5-bisphosphate levels, a PI-PLCbeta substrate; and (4) U73122, a specific inhibitor of PI-PLCbeta, abolished Ang(1-7)-induced stimulation of Na+-ATPase activity. Angiotensin(1-7) increased the protein kinase C (PKC) activity similarly to phorbol-12-myristate-13-acetate (PMA), an activator of PKC. This effect was reversed by losartan, a specific antagonist of AT1R. The stimulatory effects of Ang(1-7) and PMA on Na+-ATPase activity are similar, non-additive and reversed by calphostin C, a specific inhibitor of PKC. A catalytic subunit of PKC (PKC-M) increased the Na+-ATPase activity. These data show that Ang(1-7) stimulates Na+-ATPase activity through the AT1R-Gq protein-PI-PLCbeta-PKC pathway. This effect is similar to that described for angiotensin II, showing for the first time that these compounds could have similar effects in the renal system.

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

    PubMed

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

    2014-09-05

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

  7. Inhibitors of angiotensin-converting enzyme modulate mitosis and gene expression in pancreatic cancer cells

    SciTech Connect

    Reddy, M.K.; Baskaran, K.; Molteni, A.

    1995-12-01

    The angiotensin-converting enzyme (ACE) inhibitor captopril inhibits mitosis in several cell types that contain ACE and renin activity. In the present study, we evaluated the effect of the ACE inhibitors captopril and CGS 13945 (10{sup {minus}8} to 10{sup {minus}2}M) on proliferation and gene expression in hamster pancreatic duct carcinoma cells in culture. These cells lack renin and ACE activity. Both ACE inhibitors produced a dose-dependent reduction in tumor cell proliferation within 24 hr. Captopril at a concentration of 0.36 mM and CGS 13945 at 150 {mu}M decreased cellular growth rate to approximately half that of the control. Neither drug influenced the viability or the cell cycle distribution of the tumor cells. Slot blot analysis of mRNA for four genes, proliferation associated cell nuclear antigen (PCNA), K-ras, protein kinase C-{Beta} (PKC-{Beta}) and carbonic anhydrase II (CA II) was performed. Both ACE inhibitors increased K-ras expression by a factor of 2, and had no effect on CA II mRNA levels. Captopril also lowered PCNA by 40% and CGS 13945 lowered PKC-{Beta} gene expression to 30% of the control level. The data demonstrate that ACE inhibitors exhibit antimitotic activity and differential gene modulation in hamster pancreatic duct carcinoma cells. The absence of renin and ACE activity in these cells suggests that the antimitotic action of captopril and CGS 13945 is independent of renin-angiotensin regulation. The growth inhibition may occur through downregulation of growth-related gene expression. 27 refs., 5 figs.

  8. Susceptibility to natural killer cell-mediated lysis of colon cancer cells is enhanced by treatment with epidermal growth factor receptor inhibitors through UL16-binding protein-1 induction.

    PubMed

    Bae, Jae-Ho; Kim, So-Jung; Kim, Mi-Ju; Oh, Sae-Ock; Chung, Joo-Seop; Kim, Sun-Hee; Kang, Chi-Dug

    2012-01-01

    We have previously shown that inhibition of intracellular signaling pathways by treatment with quercetin induced the expression of natural killer cell group 2D (NKG2D) ligands on cancer cells and made the cells sensitive to natural killer (NK)-cell mediated cytotoxicity. In the present study, we investigated whether epidermal growth factor receptor (EGFR) inhibitors could induce the expression of NKG2D ligands in colon cancer cells. Treatment with EGFR inhibitors predominantly increased the levels of mRNA transcripts and surface protein of UL16-binding protein-1 (ULBP1) in various colon cancer cells, including KM12, Caco-2, HCT-15, and HT-29, which express EGFR, and increased susceptibility of these colon cancer cells to NK-92 cells. The expression of ULBP1 was not induced by inhibitors of nuclear factor-κB, phosphatidylinositol 3 kinase, and MAPK, but was induced by inhibitors of PKC, and the induction of ULBP1 expression with EGFR inhibitors was prevented by treatment with PMA in colon cancer cells. A transcription factor, activator protein-2 alpha (AP-2α), which has a suppressive effect on ULBP1 transcription, was prevented from binding to the ULBP1 promoter by treatment with EGFR inhibitors. The present study suggests that EGFR inhibitors can enhance the susceptibility to NK cell-mediated lysis of colon cancer cells by induction of ULBP1 via inhibition of the PKC pathway.

  9. Myricitrin, a nitric oxide and protein kinase C inhibitor, exerts antipsychotic-like effects in animal models.

    PubMed

    Pereira, M; Siba, I P; Chioca, L R; Correia, D; Vital, M A B F; Pizzolatti, M G; Santos, A R S; Andreatini, R

    2011-08-15

    Myricitrin is a nitric oxide (NO) and protein kinase C (PKC) inhibitor that has central nervous system activity, including anxiolytic-like action. Nitric oxide inhibitors blocked the behavioral effects of apomorphine, suggesting an antipsychotic-like effect. Furthermore, PKC inhibition reduced psychotic symptoms in acute mania patients and blocked amphetamine-induced hyperlocomotion, suggesting a potential antipsychotic-like effect. The present study evaluated the effects of myricitrin in animal models that assess antipsychotic-like effects (apomorphine-induced stereotypy and climbing and the paw test) and extrapyramidal side effects (catalepsy test and paw test). Olanzapine was used as a positive control. 7-Nitroindazole (7-NI), a NOS inhibitor, and l-arginine, a NO precursor, were used to evaluate nitrergic modulation, and tamoxifen was used to test the effect of PKC inhibition. In mice, myricitrin dose-dependently and olanzapine blocked the stereotypy and climbing induced by apomorphine at doses that did not induce catalepsy. 7-Nitroindazole also blocked apomorphine-induced stereotypy and climbing, which were reversed by l-arginine pretreatment. l-arginine only attenuated the effects of myricitrin on apomorphine's effects. Tamoxifen also blocked apomorphine-induced stereotypy and climbing. In the paw test in rats, myricitrin and olanzapine increased hindlimb retraction time at doses that did not affect forelimb reaction time, whereas haloperidol affected both parameters at the same dose. Myricitrin did not induce catalepsy in the bar test. Tamoxifen did not affect hindlimb retraction time or forelimb retraction time, whereas 7-NI significantly increased hindlimb reaction time. Thus, myricitrin exhibited an antipsychotic-like profile at doses that did not induce catalepsy, and this effect may be related to nitrergic action.

  10. PKC iota promotes ovarian tumor progression through deregulation of cyclin E

    PubMed Central

    Nanos-Webb, Angela; Bui, Tuyen; Karakas, Cansu; Zhang, Dong; Carey, Jason P.W.; Mills, Gordon B.; Hunt, Kelly K.; Keyomarsi, Khandan

    2016-01-01

    The high frequency of relapse of epithelial ovarian tumors treated with standard chemotherapy has highlighted the necessity to identify targeted therapies that can improve patient outcomes. The dynamic relationship between Cyclin E and PKCiota frequent overexpression in high-grade ovarian tumors poses a novel pathway for therapeutic investigation. We hypothesized that a PI3K dependent signaling pathway activating PKCiota perpetuates cyclin E deregulation during ovarian tumorigenesis. We observed a positive correlation between PKCiota and cyclin E in a panel of 19 ovarian cancer cell lines. Modulation of cyclin E had no effect on PKCiota knockdown/overexpression however PKCiota differentially regulated cyclin E expression. In the serous ovarian cancer cells (IGROV, OVCAR-3), shPKCiota decreased proliferation, caused a G1 arrest, and significantly prolonged overall survival in xenograft mouse models. In vitro shPKCiota decreased the ability of IGROV cells to grow under anchorage independent conditions and form aberrant acini, which was dependent upon Ad-cyclin E or Ad-LMW-E expression. RPPA analysis of PKCiota wild-type, catalytic active, dominant negative protein isoforms strengthened the association between phospho-PKCiota levels and PI3K pathway activation. Inhibitors of PI3K coordinately decreased phospho-PKCiota and Cyclin E protein levels. In conclusion, we have identified a PI3K/PKCiota/Cyclin E signaling pathway as a therapeutic target during ovarian tumorigenesis. PMID:26279297

  11. Effects of the selective protein kinase C inhibitor, Ro 31-7549, on the proliferation of cultured mouse epidermal keratinocytes.

    PubMed

    Bollag, W B; Ducote, J; Harmon, C S

    1993-03-01

    We have investigated the effects of Ro 31-7549, a selective protein kinase C (PKC) inhibitor, on DNA synthesis and proliferation in two primary mouse epidermal keratinocyte culture systems. In differentiating keratinocytes incubated in medium containing 10% serum and high calcium (approximately 0.5 mM), Ro 31-7549 blocked the inhibitory effect of the phorbol ester 12-0-tetradecanoyl-13-acetate (TPA) (a PKC activator) on keratinocyte DNA synthesis at 24 h [50% maximal response concentration (EC50) = 1 microM], consistent with inhibition of PKC-mediated differentiation. Continuous treatment of the differentiative culture system with the PKC inhibitor resulted in a marked (fourfold) stimulation of [3H]thymidine incorporation at day 7 of exposure, with an EC50 of 0.25 microM. The potencies of these effects of Ro 31-7549 are comparable to that reported for inhibition of TPA-induced platelet 47-kD protein phosphorylation [50% inhibitory concentration (IC50) = 4.4 microM]. The time course of [3H]thymidine incorporation indicated that Ro 31-7549 did not directly stimulate DNA synthesis but instead prevented the loss of proliferative capacity associated with continued culture in this medium. Maximal stimulation (2.6 times) of DNA synthesis was observed on day 4, whereas DNA synthesis at day 1 was unaffected. In a highly proliferative culture system using serum-free medium containing 25 microM calcium, TPA dose-dependently inhibited proliferation with an IC50 of approximately 0.3 mM. This antiproliferative effect of TPA was largely reversed by 0.1 microM Ro 31-7549. In the proliferative culture system, 0.75 microM Ro 31-7549 also essentially reversed the inhibition of proliferation caused by switching to high (1.0 mM) calcium. These results suggest that the loss of proliferative capacity in differentiating epidermal keratinocyte cultures may be mediated, at least in part, by PKC.

  12. ATP7B activity is stimulated by PKCɛ in porcine liver.

    PubMed

    Cardoso, Luiza H D; Britto-Borges, Thiago; Vieyra, Adalberto; Lowe, Jennifer

    2014-09-01

    Copper is necessary for all organisms since it acts as a cofactor in different enzymes, although toxic at high concentrations. ATP7B is one of two copper-transporting ATPases in humans, its vital role being manifested in Wilson disease due to a mutation in the gene that encodes this pump. Our objective has been to determine whether pathways involving protein kinase C (PKC) modulate ATP7B activity. Different isoforms of PKC (α, ɛ, ζ) were found in Golgi-enriched membrane fractions obtained from porcine liver. Cu(I)-ATPase activity was assessed in the presence of different activators and inhibitors of PKC signaling pathways. PMA (10(-8) M), a PKC activator, increased Cu(I)-ATPase activity by 60%, whereas calphostin C and U73122 (PKC and PLC inhibitors, respectively) decreased the activity by 40%. Addition of phosphatase λ decreased activity by 60%, irrespective of pre-incubation with PMA. No changes were detected with 2 μM Ca(2+), whereas PMA plus EGTA increased activity. This enhanced activity elicited by PMA decreased with a specific inhibitor of PKCɛ to levels comparable with those found after phosphatase λ treatment, showing that the ɛ isoform is essential for activation of the enzyme. This regulatory phosphorylation enhanced Vmax without modifying affinities for ATP and copper. It can be concluded that signaling pathways leading to DAG formation and PKCɛ activation stimulate the active transport of copper by ATP7B, thus evidencing a central role for this specific kinase-mediated mechanism in hepatic copper handling.

  13. Two Mechanisms Regulate Keratin K15 Expression In Keratinocytes: Role of PKC/AP-1 and FOXM1 Mediated Signalling

    PubMed Central

    Bose, Amrita; Teh, Muy-Teck; Hutchison, Iain L.; Wan, Hong; Leigh, Irene M.; Waseem, Ahmad

    2012-01-01

    Background Keratin 15 (K15) is a type I keratin that is used as a marker of stem cells. Its expression is restricted to the basal layer of stratified epithelia, and the bulge in hair follicles. However, in certain clinical situations including oral lichen planus, K15 is induced in suprabasal layers, which is inconsistent with the role of a stem cell marker. This study provides insights into the mechanisms of K15 expression in the basal and differentiating keratinocytes. Methodology/Principal Findings Human keratinocytes were differentiated by three different methods; suspension in methylcellulose, high cell density and treatment with phorbol ester. The expression of mRNA was determined by quantitative PCR and protein by western blotting and immunostaining. Keratinocytes in suspension suppressed β1-integrin expression, induced differentiation-specific markers and K15, whereas FOXM1 (a cell cycle regulated protein) and K14 were downregulated. Rescuing β1-integrin by either fibronectin or the arginine-glycine-aspartate peptide suppressed K15 but induced K14 and FOXM1 expression. Specific inhibition of PKCδ, by siRNA, and AP-1 transcription factor, by TAM67 (dominant negative c-Jun), suppressed K15 expression, suggesting that PKC/AP-1 pathway plays a role in the differentiation-specific expression of K15. The basal cell-specific K15 expression may involve FOXM1 because ectopic expression of the latter is known to induce K15. Using chromatin immunoprecipitation, we have identified a single FOXM1 binding motif in the K15 promoter. Conclusions/Significance The data suggests that K15 is induced during terminal differentiation mediated by the down regulation of β1-integrin. However, this cannot be the mechanism of basal/stem cell-specific K15 expression in stratified epithelia, because basal keratinocytes do not undergo terminal differentiation. We propose that there are two mechanisms regulating K15 expression in stratified epithelia; differentiation-specific involving

  14. 12-Deoxyphorbols Promote Adult Neurogenesis by Inducing Neural Progenitor Cell Proliferation via PKC Activation

    PubMed Central

    Geribaldi-Doldán, Noelia; Flores-Giubi, Eugenia; Murillo-Carretero, Maribel; García-Bernal, Francisco; Carrasco, Manuel; Macías-Sánchez, Antonio J.; Domínguez-Riscart, Jesús; Verástegui, Cristina; Hernández-Galán, Rosario

    2016-01-01

    Background: Neuropsychiatric and neurological disorders frequently occur after brain insults associated with neuronal loss. Strategies aimed to facilitate neuronal renewal by promoting neurogenesis constitute a promising therapeutic option to treat neuronal death-associated disorders. In the adult brain, generation of new neurons occurs physiologically throughout the entire life controlled by extracellular molecules coupled to intracellular signaling cascades. Proteins participating in these cascades within neurogenic regions constitute potential pharmacological targets to promote neuronal regeneration of injured areas of the central nervous system. Methodology: We have performed in vitro and in vivo approaches to determine neural progenitor cell proliferation to understand whether activation of kinases of the protein kinase C family facilitates neurogenesis in the adult brain. Results: We have demonstrated that protein kinase C activation by phorbol-12-myristate-13-acetate induces neural progenitor cell proliferation in vitro. We also show that the nontumorogenic protein kinase C activator prostratin exerts a proliferative effect on neural progenitor cells in vitro. This effect can be reverted by addition of the protein kinase C inhibitor G06850, demonstrating that the effect of prostratin is mediated by protein kinase C activation. Additionally, we show that prostratin treatment in vivo induces proliferation of neural progenitor cells within the dentate gyrus of the hippocampus and the subventricular zone. Finally, we describe a library of diterpenes with a 12-deoxyphorbol structure similar to that of prostratin that induces a stronger effect than prostratin on neural progenitor cell proliferation both in vitro and in vivo. Conclusions: This work suggests that protein kinase C activation is a promising strategy to expand the endogenous neural progenitor cell population to promote neurogenesis and highlights the potential of 12-deoxyphorbols as pharmaceutical

  15. Pea DNA Topoisomerase I Is Phosphorylated and Stimulated by Casein Kinase 2 and Protein Kinase C

    PubMed Central

    Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

    2003-01-01

    DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg2+-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants. PMID:12913165

  16. Exposure to 50 Hz magnetic field modulates GABAA currents in cerebellar granule neurons through an EP receptor-mediated PKC pathway

    PubMed Central

    Yang, Guang; Ren, Zhen; Mei, Yan-Ai

    2015-01-01

    Previous work from both our lab and others have indicated that exposure to 50 Hz magnetic fields (ELF-MF) was able to modify ion channel functions. However, very few studies have investigated the effects of MF on γ-aminobutyric acid (GABA) type A receptors (GABAARs) channel functioning, which are fundamental to overall neuronal excitability. Here, our major goal is to reveal the potential effects of ELF-MF on GABAARs activity in rat cerebellar granule neurons (CGNs). Our results indicated that exposing CGNs to 1 mT ELF-MF for 60 min. significantly increased GABAAR currents without modifying sensitivity to GABA. However, activation of PKA by db-cAMP failed to do so, but led to a slight decrease instead. On the other hand, PKC activation or inhibition by PMA or Bis and Docosahexaenoic acid (DHA) mimicked or eliminated the field-induced-increase of GABAAR currents. Western blot analysis indicated that the intracellular levels of phosphorylated PKC (pPKC) were significantly elevated after 60 min. of ELF-MF exposure, which was subsequently blocked by application of DHA or EP1 receptor-specific (prostaglandin E receptor 1) antagonist (SC19220), but not by EP2-EP4 receptor-specific antagonists. SC19220 also significantly inhibited the ELF-MF-induced elevation on GABAAR currents. Together, these data obviously demonstrated for the first time that neuronal GABAA currents are significantly increased by ELF-MF exposure, and also suggest that these effects are mediated via an EP1 receptor-mediated PKC pathway. Future work will focus on a more comprehensive analysis of the physiological and/or pathological consequences of these effects. PMID:26176998

  17. Heat Shock Protein 27-Targeted Heptapeptide of the PKC{Delta} Catalytic V5 Region Sensitizes Tumors With Radio- and Chemoresistance

    SciTech Connect

    Lee, Hae-June; Kim, Eun-Ho; Seo, Woo Duck; Choi, Tae Hyun; Cheon, Gi-Jeong; Lee, Yoon-Jin; Lee, Yun-Sil

    2011-05-01

    Purpose: Previous data suggest that the PKC{delta} catalytic V5 (PKC{delta}-V5) heptapeptide (HEPT) (FEQFLDI) binds HSP27 and blocks HSP27-mediated radio- or chemoresistance. Here we investigated further the in vivo function of the PKC{delta}-V5 HEPT. Methods and Materials: Labeling of HEPT with Cy5.5 or fluorescein isothiocyanate was performed to evaluate in vitro or in vivo distribution of HEPT. A clonogenic survival assay, flow cytometry, and Western blotting of cleaved caspase-3 were performed to determine in vitro sensitization effects of HEPT plus ionizing radiation (IR) versus IR alone or those of HEPT plus cisplatin(Cis) versus Cis alone. A nude mouse xenografting system was also applied to detect in vivo sensitizing effects of HEPT. Results: HEPT efficiently bound to HSP27 and showed sensitization after combined treatment with IR versus treatment with Cis alone in NCI-H1299 lung carcinoma cells, with higher HSP27 expression, which was similar to that of combined treatment with IR or with Cis alone in NCI-H460 lung carcinoma cells with lower HSP27 expression. In vivo image analysis using Cy5.5-labeled HEPT showed that HEPT was retained in HSP27-overexpressing cancer cells after xenografting to nude mice. Combined treatment of HEPT with IR versus that with Cis alone in xenografted mice showed that HEPT increased radio- or chemosensitization in NCI-H1299 cells compared to that in mice xenografted with NCI-H460 cells. Conclusions: The novel PKC{delta}-V5 HEPT may help overcome HSP27-mediated radio- or chemoresistance.

  18. Ectodomain cleavage of the EGF ligands HB-EGF, neuregulin1-beta, and TGF-alpha is specifically triggered by different stimuli and involves different PKC isoenzymes.

    PubMed

    Herrlich, Andreas; Klinman, Eva; Fu, Jonathan; Sadegh, Cameron; Lodish, Harvey

    2008-12-01

    Metalloproteinase cleavage of transmembrane proteins (ectodomain cleavage), including the epidermal growth factor (EGF) ligands heparin-binding EGF-like growth factor (HB-EGF), neuregulin (NRG), and transforming growth factor-alpha (TGF-alpha), is important in many cellular signaling pathways and is disregulated in many diseases. It is largely unknown how physiological stimuli of ectodomain cleavage--hypertonic stress, phorbol ester, or activation of G-protein-coupled receptors [e.g., by lysophosphatidic acid (LPA)]--are molecularly connected to metalloproteinase activation. To study this question, we developed a fluorescence-activated cell sorting (FACS)- based assay that measures cleavage of EGF ligands in single living cells. EGF ligands expressed in mouse lung epithelial cells are differentially and specifically cleaved depending on the stimulus. Inhibition of protein kinase C (PKC) isoenzymes or metalloproteinase inhibition by batimastat (BB94) showed that different regulatory signals are used by different stimuli and EGF substrates, suggesting differential effects that act on the substrate, the metalloproteinase, or both. For example, hypertonic stress led to strong cleavage of HB-EGF and NRG but only moderate cleavage of TGF-alpha. HB-EGF, NRG, and TGF-alpha cleavage was not dependent on PKC, and only HB-EGF and NRG cleavage were inhibited by BB94. In contrast, phorbol 12-myristate-13-acetate (TPA) -induced cleavage of HB-EGF, NRG, and TGF-alpha was dependent on PKC and sensitive to BB94 inhibition. LPA led to significant cleavage of only NRG and TGF-alpha and was inhibited by BB94; only LPA-induced NRG cleavage required PKC. Surprisingly, specific inhibition of atypical PKCs zeta and iota [not activated by diacylglycerol (DAG) and calcium] significantly enhanced TPA-induced NRG cleavage. Employed in a high-throughput cloning strategy, our cleavage assay should allow the identification of candidate proteins involved in signal transduction of different

  19. The Aspergillus fumigatus pkcA G579R Mutant Is Defective in the Activation of the Cell Wall Integrity Pathway but Is Dispensable for Virulence in a Neutropenic Mouse Infection Model.

    PubMed

    Rocha, Marina Campos; Godoy, Krissia Franco de; de Castro, Patrícia Alves; Hori, Juliana Issa; Bom, Vinícius Leite Pedro; Brown, Neil Andrew; Cunha, Anderson Ferreira da; Goldman, Gustavo Henrique; Malavazi, Iran

    2015-01-01

    Aspergillus fumigatus is an opportunistic human pathogen, which causes the life-threatening disease, invasive pulmonary aspergillosis. In fungi, cell wall homeostasis is controlled by the conserved Cell Wall Integrity (CWI) pathway. In A. fumigatus this signaling cascade is partially characterized, but the mechanisms by which it is activated are not fully elucidated. In this study we investigated the role of protein kinase C (PkcA) in this signaling cascade. Our results suggest that pkcA is an essential gene and is activated in response to cell wall stress. Subsequently, we constructed and analyzed a non-essential A. fumigatus pkcAG579R mutant, carrying a Gly579Arg substitution in the PkcA C1B regulatory domain. The pkcAG579R mutation has a reduced activation of the downstream Mitogen-Activated Protein Kinase, MpkA, resulting in the altered expression of genes encoding cell wall-related proteins, markers of endoplasmic reticulum stress and the unfolded protein response. Furthermore, PkcAG579R is involved in the formation of proper conidial architecture and protection to oxidative damage. The pkcAG579R mutant elicits increased production of TNF-α and phagocytosis but it has no impact on virulence in a murine model of invasive pulmonary aspergillosis. These results highlight the importance of PkcA to the CWI pathway but also indicated that additional regulatory circuits may be involved in the biosynthesis and/or reinforcement of the A. fumigatus cell wall during infection.

  20. The Aspergillus fumigatus pkcAG579R Mutant Is Defective in the Activation of the Cell Wall Integrity Pathway but Is Dispensable for Virulence in a Neutropenic Mouse Infection Model

    PubMed Central

    Rocha, Marina Campos; de Godoy, Krissia Franco; de Castro, Patrícia Alves; Hori, Juliana Issa; Bom, Vinícius Leite Pedro; Brown, Neil Andrew; da Cunha, Anderson Ferreira; Goldman, Gustavo Henrique; Malavazi, Iran

    2015-01-01

    Aspergillus fumigatus is an opportunistic human pathogen, which causes the life-threatening disease, invasive pulmonary aspergillosis. In fungi, cell wall homeostasis is controlled by the conserved Cell Wall Integrity (CWI) pathway. In A. fumigatus this signaling cascade is partially characterized, but the mechanisms by which it is activated are not fully elucidated. In this study we investigated the role of protein kinase C (PkcA) in this signaling cascade. Our results suggest that pkcA is an essential gene and is activated in response to cell wall stress. Subsequently, we constructed and analyzed a non-essential A. fumigatus pkcAG579R mutant, carrying a Gly579Arg substitution in the PkcA C1B regulatory domain. The pkcAG579R mutation has a reduced activation of the downstream Mitogen-Activated Protein Kinase, MpkA, resulting in the altered expression of genes encoding cell wall-related proteins, markers of endoplasmic reticulum stress and the unfolded protein response. Furthermore, PkcAG579R is involved in the formation of proper conidial architecture and protection to oxidative damage. The pkcAG579R mutant elicits increased production of TNF-α and phagocytosis but it has no impact on virulence in a murine model of invasive pulmonary aspergillosis. These results highlight the importance of PkcA to the CWI pathway but also indicated that additional regulatory circuits may be involved in the biosynthesis and/or reinforcement of the A. fumigatus cell wall during infection. PMID:26295576

  1. LDB3 splicing abnormalities are specific to skeletal muscles of patients with myotonic dystrophy type 1 and alter its PKC binding affinity.

    PubMed

    Yamashita, Yoshihiro; Matsuura, Tohru; Kurosaki, Tatsuaki; Amakusa, Yoshinobu; Kinoshita, Masanobu; Ibi, Tohru; Sahashi, Ko; Ohno, Kinji

    2014-09-01

    Myotonic dystrophy type 1 (DM1) is caused by transcription of CUG repeat RNA, which causes sequestration of muscleblind-like 1 (MBNL1) and upregulation of CUG triplet repeat RNA-binding protein (CUG-BP1). In DM1, dysregulation of these proteins contributes to many aberrant splicing events, causing various symptoms of the disorder. Here, we demonstrate the occurrence of aberrant splicing of LIM domain binding 3 (LDB3) exon 11 in DM1 skeletal muscle. Exon array surveys, RT-PCR, and western blotting studies demonstrated that exon 11 inclusion was DM1 specific and could be reproduced by transfection of a minigene containing the CTG repeat expansion. Moreover, we found that the LDB3 exon 11-positive isoform had reduced affinity for PKC compared to the exon 11-negative isoform. Since PKC exhibits hyperactivation in DM1 and stabilizes CUG-BP1 by phosphorylation, aberrant splicing of LDB3 may contribute to CUG-BP1 upregulation through changes in its affinity for PKC.

  2. Downregulated AP-1 activity is associated with inhibition of Protein-Kinase-C-dependent CD44 and ezrin localisation and upregulation of PKC theta in A431 cells.

    PubMed

    Stapleton, Genevieve; Malliri, Angeliki; Ozanne, Bradford W

    2002-07-01

    Progression to an invasive, metastatic tumour requires the coordinated expression and function of a number of gene products, as well as their regulation in the context of invasion. The transcription factor AP-1 regulates expression of many of those genes necessary for implementation of the invasion programme. Two such gene products, CD44 and ezrin, are both upregulated in fibroblasts transformed by v-fos and are commonly implicated in cell motility and invasion. Here we report that CD44 and ezrin colocalise to membrane ruffles and microvilli of A431 cells after treatment with EGF. However, A431 cells expressing dominant-negative c-Jun (TAM67), and which as a consequence fail to invade in response to EGF, also fail to correctly localise CD44 and ezrin. CD44 and ezrin are both substrates for Protein Kinase C, and we show that their EGF-dependent colocalisation requires Protein Kinase C activity. Associated with TAM67 expression and disrupted CD44 and ezrin colocalisation is the increased expression and activation of the novel PKC theta isoform. Expression of PKC theta in A431 cells results in the inhibition of cell motility and disrupted localisation of CD44 and ezrin. We propose that AP-1 regulates the integrity of Protein Kinase C signalling and identifies PKC theta as a potential suppressor of the invasion programme.

  3. STT10, a novel class-D VPS yeast gene required for osmotic integrity related to the PKC1/STT1 protein kinase pathway.

    PubMed

    Yoshida, S; Ohya, Y; Hirose, R; Nakano, A; Anraku, Y

    1995-07-04

    We report the genetic and biochemical properties of a staurosporine (ST)- and temperature-sensitive mutant, stt10, of Saccharomyces cerevisiae. The stt10 mutant shows an osmoremedial phenotype in a medium with 1 M sorbitol. ST sensitivity of the stt10 mutant was suppressed by overexpression of PKC1/STT1, showing the genetic interactions of STT10 with the PKC1/STT1 pathway. The nucleotide sequence of STT10 predicts a hydrophilic protein composed of 577 amino acids that possesses 20-25% sequence similarity with yeast Slp1/Vam5p, Sec1p and Sly1p, and nematode Unc-18. The stt10 deletion mutant is viable and shows a typical class-D vacuolar protein sorting defective (vps) phenotype. Vacuoles from stt10 cells have a normal vacuolar H(+)-ATPase activity, but are defective in vacuolar acidification. Genetic studies of yeast mutants carrying delta stt10, delta bck1, stt1/pkc1 or stt4 have revealed that their functions are phenotypically related to maintenance of cellular osmotic integrity.

  4. Down-Regulation of Desmosomes in Cultured Cells: The Roles of PKC, Microtubules and Lysosomal/Proteasomal Degradation

    PubMed Central

    McHarg, Selina; Hopkins, Gemma; Lim, Lusiana; Garrod, David

    2014-01-01

    Desmosomes are intercellular adhesive junctions of major importance for tissue integrity. To allow cell motility and migration they are down-regulated in epidermal wound healing. Electron microscopy indicates that whole desmosomes are internalised by cells in tissues, but the mechanism of down-regulation is unclear. In this paper we provide an overview of the internalisation of half-desmosomes by cultured cells induced by calcium chelation. Our results show that: (i) half desmosome internalisation is dependent on conventional PKC isoforms; (ii) microtubules transport internalised half desmosomes to the region of the centrosome by a kinesin-dependent mechanism; (iii) desmosomal proteins remain colocalised after internalisation and are not recycled to the cell surface; (iv) internalised desmosomes are degraded by the combined action of lysosomes and proteasomes. We also confirm that half desmosome internalisation is dependent upon the actin cytoskeleton. These results suggest that half desmosomes are not disassembled and recycled during or after internalisation but instead are transported to the centrosomal region where they are degraded. These findings may have significance for the down-regulation of desmosomes in wounds. PMID:25291180

  5. Protein Kinase C-θ (PKC-θ) in Natural Killer Cell Function and Anti-Tumor Immunity

    PubMed Central

    Anel, Alberto; Aguiló, Juan I.; Catalán, Elena; Garaude, Johan; Rathore, Moeez G.; Pardo, Julián; Villalba, Martín

    2012-01-01

    The protein kinase C-θ (PKCθ), which is essential for T cell function and survival, is also required for efficient anti-tumor immune surveillance. Natural killer (NK) cells, which express PKCθ, play a prominent role in this process, mainly by elimination of tumor cells with reduced or absent major histocompatibility complex class-I (MHC-I) expression. This justifies the increased interest of the use of activated NK cells in anti-tumor immunotherapy in the clinic. The in vivo development of MHC-I-deficient tumors is much favored in PKCθ−/− mice compared with wild-type mice. Recent data offer some clues on the mechanism that could explain the important role of PKCθ in NK cell-mediated anti-tumor immune surveillance: some studies show that PKCθ is implicated in signal transduction and anti-tumoral activity of NK cells elicited by interleukin (IL)-12 or IL-15, while others show that it is implicated in NK cell functional activation mediated by certain killer-activating receptors. Alternatively, the possibility that PKCθ is involved in NK cell degranulation is discussed, since recent data indicate that it is implicated in microtubule-organizing center polarization to the immune synapse in CD4+ T cells. The implication of PKC isoforms in degranulation has been more extensively studied in cytotoxic T lymphocyte, and these studies will be also summarized. PMID:22783260

  6. Cerium oxide nanoparticles promote neurogenesis and abrogate hypoxia-induced memory impairment through AMPK-PKC-CBP signaling cascade.

    PubMed

    Arya, Aditya; Gangwar, Anamika; Singh, Sushil Kumar; Roy, Manas; Das, Mainak; Sethy, Niroj Kumar; Bhargava, Kalpana

    2016-01-01

    Structural and functional integrity of the brain is adversely affected by reduced oxygen saturation, especially during chronic hypoxia exposure and often encountered by altitude travelers or dwellers. Hypoxia-induced generation of reactive nitrogen and oxygen species reportedly affects the cortex and hippocampus regions of the brain, promoting memory impairment and cognitive dysfunction. Cerium oxide nanoparticles (CNPs), also known as nanoceria, switch between +3 and +4 oxidation states and reportedly scavenge superoxide anions, hydrogen peroxide, and peroxynitrite in vivo. In the present study, we evaluated the neuroprotective as well as the cognition-enhancing activities of nanoceria during hypobaric hypoxia. Using polyethylene glycol-coated 3 nm nanoceria (PEG-CNPs), we have demonstrated efficient localization of PEG-CNPs in rodent brain. This resulted in significant reduction of oxidative stress and associated damage during hypoxia exposure. Morris water maze-based memory function tests revealed that PEG-CNPs ameliorated hypoxia-induced memory impairment. Using microscopic, flow cytometric, and histological studies, we also provide evidences that PEG-CNPs augmented hippocampus neuronal survival and promoted neurogenesis. Molecular studies revealed that PEG-CNPs promoted neurogenesis through the 5'-adenine monophosphate-activated protein kinase-protein kinase C-cyclic adenosine monophosphate response element-binding protein binding (AMPK-PKC-CBP) protein pathway. Our present study results suggest that nanoceria can be translated as promising therapeutic molecules for neurodegenerative diseases.

  7. Rb-dependent cellular senescence, multinucleation and susceptibility to oncogenic transformation through PKC scaffolding by SSeCKS/AKAP12

    PubMed Central

    Akakura, Shin; Nochajski, Peter; Gao, Lingqiu; Sotomayor, Paul; Matsui, Sei-ichi

    2010-01-01

    A subset of AKAPs (A Kinase Anchoring Proteins) regulate signaling and cytoskeletal pathways through the spaciotemporal scaffolding of multiple protein kinases (PK), such as PKC and PKA, and associations with the plasma membrane and the actin-based cytoskeleton. SSeCKS/Gravin/Akap12 expression is severely downregulated in many advanced cancers and exhibits tumor- and metastasis-suppressing activity. akap12-null (KO) mice develop prostatic hyperplasia with focal dysplasia, but the precise mechanism how Akap12 prevents oncogenic progression remains unclear. Here, we show that KO mouse embryonic fibroblasts (MEF) exhibit premature senescence marked by polyploidy and multinucleation, and by increased susceptibility to oncogenic transformation. Although p53 and Rb pathways are activated in the absence of Akap12, senescence is dependent on Rb. Senescence is driven by the activation of PKCα, which induces p16Ink4a/Rb through a MEK-dependent downregulation of Id1, and PKCδ, which downregulates Lats1/Warts, a mitotic exit network kinase required for cytokinesis. Our data strongly suggest that Akap12 controls Rb-mediated cell aging and oncogenic progression by directly scaffolding and attenuating PKCα/δ. PMID:21099353

  8. Beta-adrenergic receptor agonist decreases VEGF levels through altered eNOS and PKC signaling in diabetic retina

    PubMed Central

    Jiang, Youde; Zhang, Qiuhua; Steinle, Jena J.

    2015-01-01

    Vascular endothelial cell growth factor (VEGF) is increased in diabetic macular edema. Compound 49b, a novel β-adrenergic receptor agonist, is protective in a type 1 diabetic rat model. We questioned whether Compound 49b could decrease VEGF levels, suggesting that Compound 49b may be effective against edema. Two-month diabetic rats received topical Compound 49b for 7 days only and/or insulin-like growth factor binding protein 3 (IGFBP-3) siRNA. We also measured endothelial nitric oxide synthase (eNOS) and protein kinase C (PKC)ζ and PKCδ phosphorylation. Retinal endothelial cells (RECs) cultured in high glucose were treated with Compound 49b and IGFBP-3 siRNA for evaluation of the same signaling pathways. Compound 49b significantly decreased VEGF through increased IGFBP-3 in the diabetic retina. Compound 49b also reduced eNOS, PKCζ and PKCδ phosphorylation in the diabetic retina and REC. Compound 49b regulated a number of proteins involved in REC barrier properties. PMID:26115368

  9. Proton pump inhibitors

    MedlinePlus

    Proton pump inhibitors (PPIs) are medicines that work by reducing the amount of stomach acid made by ... Proton pump inhibitors are used to: Relieve symptoms of acid reflux, or gastroesophageal reflux disease (GERD). This ...

  10. Palmitate induces tumor necrosis factor-alpha expression in C2C12 skeletal muscle cells by a mechanism involving protein kinase C and nuclear factor-kappaB activation.

    PubMed

    Jové, Mireia; Planavila, Anna; Sánchez, Rosa M; Merlos, Manuel; Laguna, Juan Carlos; Vázquez-Carrera, Manuel

    2006-01-01

    The mechanisms responsible for increased expression of TNF-alpha in skeletal muscle cells in diabetic states are not well understood. We examined the effects of the saturated acid palmitate on TNF-alpha expression. Exposure of C2C12 skeletal muscle cells to 0.75 mm palmitate enhanced mRNA (25-fold induction, P < 0.001) and protein (2.5-fold induction) expression of the proinflammatory cytokine TNF-alpha. This induction was inversely correlated with a fall in GLUT4 mRNA levels (57% reduction, P < 0.001) and glucose uptake (34% reduction, P < 0.001). PD98059 and U0126, inhibitors of the ERK-MAPK cascade, partially prevented the palmitate-induced TNF-alpha expression. Palmitate increased nuclear factor (NF)-kappaB activation and incubation of the cells with the NF-kappaB inhibitors pyrrolidine dithiocarbamate and parthenolide partially prevented TNF-alpha expression. Incubation of palmitate-treated cells with calphostin C, a strong and specific inhibitor of protein kinase C (PKC), abolished palmitate-induced TNF-alpha expression, and restored GLUT4 mRNA levels. Palmitate treatment enhanced the expression of phospho-PKCtheta, suggesting that this PKC isoform was involved in the changes reported, and coincubation of palmitate-treated cells with the PKC inhibitor chelerythrine prevented the palmitate-induced reduction in the expression of IkappaBalpha and insulin-stimulated Akt activation. These findings suggest that enhanced TNF-alpha expression and GLUT4 down-regulation caused by palmitate are mediated through the PKC activation, confirming that this enzyme may be a target for either the prevention or the treatment of fatty acid-induced insulin resistance.

  11. Kinase inhibitors as potential therapeutics for acute and chronic neurodegenerative conditions.

    PubMed

    Cuny, G D

    2009-01-01

    Kinases, which number > 500 in humans, are a class of enzymes that participate in an array of important functions within normal cellular physiology and during various pathological conditions. Due to the key role of kinases in the regulation of all aspects of cellular signaling and the well established contribution of kinase dysregulation to the etiology of many human pathologies, the development of kinase inhibitors has emerged as a therapeutic strategy for the treatment of human disease, including most notably oncology. Difficulties generating selective inhibitors have hampered their use in other therapeutic areas with less tolerance for off-target effects. However, with an increasing understanding of kinase structures and with the advent of newer inhibitor design strategies more highly selective inhibitors are beginning to emerge. This has prompted interest in utilizing kinase inhibitors in therapeutic areas beyond oncology, including acute and chronic neurodegenerative conditions for which disease modify therapies are lacking. This review provides a background in acute (i.e. brain ischemia and traumatic brain injury) and chronic (i.e. Alzheimer's, Parkinson's, Huntington's disease, amyotrophic lateral sclerosis and multiple sclerosis) neurodegenerative conditions. Then, the role of several kinase (i.e. JNK3, p38 MAPK, ERK, PKC, ROCKII, GSK3, Cdk5, MLK, EphB3 kinase, RIP1 kinase, LRRK2, TTBK1, ASK1, CK, DAPK, and PKN1) that could serve as potential therapeutic targets for these maladies are reviewed.

  12. Rho1 GTPase and PKC ortholog Pck1 are upstream activators of the cell integrity MAPK pathway in fission yeast.

    PubMed

    Sánchez-Mir, Laura; Soto, Teresa; Franco, Alejandro; Madrid, Marisa; Viana, Raúl A; Vicente, Jero; Gacto, Mariano; Pérez, Pilar; Cansado, José

    2014-01-01

    In the fission yeast Schizosaccharomyces pombe the cell integrity pathway (CIP) orchestrates multiple biological processes like cell wall maintenance and ionic homeostasis by fine tuning activation of MAPK Pmk1 in response to various environmental conditions. The small GTPase Rho2 positively regulates the CIP through protein kinase C ortholog Pck2. However, Pmk1 retains some function in mutants lacking either Rho2 or Pck2, suggesting the existence of additional upstream regulatory elements to modulate its activity depending on the nature of the environmental stimulus. The essential GTPase Rho1 is a candidate to control the activity of the CIP by acting upstream of Pck2, whereas Pck1, a second PKC ortholog, appears to negatively regulate Pmk1 activity. However, the exact regulatory nature of these two proteins within the CIP has remained elusive. By exhaustive characterization of strains expressing a hypomorphic Rho1 allele (rho1-596) in different genetic backgrounds we show that both Rho1 and Pck1 are positive upstream regulatory members of the CIP in addition to Rho2 and Pck2. In this new model Rho1 and Rho2 control Pmk1 basal activity during vegetative growth mainly through Pck2. Notably, whereas Rho2-Pck2 elicit Pmk1 activation in response to most environmental stimuli, Rho1 drives Pmk1 activation through either Pck2 or Pck1 exclusively in response to cell wall damage. Our study reveals the intricate and complex functional architecture of the upstream elements participating in this signaling pathway as compared to similar routes from other simple eukaryotic organisms.

  13. PKC/ROS-Mediated NLRP3 Inflammasome Activation Is Attenuated by Leishmania Zinc-Metalloprotease during Infection

    PubMed Central

    Jung, Jee Yong; Chang, Kwang-Poo; Olivier, Martin

    2015-01-01

    Parasites of the Leishmania genus infect and survive within macrophages by inhibiting several microbicidal molecules, such as nitric oxide and pro-inflammatory cytokines. In this context, various species of Leishmania have been reported to inhibit or reduce the production of IL-1β both in vitro and in vivo. However, the mechanism whereby Leishmania parasites are able to affect IL-1β production and secretion by macrophages is still not fully understood. Dependent on the stimulus at hand, the maturation of IL-1β is facilitated by different inflammasome complexes. The NLRP3 inflammasome has been shown to be of pivotal importance in the detection of danger molecules such as inorganic crystals like asbestos, silica and malarial hemozoin, (HZ) as well as infectious agents. In the present work, we investigated whether Leishmania parasites modulate NLRP3 inflammasome activation. Using PMA-differentiated THP-1 cells, we demonstrate that Leishmania infection effectively inhibits macrophage IL-1β production upon stimulation. In this context, the expression and activity of the metalloprotease GP63 - a critical virulence factor expressed by all infectious Leishmania species - is a prerequisite for a Leishmania-mediated reduction of IL-1β secretion. Accordingly, L. mexicana, purified GP63 and GP63-containing exosomes, caused the inhibition of macrophage IL-1β production. Leishmania-dependent suppression of IL-1β secretion is accompanied by an inhibition of reactive oxygen species (ROS) production that has previously been shown to be associated with NLRP3 inflammasome activation. The observed loss of ROS production was due to an impaired PKC-mediated protein phosphorylation. Furthermore, ROS-independent inflammasome activation was inhibited, possibly due to an observed GP63-dependent cleavage of inflammasome and inflammasome-related proteins. Collectively for the first time, we herein provide evidence that the protozoan parasite Leishmania, through its surface

  14. Effects of activation of protein kinase C (PKC) on the hormonal stimulation and inhibition of cAMP formation in intact human platelets

    SciTech Connect

    Williams, K.A.; Haslam, R.J.

    1986-05-01

    Washed platelets, labelled by preincubation with (/sup 3/H)adenine and (/sup 32/P)P/sub i/, were studied in the presence of indomethacin, phosphocreatine and creatine phosphokinase to block thromboxane A/sub 2/ formation and inhibitory effects of released ADP. Addition of phorbol 12-myristate 13-acetate (PMA) or 1,2-dioctanoyl-glycerol (diC/sub 8/) decreased the initial rate of accumulation of (/sup 3/H)cAMP observed with PGE/sub 1/ and 3-isobutyl 1- methylxanthine. Maximal decreases of 31% (1 ..mu..M PMA) and 42% (100 ..mu..M diC/sub 8/) were obtained. Also, the inhibition of (/sup 3/H)cAMP formation by epinephrine (5 ..mu..M) was decreased from 68% to 16% and 31% by 1..mu..M PMA and 100 ..mu..M diC/sub 8/, respectively. The effects of increasing concentrations of PMA and diC/sub 8/ on the stimulation of (/sup 3/H)cAMp formation by PGE/sub 1/ and on the inhibitory action of epinephrine correlated with increases in /sup 32/P incorporation into the major substrate of PKC (P47) and into two other polypeptides (P41 and P20). These results suggested that activation of PKC might explain the failure of some aggregating agents (e.g. PAF and vasopressin) to inhibit adenylate cyclase in intact platelets, although they are inhibitory with isolated membranes. However, comparison of the effects of PMA and these aggregating agents on the phosphorylation of platelet polypeptides indicated that activation of PKC by aggregating agents is inadequate to block their inhibitory effects on adenylate cyclase, when PGE/sub 1/ is present.

  15. African Swine Fever Virus Blocks the Host Cell Antiviral Inflammatory Response through a Direct Inhibition of PKC-θ-Mediated p300 Transactivation▿

    PubMed Central

    Granja, Aitor G.; Sánchez, Elena G.; Sabina, Prado; Fresno, Manuel; Revilla, Yolanda

    2009-01-01

    During a viral infection, reprogramming of the host cell gene expression pattern is required to establish an adequate antiviral response. The transcriptional coactivators p300 and CREB binding protein (CBP) play a central role in this regulation by promoting the assembly of transcription enhancer complexes to specific promoters of immune and proinflammatory genes. Here we show that the protein A238L encoded by African swine fever virus counteracts the host cell inflammatory response through the control of p300 transactivation during the viral infection. We demonstrate that A238L inhibits the expression of the inflammatory regulators cyclooxygenase-2 (COX-2) and tumor necrosis factor alpha (TNF-α) by preventing the recruitment of p300 to the enhanceosomes formed on their promoters. Furthermore, we report that A238L inhibits p300 activity during the viral infection and that its amino-terminal transactivation domain is essential in the A238L-mediated inhibition of the inflammatory response. Importantly, we found that the residue serine 384 of p300 is required for the viral protein to accomplish its inhibitory function and that ectopically expressed PKC-θ completely reverts this inhibition, thus indicating that this signaling pathway is disrupted by A238L during the viral infection. Furthermore, we show here that A238L does not affect PKC-θ enzymatic activity, but the molecular mechanism of this viral inhibition relies on the lack of interaction between PKC-θ and p300. These findings shed new light on how viruses alter the host cell antiviral gene expression pattern through the blockade of the p300 activity, which represents a new and sophisticated viral mechanism to evade the inflammatory and immune defense responses. PMID:19004945

  16. Epidermal growth factor (EGF) ligand release by substrate-specific a disintegrin and metalloproteases (ADAMs) involves different protein kinase C (PKC) isoenzymes depending on the stimulus.

    PubMed

    Dang, Michelle; Dubbin, Karen; D'Aiello, Antonio; Hartmann, Monika; Lodish, Harvey; Herrlich, Andreas

    2011-05-20

    The dysregulation of EGF family ligand cleavage has severe consequences for the developing as well as the adult organism. Therefore, their production is highly regulated. The limiting step is the ectodomain cleavage of membrane-bound precursors by one of several a disintegrin and metalloprotease (ADAM) metalloproteases, and understanding the regulation of cleavage is an important goal of current research. We have previously reported that in mouse lung epithelial cells, the pro-EGF ligands TGFα, neuregulin 1β (NRG), and heparin-binding EGF are differentially cleaved depending on the cleavage stimulus (Herrlich, A., Klinman, E., Fu, J., Sadegh, C., and Lodish, H. (2008) FASEB J.). In this study in mouse embryonic fibroblasts that lack different ADAMs, we show that induced cleavage of EGF ligands can involve the same substrate-specific metalloprotease but does require different stimulus-dependent signaling pathways. Cleavage was stimulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA), a mimic of diacylglycerol and PKC activator), hypertonic stress, lysophosphatidic acid (LPA)-induced G protein-coupled receptor activation, or by ionomycin-induced intracellular calcium release. Although ADAMs showed substrate preference (ADAM17, TGFα and heparin-binding EGF; and ADAM9, NRG), substrate cleavage differed substantially with the stimulus, and cleavage of the same substrate depended on the presence of different, sometimes multiple, PKC isoforms. For instance, classical PKC was required for TPA-induced but not hypertonic stress-induced cleavage of all EGF family ligands. Inhibition of PKCζ enhanced NRG release upon TPA stimulation, but it blocked NRG release in response to hypertonic stress. Our results suggest a model in which substantial regulation of ectodomain cleavage occurs not only on the metalloprotease level but also on the level of the substrate or of a third protein.

  17. Modulation of PKC signaling and induction of apoptosis through suppression of reactive oxygen species and tumor necrosis factor receptor 1 (TNFR1): key role of quercetin in cancer prevention.

    PubMed

    Maurya, Akhilendra Kumar; Vinayak, Manjula

    2015-11-01

    Cancer cells are characterized by increased production of reactive oxygen species (ROS) and an altered redox environment as compared to normal cells. Continuous accumulation of ROS triggers oxidative stress leading to hyper-activation of signaling pathways that promote cell proliferation, survival, and metabolic adaptation to the tumor microenvironment. Therefore, antioxidants are proposed to contribute to cancer prevention. Protein kinase C (PKC) is a crucial regulator of diverse cellular processes and contributes to cancer progression. The activation of PKC is partially dependent on ROS signaling. In the present study, cancer preventive activity of natural flavonoid quercetin is analyzed in ascite cells of Dalton's lymphoma-bearing mice. The total ROS level and activity of PKC were downregulated after quercetin treatment in lymphoma-bearing mice. Quercetin modulates the expression of almost all isozymes of classical, novel, and atypical PKC as well as downregulates the level and expression of PKCα. Further, quercetin improves apoptotic potential, as observed by the levels of caspase 3, caspase 9, PARP, PKCδ, and nuclear condensation. Additionally, quercetin reduces cell survival and promotes death receptor-mediated apoptosis via differential localization of the TNFR1 level in ascite cells. The overall result suggests the cancer preventive activity of quercetin via the induction of apoptosis and modulates PKC signaling with the reduction of oxidative stress in ascite cells of lymphoma-bearing mice.

  18. Scaffold State Switching Amplifies, Accelerates, and Insulates Protein Kinase C Signaling*

    PubMed Central

    Greenwald, Eric C.; Redden, John M.; Dodge-Kafka, Kimberly L.; Saucerman, Jeffrey J.

    2014-01-01

    Scaffold proteins localize two or more signaling enzymes in close proximity to their downstream effectors. A-kinase-anchoring proteins (AKAPs) are a canonical family of scaffold proteins known to bind protein kinase A (PKA) and other enzymes. Several AKAPs have been shown to accelerate, amplify, and specify signal transduction to dynamically regulate numerous cellular processes. However, there is little theory available to mechanistically explain how signaling on protein scaffolds differs from solution biochemistry. In our present study, we propose a novel kinetic mechanism for enzymatic reactions on protein scaffolds to explain these phenomena, wherein the enzyme-substrate-scaffold complex undergoes stochastic state switching to reach an active state. This model predicted anchored enzymatic reactions to be accelerated, amplified, and insulated from inhibition compared with those occurring in solution. We exploited a direct interaction between protein kinase C (PKC) and AKAP7α as a model to validate these predictions experimentally. Using a genetically encoded PKC activity reporter, we found that both the strength and speed of substrate phosphorylation were enhanced by AKAP7α. PKC tethered to AKAP7α was less susceptible to inhibition from the ATP-competitive inhibitor Gö6976 and the substrate-competitive inhibitor PKC 20-28, but not the activation-competitive inhibitor calphostin C. Model predictions and experimental validation demonstrated that insulation is a general property of scaffold tethering. Sensitivity analysis indicated that these findings may be applicable to many other scaffolds as well. Collectively, our findings provide theoretical and experimental evidence that scaffold proteins can amplify, accelerate, and insulate signal transduction. PMID:24302730

  19. Searching for disease modifiers-PKC activation and HDAC inhibition - a dual drug approach to Alzheimer's disease that decreases Abeta production while blocking oxidative stress.

    PubMed

    Kozikowski, Alan P; Chen, Yihua; Subhasish, Tapadar; Lewin, Nancy E; Blumberg, Peter M; Zhong, Zhenyu; D'Annibale, Melissa A; Wang, Weng-Long; Shen, Yong; Langley, Brett

    2009-07-01

    A series of benzolactam compounds were synthesized, some of which caused a concentration-dependent increase in sAPPalpha and decrease in Abeta production in the concentration range of 0.1-10 microM. Moreover, some compounds showed neuroprotective effects in the 10-20 microM range in the HCA cortical neuron model of oxidative stress and no toxicity in measurements of neuron viability by MTT assay, even at the highest concentrations tested (20 microM). Alzheimer's disease (AD) is a well-studied neurodegenerative process characterized by the presence of amyloid plaques and neurofibrillary tangles. In this study, a series of protein kinase C (PKC) activators were investigated, some of which also exhibit histone deacetylase (HDAC) inhibitory activity, under the hypothesis that such compounds might provide a new path forward in the discovery of drugs for the treatment of AD. The PKC-activating properties of these drugs were expected to enhance the alpha-secretase pathway in the processing of amyloid precursor protein (APP), while their HDAC inhibition was anticipated to confer neuroprotective activity. We found that benzolactams 9 and 11-14 caused a concentration-dependent increase in sAPPalpha and decrease in beta-amyloid (Abeta) production in the concentration range of 0.1-10 microM, consistent with a shift of APP metabolism toward the alpha-secretase-processing pathway. Moreover, compounds 9-14 showed neuroprotective effects in the 10-20 microM range in the homocysteate (HCA) cortical neuron model of oxidative stress. In parallel, we found that the most neuroprotective compounds caused increased levels of histone acetylation (H4), thus indicating their likely ability to inhibit HDAC activity. As the majority of the compounds studied also show nanomolar binding affinities for PKC, we conclude that it is possible to design, de novo, agents that combine both PKC-activating properties along with HDAC inhibitory properties. Such agents would be capable of modulating

  20. Inhibitors of Pyruvate Carboxylase

    PubMed Central

    Zeczycki, Tonya N.; Maurice, Martin St.; Attwood, Paul V.

    2010-01-01

    This review aims to discuss the varied types of inhibitors of biotin-dependent carboxylases, with an emphasis on the inhibitors of pyruvate carboxylase. Some of these inhibitors are physiologically relevant, in that they provide ways of regulating the cellular activities of the enzymes e.g. aspartate and prohibitin inhibition of pyruvate carboxylase. Most of the inhibitors that will be discussed have been used to probe various aspects of the structure and function of these enzymes. They target particular parts of the structure e.g. avidin – biotin, FTP – ATP binding site, oxamate – pyruvate binding site, phosphonoacetate – binding site of the putative carboxyphosphate intermediate. PMID:22180764

  1. Acquired Factor V Inhibitor

    PubMed Central

    Hirai, Daisuke; Yamashita, Yugo; Masunaga, Nobutoyo; Katsura, Toshiaki; Akao, Masaharu; Okuno, Yoshiaki; Koyama, Hiroshi

    2016-01-01

    Inhibitors directed against factor V rarely occur, and the clinical symptoms vary. We herein report the case of a patient who presented with a decreased factor V activity that had decreased to <3 %. We administered vitamin K and 6 units of fresh frozen plasma, but she thereafter developed an intracerebral hemorrhage. It is unclear whether surgery >10 years earlier might have caused the development of a factor V inhibitor. The treatment of acquired factor V inhibitors is mainly the transfusion of platelet concentrates and corticosteroids. Both early detection and the early initiation of the treatment of factor V inhibitor are thus considered to be important. PMID:27746446

  2. Angiotensin II inhibits the ROMK-like small conductance K channel in renal cortical collecting duct during dietary potassium restriction.

    PubMed

    Wei, Yuan; Zavilowitz, Beth; Satlin, Lisa M; Wang, Wen-Hui

    2007-03-02

    Base-line urinary potassium secretion in the distal nephron is mediated by small conductance rat outer medullary K (ROMK)-like channels. We used the patch clamp technique applied to split-open cortical collecting ducts (CCDs) isolated from rats fed a normal potassium (NK) or low potassium (LK) diet to test the hypothesis that AngII directly inhibits ROMK channel activity. We found that AngII inhibited ROMK channel activity in LK but not NK rats in a dose-dependent manner. The AngII-induced reduction in channel activity was mediated by AT1 receptor (AT1R) binding, because pretreatment of CCDs with losartan but not PD123319 AT1 and AT2 receptor antagonists, respectively, blocked the response. Pretreatment of CCDs with U73122 and calphostin C, inhibitors of phospholipase C (PLC) and protein kinase C (PKC), respectively, abolished the AngII-induced decrease in ROMK channel activity, confirming a role of the PLC-PKC pathway in this response. Studies by others suggest that AngII stimulates an Src family protein-tyrosine kinase (PTK) via PKC-NADPH oxidase. PTK has been shown to regulate the ROMK channel. Inhibition of NADPH oxidase with diphenyliodonium abolished the inhibitory effect of AngII or the PKC activator phorbol 12-myristate 13-acetate on ROMK channels. Suppression of PTK by herbimycin A significantly attenuated the inhibitory effect of AngII on ROMK channel activity. We conclude that AngII inhibits ROMK channel activity through PKC-, NADPH oxidase-, and PTK-dependent pathways under conditions of dietary potassium restriction.

  3. Angiotensin II Inhibits the ROMK-like Small Conductance K Channel in Renal Cortical Collecting Duct during Dietary Potassium Restriction*

    PubMed Central

    Wei, Yuan; Zavilowitz, Beth; Satlin, Lisa M.; Wang, Wen-Hui

    2010-01-01

    Base-line urinary potassium secretion in the distal nephron is mediated by small conductance rat outer medullary K (ROMK)-like channels. We used the patch clamp technique applied to split-open cortical collecting ducts (CCDs) isolated from rats fed a normal potassium (NK) or low potassium (LK) diet to test the hypothesis that AngII directly inhibits ROMK channel activity. We found that AngII inhibited ROMK channel activity in LK but not NK rats in a dose-dependent manner. The AngII-induced reduction in channel activity was mediated by AT1 receptor (AT1R) binding, because pretreatment of CCDs with losartan but not PD123319 AT1 and AT2 receptor antagonists, respectively, blocked the response. Pretreatment of CCDs with U73122 and calphostin C, inhibitors of phospholipase C (PLC) and protein kinase C (PKC), respectively, abolished the AngII-induced decrease in ROMK channel activity, confirming a role of the PLC-PKC pathway in this response. Studies by others suggest that AngII stimulates an Src family protein-tyrosine kinase (PTK) via PKC-NADPH oxidase. PTK has been shown to regulate the ROMK channel. Inhibition of NADPH oxidase with diphenyliodonium abolished the inhibitory effect of AngII or the PKC activator phorbol 12-myristate 13-acetate on ROMK channels. Suppression of PTK by herbimycin A significantly attenuated the inhibitory effect of AngII on ROMK channel activity. We conclude that AngII inhibits ROMK channel activity through PKC-, NADPH oxidase-, and PTK-dependent pathways under conditions of dietary potassium restriction. PMID:17194699

  4. Induction by staurosporine of nitric oxide synthase expression in vascular smooth muscle cells: role of NF-κB, CREB and C/EBPβ

    PubMed Central

    Hecker, Markus; Preiß, Christiane; Schini-Kerth, Valérie B

    1997-01-01

    The effect of different protein kinase inhibitors on the expression of the inducible isoform of nitric oxide (NO) synthase (iNOS) was investigated in cultured vascular smooth muscle cells (VSMC) isolated from the rat aorta.The non-selective protein kinase C (PKC) inhibitor, staurosporine, but not the more selective PKC inhibitors, calphostin C and Ro 31-8220, or the tyrosine kinase inhibitors, genistein and erbstatin analogue (erbstatin A), elicited a distinct (up to six fold) up-regulation of iNOS gene expression in these cells, as demonstrated by a parallel increase in iNOS mRNA and protein abundance as well as an accumulation of nitrite (NO2−) in the conditioned medium. Actinomycin D and cycloheximide inhibited the effect of staurosporine, suggesting an involvement of both DNA transcription and de novo protein synthesis.Staurosporine also synergistically potentiated the stimulating effect of interleukin-1β (IL-1β), but not that of the adenylyl cyclase activator, forskolin, on NO2− production and iNOS protein abundance. Staurosporine, on the other hand, had no effect on the IL-1β-mediated increase in iNOS mRNA abundance. The effect of staurosporine on both basal and IL-1β-stimulated NO2− production was concentration-dependent with an apparent maximum at 3 nM. Among the other protein kinase inhibitors tested, only calphostin C also enhanced the stimulant effect of IL-1β approximately two fold, while genistein, erbstatin A and Ro 31-8220 inhibited rather than potentiated it.Staurosporine did not influence basal activity of the transcription factors CREB and nuclear factor κB (NF-κB), but increased that of C/EBP. Moreover, staurosporine significantly augmented the activation of C/EBP by IL-1β and forskolin.These findings suggest that in cultured VSMC a staurosporine-sensitive protein kinase exists, which is unlikely to be related to PKC, that prevents iNOS gene expression presumably by suppressing basal C/EBP activity. They also indicate that NF

  5. Allium cepa Extract and Quercetin Protect Neuronal Cells from Oxidative Stress via PKC-ε Inactivation/ERK1/2 Activation

    PubMed Central

    2016-01-01

    Oxidative stress plays an important role in the pathophysiology of various neurologic disorders. Allium cepa extract (ACE) and their main flavonoid component quercetin (QCT) possess antioxidant activities and protect neurons from oxidative stress. We investigated the underlying molecular mechanisms, particularly those linked to the antioxidant effects of the ACE. Primary cortical neuronal cells derived from mouse embryos were preincubated with ACE or QCT for 30 min and exposed to L-buthionine sulfoximine for 4~24 h. We found that ACE and QCT significantly decreased neuronal death and the ROS increase induced by L-buthionine-S, R-sulfoximine (BSO) in a concentration-dependent manner. Furthermore, ACE and QCT activated extracellular signal-regulated kinase 1/2 (ERK1/2), leading to downregulation of protein kinase C-ε (PKC-ε) in BSO-stimulated neuronal cells. In addition, ACE and QCT decreased the phosphorylated levels of p38 mitogen-activated protein kinase. Our results provide new insight into the protective mechanism of ACE and QCT against oxidative stress in neuronal cells. The results suggest that the inactivation of PKC-ε induced by phosphorylating ERK1/2 is responsible for the neuroprotective effect of ACE and QCT against BSO-induced oxidative stress. PMID:27668036

  6. 6-Gingerol inhibits ROS and iNOS through the suppression of PKC-{alpha} and NF-{kappa}B pathways in lipopolysaccharide-stimulated mouse macrophages

    SciTech Connect

    Lee, Tzung-Yan; Lee, Ko-Chen; Chen, Shih-Yuan; Chang, Hen-Hong

    2009-04-24

    Inflammation is involved in numerous diseases, including chronic inflammatory diseases and the development of cancer. Many plants possess a variety of biological activities, including antifungal, antibacterial and anti-inflammatory activities. However, our understanding of the anti-inflammatory effects of 6-gingerol is very limited. We used lipopolysaccharide (LPS)-stimulated macrophages as a model of inflammation to investigate the anti-inflammatory effects of 6-gingerol, which contains phenolic structure. We found that 6-gingerol exhibited an anti-inflammatory effect. 6-Gingerol could decrease inducible nitric oxide synthase and TNF-{alpha} expression through suppression of I-{kappa}B{alpha} phosphorylation, NF-{kappa}B nuclear activation and PKC-{alpha} translocation, which in turn inhibits Ca{sup 2+} mobilization and disruption of mitochondrial membrane potential in LPS-stimulated macrophages. Here, we demonstrate that 6-gingerol acts as an anti-inflammatory agent by blocking NF-{kappa}B and PKC signaling, and may be developed as a useful agent for the chemoprevention of cancer or inflammatory diseases.

  7. Binding of FGF2 to FGFR2 in an autocrine mode in trophectoderm cells is indispensable for mouse blastocyst formation through PKC-p38 pathway

    PubMed Central

    Yang, Jing; Zhang, Dan; Yu, Ying; Zhang, Run-Ju; Hu, Xiao-Ling; Huang, He-Feng; Lu, Yong-Chao

    2015-01-01

    Fibroblast growth factors (FGF1, FGF2 and FGF4) and fibroblast growth factor receptors (FGFR1, FGFR2, FGFR3 and FGFR4) have been reported to be expressed in preimplantation embryos and be required for their development. However, the functions of these molecules in trophectoderm cells (TEs) that lead to the formation of the blastocyst as well as the underlying mechanism have not been elucidated. The present study has demonstrated for the first time that endogenous FGF2 secreted by TEs can regulate protein expression and distribution in TEs via the FGFR2-mediated activation of PKC and p38, which are important for the development of expanded blastocysts. This finding provides the first explanation for the long-observed phenomenon that only high concentrations of exogenous FGFs have effects on embryonic development, but in vivo the amount of endogenous FGFs are trace. Besides, the present results suggest that FGF2/FGFR2 may act in an autocrine fashion and activate the downstream PKC/p38 pathway in TEs during expanded blastocyst formation. PMID:26378412

  8. Structural insight with mutational impact on tyrosinase and PKC-β interaction from Homo sapiens: Molecular modeling and docking studies for melanogenesis, albinism and increased risk for melanoma.

    PubMed

    Banerjee, Arundhati; Ray, Sujay

    2016-10-30

    Human tyrosinase, is an important protein for biosynthetic pathway of melanin. It was studied to be phosphorylated and activated by protein kinase-C, β-subunit (PKC-β) through earlier experimentations with in vivo evidences. Documentation documents that mutation in two essentially vital serine residues in C-terminal end of tyrosinase leads to albinism. Due to the deficiency of protective shield like enzyme; melanin, albinos are at an increased peril for melanoma and other skin cancers. So, computational and residue-level insight including a mutational exploration with evolutionary importance into this mechanism lies obligatory for future pathological and therapeutic developments. Therefore, functional tertiary models of the relevant proteins were analyzed after satisfying their stereo-chemical features. Evolutionarily paramount residues for the activation of tyrosinase were perceived via multiple sequence alignment phenomena. Mutant-type tyrosinase protein (S98A and S102A) was thereby modeled, maintaining the wild-type proteins' functionality. Furthermore, this present comparative study discloses the variation in the stable residual participation (for mutant-type and wild-type tyrosinase-PKCβ complex). Mainly, an increased number of polar negatively charged residues from the wild-type tyrosinase participated with PKC-β, predominantly. Fascinatingly supported by evaluation of statistical significances, mutation even led to a destabilizing impact in tyrosinase accompanied by conformational switches with a helix-to-coil transition in the mutated protein. Even the allosteric sites in the protein got poorly hampered upon mutation leading to weaker tendency for binding partners to interact.

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

    PubMed

    Maurya, Akhilendra Kumar; Vinayak, Manjula

    2015-09-01

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

  10. Novel corrosion inhibitor technology

    SciTech Connect

    Van de Ven, P.; Fritz, P.; Pellet, R.

    1999-11-01

    A novel, patented corrosion inhibitor technology has been identified for use in heat transfer applications such as automotive and heavy-duty coolant. The new technology is based on a low-toxic, virtually depletion-free carboxylic acid corrosion inhibitor package that performs equally well in mono ethylene glycol and in less toxic propylene glycol coolants. An aqueous inhibitor concentrate is available to provide corrosion protection where freezing protection is not an issue. In the present paper, this inhibitor package is evaluated in the different base fluids: mono ethylene glycol, mono propylene glycol and water. Results are obtained in both standardized and specific corrosion tests as well as in selected field trials. These results indicate that the inhibitor package remains effective and retains the benefits previously identified in automotive engine coolant applications: excellent corrosion protection under localized conditions, general corrosion conditions as well as at high temperature.

  11. Mucin-like glycoprotein secretion is mediated by cyclic-AMP and protein kinase C signal transduction pathways in rat corneal epithelium.

    PubMed

    Nakamura, M; Endo, K; Nakata, K

    1998-05-01

    Ocular surface mucin is secreted from both goblet cells in the conjunctival epithelium and corneal epithelial cells. To clarify its mechanism of secretion in corneal epithelial cells, a rat cornea organ culture system was used to evaluate the second messenger roles of cyclic-AMP (cAMP), cyclic-GMP (cGMP) and protein kinase C (PKC) in modulating mucin-like glycoprotein secretion. Rat cornea sections (3 mm diameter) were cultured in TC-199 medium, and radiolabeled with sodium sulfate for 18 hr. After washing, the corneas were treated with various second messenger modulating agents for 30 min. The culture media were reacted with Dolichos biflorus (DBA)-lectin, and mucin-like glycoprotein was isolated. Then the radioactivity of DBA-binding mucin-like glycoprotein was isolated. Then the radioactivity of DBA-binding mucin-like glycoprotein was measured. There was a time-dependent increase in mucin-like glycoprotein was measured. There was a time-dependent increase in mucin-like glycoprotein secretion, whereas after corneal epithelial debridement the secretion was markedly inhibited by 81%. Mucin-like glycoprotein secretion was stimulated in a dose-dependent manner following elevation of cAMP levels by exposure to either forskolin, dibutyryl cAMP or 3-isobutyl-1-methylxanthine. Concomitant exposure to the cAMP dependent protein kinase inhibitor, KT5720 completely inhibited their stimulatory effects. Neither exposure to dibutyryl cGMP nor nitroprusside affected mucin-like glycoprotein secretion. Stimulation by PKC, phorbol 12, 13-dibutyrate (PDBu) also increased mucin-like glycoprotein secretion in a dose-dependent fashion. The PKC inhibitor, calphostin C completely inhibited the stimulation by PDBu of mucine-like glycoprotein secretion. These results demonstrate that corneal epithelial cells secrete mucin-like glycoprotein, which is mediated by cAMP and PKC signal transduction pathways.

  12. Metformin inhibits angiotensin II-induced differentiation of cardiac fibroblasts into myofibroblasts.

    PubMed

    Bai, Jian; Zhang, Na; Hua, Ying; Wang, Bingjian; Ling, Lin; Ferro, Albert; Xu, Biao

    2013-01-01

    Differentiation of cardiac fibroblasts into myofibroblasts is a critical event in the progression of cardiac fibrosis that leads to pathological cardiac remodeling. Metformin, an antidiabetic agent, exhibits a number of cardioprotective properties. However, much less is known regarding the effect of metformin on cardiac fibroblast differentiation. Thus, in the present study, we examined the effect of metformin on angiotensin (Ang) II-induced differentiation of cardiac fibroblasts into myofibroblasts and its underlying mechanism. Adult rat cardiac fibroblasts were stimulated with Ang II (100 nM) in the presence or absence of metformin (10-200 µM). Ang II stimulation induced the differentiation of cardiac fibroblasts into myofibroblasts, as indicated by increased expression of α-smooth muscle actin (α-SMA) and collagen types I and III, and this effect of Ang II was inhibited by pretreatment of cardiac fibroblasts with metformin. Metformin also decreased Ang II-induced reactive oxygen species (ROS) generation in cardiac fibroblasts via inhibiting the activation of the PKC-NADPH oxidase pathway. Further experiments using PKC inhibitor calphostin C and NADPH oxidase inhibitor apocynin confirmed that inhibition of the PKC-NADPH oxidase pathway markedly attenuated Ang II-induced ROS generation and myofibroblast differentiation. These data indicate that metformin inhibits Ang II-induced myofibroblast differentiation by suppressing ROS generation via the inhibition of the PKC-NADPH oxidase pathway in adult rat cardiac fibroblasts. Our results provide new mechanistic insights regarding the cardioprotective effects of metformin and provide an efficient therapeutic strategy to attenuate cardiac fibrosis.

  13. CRYSTALLINE SOYBEAN TRYPSIN INHIBITOR

    PubMed Central

    Kunitz, M.

    1947-01-01

    A study has been made of the general properties of crystalline soybean trypsin inhibitor. The soy inhibitor is a stable protein of the globulin type of a molecular weight of about 24,000. Its isoelectric point is at pH 4.5. It inhibits the proteolytic action approximately of an equal weight of crystalline trypsin by combining with trypsin to form a stable compound. Chymotrypsin is only slightly inhibited by soy inhibitor. The reaction between chymotrypsin and the soy inhibitor consists in the formation of a reversibly dissociable compound. The inhibitor has no effect on pepsin. The inhibiting action of the soybean inhibitor is associated with the native state of the protein molecule. Denaturation of the soy protein by heat or acid or alkali brings about a proportional decrease in its inhibiting action on trypsin. Reversal of denaturation results in a proportional gain in the inhibiting activity. Crystalline soy protein when denatured is readily digestible by pepsin, and less readily by chymotrypsin and by trypsin. Methods are given for measuring trypsin and inhibitor activity and also protein concentration with the aid of spectrophotometric density measurements at 280 mµ. PMID:19873496

  14. The STAT5 Inhibitor Pimozide Displays Efficacy in Models of Acute Myelogenous Leukemia Driven by FLT3 Mutations

    PubMed Central

    Nelson, Erik A.; Walker, Sarah R.; Xiang, Michael; Weisberg, Ellen; Bar-Natan, Michal; Barrett, Rosemary; Liu, Suiyang; Kharbanda, Surender; Christie, Amanda L.; Nicolais, Maria; Griffin, James D.; Stone, Richard M.; Kung, Andrew L.

    2012-01-01

    Activation of the transcription factor STAT5 is essential for the pathogenesis of acute myelogenous leukemia (AML) containing the FLT3 internal tandem duplication (ITD) mutation. FLT3 ITD is a constitutively active tyrosine kinase that drives the activation of STAT5, leading to the growth and survival of AML cells. Although there has been some success in identifying tyrosine kinase inhibitors that block the function of FLT3 ITD, there remains a continued need for effective treatment of this disease. We have identified the psychotropic drug pimozide as an effective inhibitor of STAT5 function. Pimozide inhibits the tyrosine phosphorylation of STAT5, leading to the death of AML cells through the induction of apoptosis. Pimozide shows a combinatorial effect with the tyrosine kinase inhibitors midostaurin (PKC412) and sunitinib in the inhibition of STAT5 tyrosine phosphorylation and the induction of apoptosis. Significantly, pimozide reduces the tumor burden in a mouse model of FLT3-driven AML. Therefore, identifying STAT5 inhibitors may provide a new avenue for the treatment of AML, and these may be effective alone or in combination with tyrosine kinase inhibitors. PMID:23264850

  15. PKC-dependent ERK phosphorylation is essential for P2X7 receptor-mediated neuronal differentiation of neural progenitor cells

    PubMed Central

    Tsao, H-K; Chiu, P-H; Sun, S H

    2013-01-01

    Purinergic receptors have been shown to be involved in neuronal development, but the functions of specific subtypes of P2 receptors during neuronal development remain elusive. In this study we investigate the distribution of P2X7 receptors (P2X7Rs) in the embryonic rat brain using in situ hybridization. At E15.5, P2X7R mRNA was observed in the ventricular zone and subventricular zone, and colocalized with nestin, indicating that P2X7R might be expressed in neural progenitor cells (NPCs). P2X7R mRNA was also detected in the subgranular zone and dentate gyrus of the E18.5 and P4 brain. To investigate the roles of P2X7R and elucidate its mechanism, we established NPC cultures from the E15.5 rat brain. Stimulation of P2X7Rs induced Ca2+ influx, inhibited proliferation, altered cell cycle progression and enhanced the expression of neuronal markers, such as TUJ1 and MAP2. Similarly, knockdown of P2X7R by shRNA nearly abolished the agonist-stimulated increases in intracellular Ca2+ concentration and the expression of TUJ1 and NeuN. Furthermore, stimulation of P2X7R induced activation of ERK1/2, which was inhibited by the removal of extracellular Ca2+ and treatment with blockers for P2X7R and PKC activity. Stimulation of P2X7R also induced translocation of PKCα and PKCγ, but not of PKCβ, whereas knockdown of either PKCα or PKCγ inhibited ERK1/2 activation. Inhibition of PKC or p-ERK1/2 also caused a decrease in the number of TUJ1-positive cells and a concomitant increase in the number of GFAP-positive cells. Taken together, the activation of P2X7R in NPCs induced neuronal differentiation through a PKC-ERK1/2 signaling pathway. PMID:23907465

  16. Artocarpol A stimulation of superoxide anion generation in neutrophils involved the activation of PLC, PKC and p38 mitogen-activated PK signaling pathways.

    PubMed

    Kuan, Yu-Hsiang; Lin, Ruey-Hseng; Tsao, Lo-Ti; Lin, Chun-Nan; Wang, Jih-Pyang

    2005-06-01

    1 Artocarpol A (ART), a natural phenolic compound isolated from Artocarpus rigida, stimulated a slow onset and long-lasting superoxide anion generation in rat neutrophils, whereas only slightly activated the NADPH oxidase in a cell-free system. 2 Pretreatment of neutrophils with pertussis toxin (1 microg ml(-1)), 50 microM 2'-amino-3'-methoxyflavone (PD 98059), or 1 microM 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126) had no effect on ART-stimulated superoxide anion generation. ART (30 microM) did not induce extracellular signal-regulated kinase (ERK) phosphorylation. 3 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580) markedly attenuated the ART-stimulated superoxide anion generation (IC50 value of 4.3+/-0.3 microM). Moreover, ART induced p38 mitogen-activated PK (MAPK) phosphorylation and activation. 4 The superoxide anion generation in response to ART was also substantially inhibited in a Ca2+-free medium, and by pretreatment with 1 microM 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122) and 100 microM 2-aminoethyldiphenyl borate (2-APB). ART (30 microM) stimulated the [Ca2+]i elevation in the presence or absence of external Ca2+, and also increased the D-myo-inositol 1,4,5-trisphosphate formation. 5 2-[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X) greatly inhibited the ART-stimulated superoxide anion generation (IC50 value of 7.8+/-1.0 nM). ART increased the recruitment of PKC-alpha, -betaI, and -betaII to the plasma membrane of neutrophils, and stimulated Ca2+-dependent PKC activation in the cytosol preparation. 6 ART induced the phosphorylation of p47phox, which was attenuated by GF 109203X. Moreover, ART evoked the membrane association of p47(phox), which was inhibited by GF 109203X and SB 203580. 7 These results indicate that the ART stimulation of superoxide anion generation involved the activation of p38 MAPK, PLC/Ca2

  17. Cadmium exposure activates the ERK signaling pathway leading to altered osteoblast gene expression and apoptotic death in Saos-2 cells.

    PubMed

    Arbon, Kate S; Christensen, Cody M; Harvey, Wendy A; Heggland, Sara J

    2012-02-01

    Recent reports of cadmium in electronic waste and jewelry have increased public awareness regarding this toxic metal. Human exposure to cadmium is associated with the development of osteoporosis. We previously reported cadmium induces apoptosis in human tumor-derived Saos-2 osteoblasts. In this study, we examine the extracellular signal-regulated protein kinase (ERK) and protein kinase C (PKC) pathways in cadmium-induced apoptosis and altered osteoblast gene expression. Saos-2 osteoblasts were cultured in the presence or absence of 10μM CdCl(2) for 2-72h. We detected significant ERK activation in response to CdCl(2) and pretreatment with the ERK inhibitor PD98059 attenuated cadmium-induced apoptosis. However, PKCα activation was not observed after exposure to CdCl(2) and pretreatment with the PKC inhibitor, Calphostin C, was unable to rescue cells from cadmium-induced apoptosis. Gene expression studies were conducted using qPCR. Cells exposed to CdCl(2) exhibited a significant decrease in the bone-forming genes osteopontin (OPN) and alkaline phosphatase (ALP) mRNA. In contrast, SOST, whose protein product inhibits bone formation, significantly increased in response to CdCl(2). Pretreatment with PD98059 had a recovery effect on cadmium-induced changes in gene expression. This research demonstrates cadmium can directly inhibit osteoblasts via ERK signaling pathway and identifies SOST as a target for cadmium-induced osteotoxicity.

  18. Extracellular simian virus 40 induces an ERK/MAP kinase-independent signalling pathway that activates primary response genes and promotes virus entry.

    PubMed

    Dangoria, N S; Breau, W C; Anderson, H A; Cishek, D M; Norkin, L C

    1996-09-01

    Simian virus 40 (SV40) binding to growth-arrested cells activated an intracellular signalling pathway that induced the up-regulation of the primary response genes c-myc, c-jun and c-sis within 30 min and of JE within 90 min. The up-regulation of the primary response genes occurred in the presence of cycloheximide and when UV-inactivated SV40 was adsorbed to cells. SV40 binding did not activate Raf or mitogen-activated protein kinase (MAP/ERK1), or mobilize intracellular Ca2+. The SV40-induced up-regulation of c-myc and c-jun was blocked by the tyrosine kinase inhibitor, genistein, and by the protein kinase C (PKC) inhibitor, calphostin C, but not by expression of the MAP kinase-specific phosphatase, MKP-1. These results suggest that the SV40-induced signalling pathway includes the activities of a tyrosine kinase and a Ca(2+)-independent isoform of PKC, but not of Raf or MAP kinase. Finally, SV40 infectious entry into cells was specifically and reversibly blocked by genistein.

  19. The Acid-sensitive, Anesthetic-activated Potassium Leak Channel, KCNK3, Is Regulated by 14-3-3β-dependent, Protein Kinase C (PKC)-mediated Endocytic Trafficking*

    PubMed Central

    Gabriel, Luke; Lvov, Anatoli; Orthodoxou, Demetra; Rittenhouse, Ann R.; Kobertz, William R.; Melikian, Haley E.

    2012-01-01

    The acid-sensitive neuronal potassium leak channel, KCNK3, is vital for setting the resting membrane potential and is the primary target for volatile anesthetics. Recent reports demonstrate that KCNK3 activity is down-regulated by PKC; however, the mechanisms responsible for PKC-induced KCNK3 down-regulation are undefined. Here, we report that endocytic trafficking dynamically regulates KCNK3 activity. Phorbol esters and Group I metabotropic glutamate receptor (mGluR) activation acutely decreased both native and recombinant KCNK3 currents with concomitant KCNK3 surface losses in cerebellar granule neurons and cell lines. PKC-mediated KCNK3 internalization required the presence of both 14-3-3β and a novel potassium channel endocytic motif, because depleting either 14-3-3β protein levels or ablating the endocytic motif completely abrogated PKC-regulated KCNK3 trafficking. These results demonstrate that neuronal potassium leak channels are not static membrane residents but are subject to 14-3-3β-dependent regulated trafficking, providing a straightforward mechanism to modulate neuronal excitability and synaptic plasticity by Group I mGluRs. PMID:22846993

  20. The Effects of Lactobacillus acidophilus on the Intestinal Smooth Muscle Contraction through PKC/MLCK/MLC Signaling Pathway in TBI Mouse Model.

    PubMed

    Sun, Bo; Hu, Chen; Fang, Huan; Zhu, Lina; Gao, Ning; Zhu, Jingci

    2015-01-01

    Clinical studies have shown that probiotics influence gastrointestinal motility. However, the molecular mechanisms by which probiotic Lactobacillus modulates intestinal motility in traumatic brain injury (TBI) mouse model have not been explored. In the present study, we provided evidence showing that treatment of TBI mice with Lactobacillus acidophilus significantly improved the terminal ileum villus morphology, restored the impaired interstitial cells of Cajal (ICC) and the disrupted ICC networks after TBI, and prevented TBI-mediated inhibition of contractile activity in intestinal smooth muscle. Mechanistically, the decreased concentration of MLCK, phospho-MLC20 and phospho-MYPT1 and increased concentration of MLCP and PKC were observed after TBI, and these events mediated by TBI were efficiently prevented by Lactobacillus acidophilus application. These findings may provide a novel mechanistic basis for the application of Lactobacillus acidophilus in the treatment of TBI.

  1. Activation of bitter taste receptors in pulmonary nociceptors sensitizes TRPV1 channels through the PLC and PKC signaling pathway.

    PubMed

    Gu, Qihai David; Joe, Deanna S; Gilbert, Carolyn A

    2017-03-01

    Bitter taste receptors (T2Rs), a G protein-coupled receptor family capable of detecting numerous bitter-tasting compounds, have recently been shown to be expressed and play diverse roles in many extraoral tissues. Here we report the functional expression of T2Rs in rat pulmonary sensory neurons. In anesthetized spontaneously breathing rats, intratracheal instillation of T2R agonist chloroquine (10 mM, 0.1 ml) significantly augmented chemoreflexes evoked by right-atrial injection of capsaicin, a specific activator for transient receptor potential vanilloid receptor 1 (TRPV1), whereas intravenous infusion of chloroquine failed to significantly affect capsaicin-evoked reflexes. In patch-clamp recordings with isolated rat vagal pulmonary sensory neurons, pretreatment with chloroquine (1-1,000 µM, 90 s) concentration dependently potentiated capsaicin-induced TRPV1-mediated inward currents. Preincubating with diphenitol and denatonium (1 mM, 90 s), two other T2R activators, also enhanced capsaicin currents in these neurons but to a lesser extent. The sensitizing effect of chloroquine was effectively prevented by the phospholipase C inhibitor U73122 (1 µM) or by the protein kinase C inhibitor chelerythrine (10 µM). In summary, our study showed that activation of T2Rs augments capsaicin-evoked TRPV1 responses in rat pulmonary nociceptors through the phospholipase C and protein kinase C signaling pathway.

  2. Ketamine plus imipramine treatment induces antidepressant-like behavior and increases CREB and BDNF protein levels and PKA and PKC phosphorylation in rat brain.

    PubMed

    Réus, Gislaine Z; Stringari, Roberto B; Ribeiro, Karine F; Ferraro, Ana K; Vitto, Marcelo F; Cesconetto, Patrícia; Souza, Claúdio T; Quevedo, João

    2011-08-01

    A growing body of evidence has pointed to the N-methyl-d-aspartate (NMDA) receptor antagonists as a potential therapeutic target for the treatment of major depression. The present study investigated the possibility of synergistic interactions between antidepressant imipramine with the uncompetitive NMDA receptor antagonist ketamine. Wistar rats were acutely treated with ketamine (5 and 10mg/kg) and imipramine (10 and 20mg/kg) and then subjected to forced swimming tests. The cAMP response element bindig (CREB) and brain-derived neurotrophic factor (BDNF) protein levels and protein kinase C (PKC) and protein kinase A (PKA) phosphorylation were assessed in the prefrontal cortex, hippocampus and amygdala by imunoblot. Imipramine at the dose of 10mg/kg and ketamine at the dose of 5mg/kg did not have effect on the immobility time; however, the effect of imipramine (10 and 20mg/kg) was enhanced by both doses of ketamine. Ketamine and imipramine alone or in combination at all doses tested did not modify locomotor activity. Combined treatment with ketamine and imipramine produced stronger increases of CREB and BDNF protein levels in the prefrontal cortex, hippocampus and amygdala, and PKA phosphorylation in the hippocampus and amygdala and PKC phosphorylation in prefrontal cortex. The results described indicate that co-administration of antidepressant imipramine with ketamine may induce a more pronounced antidepressant activity than treatment with each antidepressant alone. This finding may be of particular importance in the case of drug-resistant patients and could suggest a method of obtaining significant antidepressant actions whilst limiting side effects.

  3. par-1, Atypical pkc, and PP2A/B55 sur-6 Are Implicated in the Regulation of Exocyst-Mediated Membrane Trafficking in Caenorhabditis elegans

    PubMed Central

    Jiu, Yaming; Hasygar, Kiran; Tang, Lois; Liu, Yanbo; Holmberg, Carina I.; Bürglin, Thomas R.; Hietakangas, Ville; Jäntti, Jussi

    2013-01-01

    The exocyst is a conserved protein complex that is involved in tethering secretory vesicles to the plasma membrane and regulating cell polarity. Despite a large body of work, little is known how exocyst function is controlled. To identify regulators for exocyst function, we performed a targeted RNA interference (RNAi) screen in Caenorhabditis elegans to uncover kinases and phosphatases that genetically interact with the exocyst. We identified seven kinase and seven phosphatase genes that display enhanced phenotypes when combined with hypomorphic alleles of exoc-7 (exo70), exoc-8 (exo84), or an exoc-7;exoc-8 double mutant. We show that in line with its reported role in exocytotic membrane trafficking, a defective exoc-8 caused accumulation of exocytotic soluble NSF attachment protein receptor (SNARE) proteins in both intestinal and neuronal cells in C. elegans. Down-regulation of the phosphatase protein phosphatase 2A (PP2A) phosphatase regulatory subunit sur-6/B55 gene resulted in accumulation of exocytic SNARE proteins SNB-1 and SNAP-29 in wild-type and in exoc-8 mutant animals. In contrast, RNAi of the kinase par-1 caused reduced intracellular green fluorescent protein signal for the same proteins. Double RNAi experiments for par-1, pkc-3, and sur-6/B55 in C. elegans suggest a possible cooperation and involvement in postembryo lethality, developmental timing, as well as SNARE protein trafficking. Functional analysis of the homologous kinases and phosphatases in Drosophila median neurosecretory cells showed that atypical protein kinase C kinase and phosphatase PP2A regulate exocyst-dependent, insulin-like peptide secretion. Collectively, these results characterize kinases and phosphatases implicated in the regulation of exocyst function, and suggest the possibility for interplay between the par-1 and pkc-3 kinases and the PP2A phosphatase regulatory subunit sur-6 in this process. PMID:24192838

  4. Snail regulated by PKC/GSK-3β pathway is crucial for EGF-induced epithelial-mesenchymal transition (EMT) of cancer cells.

    PubMed

    Liu, Zong-cai; Chen, Xiao-hui; Song, Hai-xing; Wang, Hong-sheng; Zhang, Ge; Wang, Hao; Chen, Dan-yang; Fang, Rui; Liu, Hao; Cai, Shao-hui; Du, Jun

    2014-11-01

    Cancer metastasis is considered a major challenge in cancer therapy. Recently, epidermal growth factor (EGF)/epidermal growth factor receptor (EGFR) signaling has been shown to induce epithelial-mesenchymal transition (EMT) and thereby to promote cancer metastasis. However, the underlying mechanism has not been fully elucidated. We demonstrate that EGF can induce EMT in human prostate and lung cancer cells and thus promote invasion and migration. EGF-induced EMT has been characterized by the cells acquiring mesenchymal spindle-like morphology and increasing their expression of N-cadherin and fibronectin, with a concomitant decrease of E-cadherin. Both protein and mRNA expression of transcription factor Snail rapidly increases after EGF treatment. The knockdown of Snail significantly attenuates EGF-induced EMT, suggesting that Snail is crucial for this process. To determine the way that Snail is accumulated, we demonstrate (1) that EGF promotes the stability of Snail via inhibiting the activity of glycogen synthase kinase 3 beta (GSK-3β), (2) that protein kinase C (PKC) rather than the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is responsible for GSK-3β inhibition and (3) that GSK-3β inhibition promotes the transcription of Snail. Taken together, these results reveal that the PKC/GSK-3β signaling pathway controls both the stability and transcription of Snail, which is crucial for EMT induced by EGF in PC-3 and A549 cells. Our study suggests a novel signaling pathway for Snail regulation and provides a better understanding of growth-factor-induced tumor EMT and metastasis.

  5. SGLT2 inhibitors.

    PubMed

    Dardi, I; Kouvatsos, T; Jabbour, S A

    2016-02-01

    Diabetes mellitus is a serious health issue and an economic burden, rising in epidemic proportions over the last few decades worldwide. Although several treatment options are available, only half of the global diabetic population achieves the recommended or individualized glycemic targets. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of antidiabetic agents with a novel insulin-independent action. SGLT2 is a transporter found in the proximal renal tubules, responsible for the reabsorption of most of the glucose filtered by the kidney. Inhibition of SGLT2 lowers the blood glucose level by promoting the urinary excretion of excess glucose. Due to their insulin-independent action, SGLT2 inhibitors can be used with any degree of beta-cell dysfunction or insulin resistance, related to a very low risk of hypoglycemia. In addition to improving glycemic control, SGLT2 inhibitors have been associated with a reduction in weight and blood pressure when used as monotherapy or in combination with other antidiabetic agents in patients with type 2 diabetes mellitus (T2DM). Treatment with SGLT2 inhibitors is usually well tolerated; however, they have been associated with an increased incidence of urinary tract and genital infections, although these infections are usually mild and easy to treat. SGLT2 inhibitors are a promising new option in the armamentarium of drugs for patients with T2DM.

  6. [Acquired coagulant factor inhibitors].

    PubMed

    Nogami, Keiji

    2015-02-01

    Acquired coagulation factor inhibitors are an autoimmune disease causing bleeding symptoms due to decreases in the corresponding factor (s) which result from the appearance of autoantibodies against coagulation factors (inhibitor). This disease is quite different from congenital coagulation factor deficiencies based on genetic abnormalities. In recent years, cases with this disease have been increasing, and most have anti-factor VIII autoantibodies. The breakdown of the immune control mechanism is speculated to cause this disease since it is common in the elderly, but the pathology and pathogenesis are presently unclear. We herein describe the pathology and pathogenesis of factor VIII and factor V inhibitors. Characterization of these inhibitors leads to further analysis of the coagulation process and the activation mechanisms of clotting factors. In the future, with the development of new clotting examination method (s), we anticipate that further novel findings will be obtained in this field through inhibitor analysis. In addition, detailed elucidation of the coagulation inhibitory mechanism possibly leading to hemostatic treatment strategies for acquired coagulation factor disorders will be developed.

  7. Cholinesterase inhibitors from botanicals

    PubMed Central

    Ahmed, Faiyaz; Ghalib, Raza Murad; Sasikala, P.; Ahmed, K. K. Mueen

    2013-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease, wherein a progressive loss of cholinergic synapses occurs in hippocampus and neocortex. Decreased concentration of the neurotransmitter, acetylcholine (ACh), appears to be critical element in the development of dementia, and the most appropriate therapeutic approach to treat AD and other form of dementia is to restore acetylcholine levels by inhibiting both major form of cholinesterase: Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Consequently, researches have focused their attention towards finding cholinesterase inhibitors from natural products. A large number of such inhibitors have been isolated from medicinal plants. This review presents a comprehensive account of the advances in field of cholinesterase inhibitor phytoconstituents. The structures of some important phytoconstituents (collected through www.Chemspider.com) are also presented and the scope for future research is discussed. PMID:24347920

  8. Angiotensin II stimulates basolateral 50-pS K channels in the thick ascending limb.

    PubMed

    Wang, Mingxiao; Luan, Haiyan; Wu, Peng; Fan, Lili; Wang, Lijun; Duan, Xinpeng; Zhang, Dandan; Wang, Wen-Hui; Gu, Ruimin

    2014-03-01

    We used the patch-clamp technique to examine the effect of angiotensin II (ANG II) on the basolateral K channels in the thick ascending limb (TAL) of the rat kidney. Application of ANG II increased the channel activity and the current amplitude of the basolateral 50-pS K channel. The stimulatory effect of ANG II on the K channels was completely abolished by losartan, an inhibitor of type 1 angiotensin receptor (AT1R), but not by PD123319, an AT2R antagonist. Moreover, inhibition of phospholipase C (PLC) and protein kinase C (PKC) also abrogated the stimulatory effect of ANG II on the basolateral K channels in the TAL. This suggests that the stimulatory effect of ANG II on the K channels was induced by activating PLC and PKC pathways. Western blotting demonstrated that ANG II increased the phosphorylation of c-Src at tyrosine residue 416, an indication of c-Src activation. This effect was mimicked by PKC stimulator but abolished by calphostin C. Moreover, inhibition of NADPH oxidase (NOX) also blocked the effect of ANG II on c-Src tyrosine phosphorylation. The role of Src-family protein tyrosine kinase (SFK) in mediating the effect of ANG II on the basolateral K channel was further suggested by the experiments in which inhibition of SFK abrogated the stimulatory effect of ANG II on the basolateral 50-pS K channel. We conclude that ANG II increases basolateral 50-pS K channel activity via AT1R and that activation of AT1R stimulates SFK by a PLC-PKC-NOX-dependent mechanism.

  9. Paradoxical stimulation and inhibition by protein kinase C modulating agents of lipopolysaccharide evoked production of tumour necrosis factor in human monocytes.

    PubMed Central

    Coffey, R G; Weakland, L L; Alberts, V A

    1992-01-01

    Human blood monocytes were activated by bacterial lipopolysaccharide endotoxin (LPS) (10 ng/ml) for cytotoxicity of WEHI-164 mouse fibrosarcoma cells, determined by release of 51Cr from WEHI-164 tumour cells incubated with monocyte supernatants. The chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) augmented LPS-induced cytotoxicity but had no effect alone. FMLP but not LPS stimulated phospholipase C (PLC), determined by the release of [3H]inositol phosphates. Addition of tumour promoter and protein kinase C stimulant, phorbol-12-myristate-13-acetate (PMA) at concentrations of 3 x 10(-10) M to 3 x 10(-9) M, resulted in an augmentation of 30-200% in LPS-evoked cytotoxicity. The effects of FMLP and PMA, like the effect of LPS alone, were completely blocked by antibody to recombinant human tumour necrosis factor-alpha (TNF-alpha), indicating that cytotoxicity induced by LPS, FMLP, and PMA were due solely to TNF release. Concentrations of PMA greater than 3 x 10(-9) M caused inhibition of TNF release. Okadaic acid (20 ng/ml), an inhibitor of phosphatases I and IIa, augmented the effects of LPS and the stimulatory effects of low levels of PMA, suggesting that phosphorylation was important in the actions of both LPS and PMA. The effects of LPS and of low levels of PMA were augmented by the protein kinase C (PKC) inhibitors H-7 (10-30 microM), staurosporine (2-10 nM) and calphostin C (0.1 microM). Higher concentrations of the inhibitors prevented LPS-evoked TNF release and its augmentation by low levels of PMA. However, they did not prevent the inhibition by high levels of PMA. One possible explanation for the results is that different isozymes of PKC may mediate the stimulatory as compared to the inhibitory effects of PKC on TNF production. PMID:1628900

  10. Evidence that simulated microgravity may alter the vascular nonreceptor tyrosine kinase second messenger pathway

    NASA Technical Reports Server (NTRS)

    Kahwaji, C. I.; Sheibani, S.; Han, S.; Siu, W. O.; Kaka, A. H.; Fathy, T. M.; el-Abbadi, N. H.; Purdy, R. E.

    2000-01-01

    Simulated microgravity (hind limb unweighting; HU) reduces maximal contractile capacity to norepinephrine (NE) but not 5-hydroxytryptamine (5-HT) in the rat abdominal aorta of male Wistar rats. Our earlier study showed that voltage-operated calcium channels, the MAPK pathway [1], and vasoconstrictive prostaglandins contribute to the NE-induced contraction of control (C) but not HU, aorta rings. Genistein, a general tyrosine kinase inhibitor, caused a significant reduction in vascular contractility in C but not HU arteries. The present study explored the role of protein kinase C (PKC) and extracellular receptor-activated kinase 1 and 2 (ERK1/2) in the HU-induced vascular hyporesponsiveness to NE. Microgravity was simulated in Wistar rats by 20 day HU. The abdominal aorta was removed from control and HU rats, cut into 3 mm rings, and mounted in tissue baths to measure isometric contraction. Protein levels were determined using Western blot analysis. PD98059, a selective MAPKK inhibitor, caused a marked inhibition of NE-induced contraction in both C and HU arteries. Calphostin C, a PKC inhibitor, completely abolished the contractile response to NE in both C and HU tissues. Phosphorylated (activated) ERK1/2 protein mass was greater in C, compared to HU, aortas, and was reduced by genistein only in C tissues. MAPK total protein levels in the rat aorta were increased in the HU-treated, compared to C, animals. These results indicate that PKC represents an early transduction step in the contractile response to NE in the rat abdominal aorta. That inhibition of the step immediately before activation of MAPK reduced contraction in both C and HU tissues, while general tyrosine kinase inhibition with genistein blocked only the control responses, suggests that a nonreceptor tyrosine kinase may be involved in HU-induced vascular hyporesponsiveness to NE.

  11. Characterization of the transduction pathway involved in c-fos and c-jun expression induced by Aggregatibacter actinomycetemcomitans lipopolysaccharides in human gingival fibroblasts.

    PubMed

    Gutiérrez-Venegas, Gloria; Castillo-Alemán, Ramiro

    2008-11-01

    Periodontal disease is an inflammatory disease caused by infection with oral bacteria that results in tooth exfoliation. Lipopolysaccharides (LPS) are a major component of the outer membrane of Gram-negative microorganisms and are involved in the inflammatory response. c-fos and c-jun are involved in pathological conditions such as inflammatory disorders. Inflammatory signaling cascades leading to c-fos activation in human gingival fibroblasts (HGFs) are not well characterized. Thus, we have investigated the kinase pathways involved in c-fos and c-jun activation induced by LPS in human gingival fibroblasts. LPS promoted a dose- and time-dependent increase in c-fos transcription. Phosphoinositide-phospholipase C inhibitor (U-73122), protein kinase A inhibitor (H89), MEK1 inhibitor (PD 98,059), p38 inhibitor (SB203580), and tyrosine kinase inhibitors (genistein and herbimycin) attenuated the LPS effect, while the PI-3 K inhibitor (Wortmannin) had no effect on LPS-induced c-fos transcription. Protein kinase C inhibitors (Ro 31-8220, calphostin C, staurosporine, and chelerythrine chloride) also inhibited LPS-induced c-fos transcription. However, long-term treatment (24 -h) with the PKC activator tetradecanoyl phorbol-13-acetate (PMA) had no significant effect on LPS-induced transcription in HGFs. We also found an increase in c-jun expression in HGF stimulated with LPS. In addition, experiments using pharmacological inhibitors of individual mitogen-activated protein kinases (MAPK) and of protein kinase C (PKC) revealed that p38, ERK 1/2, and PKC are involved in LPS-induced c-jun expression. Our results indicate that LPS-induced c-fos and c-jun expressions are mediated by two different signaling pathways: one through phosphoinositide-phospholipase C via an upstream protein tyrosine kinase, which activates PKC isoforms that are insensitive to phorbol stress, and the second through activation of protein kinase A (PKA). Both kinases regulate the phosphorylation of the

  12. H-7, a protein kinase C inhibitor, inhibits spontaneous tone and spasmogenic responses in normal and sensitized guinea pig trachea.

    PubMed

    de Diego, A; Cortijo, J; Villagrasa, V; Perpina, M; Morcillo, E J

    1995-12-01

    1. H-7, a protein kinase C inhibitor, fully inhibited the spontaneous and stimulated (KCl 20 mM or histamine 0.5 mM) tone of trachea from normal and sensitized guinea pig. 2. H-7 depressed the concentration-contraction curves to KCl, histamine or 5-hydroxytryptamine in epithelium-denuded, indomethacin-treated, trachea from normal and sensitized guinea pigs while responses to CaCl2 (in Ca2+ -free, K+ -depolarized tissues) and acetylcholine were not affected. 3. H-7 (100 microM did not depress Ca2+ (20 microM-induced contraction of Triton X-100 skinned trachea. 4. These results suggest the involvement of PKC in the maintenance of spontaneous tone and spasmogenic responses of guinea pig trachea.

  13. Prevention of Remifentanil Induced Postoperative Hyperalgesia by Dexmedetomidine via Regulating the Trafficking and Function of Spinal NMDA Receptors as well as PKC and CaMKII Level In Vivo and In Vitro

    PubMed Central

    Chen, Yi; Zheng, Yuxin; Xie, Ke-liang; He, Ying; Wang, Zhifen; Wang, Guo-lin; Yu, Yong-hao

    2017-01-01

    Remifentanil-induced secondary hyperalgesia has been demonstrated in both animal experiments and clinical trials. Enhancement of N-methyl-D-aspartate (NMDA) receptor trafficking as well as protein kinase C (PKC) and calmodulin-dependent protein kinase II (CaMKII) have been reported to be involved in the induction and maintenance of central sensitization. In the current study, it was demonstrated that dexmedetomidine could prevent remifentanil-induced hyperalgesia (RIH) via regulating spinal NMDAR-PKC-Ca2+/ CaMKII pathway in vivo and in vitro. We firstly investigated the effect of dexmedetomidine, a highly selective α2-adrenergic receptor agonist, on mechanical and thermal hyperalgesia using a rat model of RIH. NMDA receptor subunits (NR1, NR2A and NR2B) expression and membrane trafficking as well as PKC and CaMKII expression in spinal cord L4–L5 segments were measured by Western blot analysis. The expression of NMDA receptor subunits (NR1, NR2A and NR2B) were also detected by immunohistochemistry. Further more, the effect of dexmedetomidine on NMDA receptor current amplitude and frequency in spinal cord slices were investigated by whole-cell patch-clamp recording. We found that remifentail infusion at 1.2 μg.kg−1.min−1 for 90 min caused mechanical and thermal hyperalgesia, up-regulated NMDA receptor subunits NR1 and NR2B expression in both membrane fraction and total lysate as well as increased PKC and CaMKII expression in spinal cord dorsal horn. Subcutaneously injection of dexmedetomidine at the dose of 50 μg/kg at 30 min before plantar incision significantly attenuated remifentanil-induced mechanical and thermal hyperalgesia from 2 h to 48 h after infusion, and this was associated with reversal of up-regulated NR1 and NR2B subunits in both membrane fraction and total lysate as well as increased PKC and CaMKII expression in spinal cord dorsal horn. Furthermore, remifentanil incubation increased amplitude and frequency of NMDA receptor-induced current in

  14. Thrombin inhibitor design.

    PubMed

    Sanderson, P E; Naylor-Olsen, A M

    1998-08-01

    Recently, iv formulated direct thrombin inhibitors have been shown to be safe and efficacious alternatives to heparin. These results have fueled the hopes for an orally active compound. Such a compound could be a significant advance over warfarin if it had predictable pharmacokinetics and a duration of action sufficient for once or twice a day dosing. In order to develop an orally active compound which meets these criteria, the deficiencies of the prototype inhibitor efegatran have had to be addressed. First, using a combination of structure based design and empirical structure optimization, more selective compounds have been identified by modifying the P1 group or by incorporating different peptidomimetic P2/P3 scaffolds. Secondly, this optimization has resulted in the development of potent and selective non-covalent inhibitors, thus bypassing the liabilities of the serine trap. Thirdly, oral bioavailability has been achieved while maintaining selectivity and efficacy through the incorporation of progressively less basic P1 groups. The duration of action of these compounds remains to be optimized. Other advances in thrombin inhibitor design have included the development of uncharged P1 groups and the discovery of two non-peptide templates.

  15. MAG-EPA and 17,18-EpETE target cytoplasmic signalling pathways to reduce short-term airway hyperresponsiveness.

    PubMed

    Khaddaj-Mallat, Rayan; Rousseau, Éric

    2015-07-01

    This study was aimed to investigate the role of eicosapentaenoic acid monoacylglyceride (MAG-EPA) and 17,18-epoxyeicosatetraenoic acid (17,18-EpETE) on the regulation of contractile reactivity and nuclear protein expression in 72-h-cultured and TNF-α-treated guinea pig tracheal rings. Tension measurements performed on native tissues demonstrated that the cytochrome P-450 epoxygenase (CYP450)-dependent EPA metabolite, 17,18-EpETE, displayed a higher potency than MAG-EPA in inhibiting U-46619-induced tone. Calphostin C (a PKC inhibitor), whether in association or not with MAG-EPA or 17,18-EpETE, had no further effect, while 17,18-EpETE and Y-27632 (a Rho kinase inhibitor) yielded additive effects. Of note, MAG-EPA and 17,18-EpETE pre-treatments normalized the contractile responses to broncho-constrictive agents in 72-h-cultured trachea. The enhanced expression of TNF-α, P-p65-nuclear factor kappaB (NF)-κB, c-fos and c-Jun in 72-h-cultured tissues likely contributed to the hyperresponsiveness. β-Escin-permeabilized preparations demonstrated that 17,18-EpETE abolished Ca(2+) hypersensitivity, suggesting a blunting of PKC and/or Rho kinase activation. Lastly, activation of NF-κB and activating protein-1 (AP-1) signalling by exogenous TNF-α markedly increased the contractile response to MCh, through an increase in 17-kDa PKC-potentiated inhibitory protein of PP1 (CPI-17) phosphorylation and IκBα degradation. Dual incubation of 17,18-EpETE with calphostin C or Y-27632 induced cumulative inhibitory effects on MCh responses in TNF-α-incubated tracheal rings. 17,18-EpETE also reduced the detection level of P-p65-NF-κB and AP-1 subunits. The present data provide evidence that MAG-EPA, through its bioactive metabolite, represents a prospective pharmacological target in respiratory diseases.

  16. Evidence for the involvement of GPR40 and NADPH oxidase in palmitic acid-induced superoxide production and insulin secretion.

    PubMed

    Graciano, Maria Fernanda; Valle, Maíra Mello; Curi, Rui; Carpinelli, Angelo Rafael

    2013-01-01

    G protein coupled receptor 40 (GPR40) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex have been shown to be involved in the fatty acid amplification of glucose-stimulated insulin secretion (GSIS). The effect of palmitic acid on superoxide production and insulin secretion by INS-1E cells and the possible involvement of GPR40 and NADPH oxidase in these processes were examined in this study. Cells were incubated during 1 h with palmitic acid in low and high glucose concentrations, a GPR40 agonist (GW9508) and inhibitors of NADPH oxidase (diphenyleneiodonium, DPI) and PKC (calphostin C). GW9508 induced superoxide production at 2.8 and 5.6 mM glucose concentrations and stimulated insulin secretion at 16.7 mM glucose concentration involving both PKC and NADPH oxidase activation. Palmitic acid induced superoxide production through NADPH oxidase and GPR40-dependent pathways and the stimulation of insulin secretion in the presence of a high glucose concentration was reduced by knockdown of GPR40 using siRNA. Our results suggest that palmitic acid induces superoxide production and potentiates GSIS through NADPH oxidase and GPR40 pathways in pancreatic ? cells.

  17. Domain-specific phosphomimetic mutation allows dissection of different protein kinase C (PKC) isotype-triggered activities of the RNA binding protein HuR.

    PubMed

    Schulz, Sebastian; Doller, Anke; Pendini, Nicole R; Wilce, Jacqueline A; Pfeilschifter, Josef; Eberhardt, Wolfgang

    2013-12-01

    The ubiquitous mRNA binding protein human antigen R (HuR) participates in the post-transcriptional regulation of many AU-rich element (ARE)-bearing mRNAs. Previously, by using in vitro kinase assay, we have identified serines (Ser) 158, 221 and 318 as targets of protein kinase C (PKC)-triggered phosphorylation. In this study, we tested whether GFP- or GST-tagged HuR constructs bearing a phosphomimetic Ser (S)-to-Asp (D) substitution at the different PKC target sites, would affect different HuR functions including HuR nucleo-cytoplasmic redistribution and binding to different types of ARE-containing mRNAs. The phosphomimetic GFP-tagged HuR protein bearing a phosphomimetic substitution in the hinge region of HuR (HuR-S221D) showed an increased cytoplasmic abundance when compared to wild-type HuR. Conversely, data from in vitro kinase assay and electrophoretic mobility shift assay (EMSA), implicates that phosphorylation at Ser 221 is not relevant for mRNA binding of HuR. Quantification of in vitro binding affinities of GST-tagged wild-type HuR and corresponding HuR proteins bearing a phosphomimetic substitution in either RRM2 (HuR-S158D) or in RRM3 (HuR-S318D) by microscale thermophoresis (MST) indicates a specific binding of wild-type HuR to type I, II or type III-ARE-oligonucleotides in the high nanomolar range. Interestingly, phosphomimetic mutation at position 158 or 318 had a negative influence on HuR binding to type I- and type II-ARE-mRNAs whereas it significantly enhanced HuR affinity to a type III-ARE substrate. Our data suggest that differential phosphorylation of HuR by PKCs at different HuR domains coordinates subcellular HuR distribution and leads to a preferential binding to U-rich bearing target mRNA.

  18. Acyclic peptide inhibitors of amylases.

    PubMed

    Pohl, Nicola

    2005-12-01

    In this issue of Chemistry and Biology, a library screening approach reveals a linear octapeptide inhibitor of alpha-amylases reached by de novo design . The selected molecule shares characteristics with naturally occurring protein inhibitors -- a result that suggests general rules for the design of peptide-based amylase inhibitors may be achievable.

  19. Toxic effects of TiO2 nanoparticles in primary cultured rat sertoli cells are mediated via a dysregulated Ca(2+) /PKC/p38 MAPK/NF-κB cascade.

    PubMed

    Ye, Lingqun; Hong, Fashui; Ze, Xiao; Li, Lingjuan; Zhou, Yaoming; Ze, Yuguan

    2017-02-11

    Although numerous studies have demonstrated that titanium dioxide nanoparticles (TiO2 NPs) can be accumulated in various animal organs and can cause toxicity, there is currently only limited data regarding reproductive toxicity especially on the toxic mechanisms of TiO2 NPs in Sertoli cells. In order to investigate the mechanism of reproductive toxicity, primary cultured rat Sertoli cells were exposed to 5, 15, or 30 μg/mL TiO2 NPs for 24 h, and TiO2 NPs internalization, expression of PKC (p-PKC) and p38 MAPK (p-p38 MAPK) as well as calcium homeostasis were examined. Our findings demonstrated that TiO2 NPs crossed the membrane into the cytoplasm or nucleus, and significantly suppressed cell viability of primary cultured rat Sertoli cells in a concentration-dependent manner. Furthermore, immunological dysfunction caused by TiO2 NPs was involved in the increased expression of NF-κB, TNF-α, and IL-1β, and decreased IκB expression. TiO2 NPs significantly decreased Ca(2+) -ATPase and Ca(2+) /Mg(2+) -ATPase activity and enhanced intracellular Ca(2+) levels, and up-regulated the expression of p-PKC and p-p38 MAPK in a dose-dependent manner in primary cultured rat Sertoli cells. Taken together, these findings indicate that TiO2 NPs may induce immunological dysfunction of primary cultured rat Sertoli cells by stimulating the Ca(2+) /PKC/p38 MAPK cascade, which triggers NF-κB activation and ultimately induces the expression of inflammatory cytokines in primary cultured rat Sertoli cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2017.

  20. [SGLT2 inhibitor].

    PubMed

    Kubota, Naoto; Kadowaki, Takashi

    2015-12-01

    SGLT2 is a glucose transporter which plays an important role for reabsorption of urinary glucose depending on the sodium concentration gradient. SGLT2 is mainly present in apical site of S1 segment of renal proximal tubule and accounts for approximately 90% of total urinary glucose reabsorption. SLC5a2, which codes SGLT2, is also known as the causative gene of familial renal glucosuria. SGLT2 inhibitors are attracting attention as newly developed oral anti-diabetic agents which improve glucose intolerance and also have an anti-obese effect by promoting urinary glucose excretion (UGE), which is a different pharmacological effect from other conventional anti-diabetic agents. In this review, we will discuss the effect of SGLT2 inhibitor on the regulation of glucose and lipid metabolism in type 2 diabetes.

  1. Development of scale inhibitors

    SciTech Connect

    Gill, J.S.

    1996-12-01

    During the last fifty years, scale inhibition has gone from an art to a science. Scale inhibition has changed from simple pH adjustment to the use of optimized dose of designer polymers from multiple monomers. The water-treatment industry faces many challenges due to the need to conserve water, availability of only low quality water, increasing environmental regulations of the water discharge, and concern for human safety when using acid. Natural materials such as starch, lignin, tannin, etc., have been replaced with hydrolytically stable organic phosphates and synthetic polymers. Most progress in scale inhibition has come from the use of synergistic mixtures and copolymerizing different functionalities to achieve specific goals. Development of scale inhibitors requires an understanding of the mechanism of crystal growth and its inhibition. This paper discusses the historic perspective of scale inhibition and the development of new inhibitors based on the understanding of the mechanism of crystal growth and the use of powerful tools like molecular modeling to visualize crystal-inhibitor interactions.

  2. Parathyroid hormone regulates osterix and Runx2 mRNA expression predominantly through protein kinase A signaling in osteoblast-like cells.

    PubMed

    Wang, B L; Dai, C L; Quan, J X; Zhu, Z F; Zheng, F; Zhang, H X; Guo, S Y; Guo, G; Zhang, J Y; Qiu, M C

    2006-02-01

    Runt-related transcription factor 2 (Runx2) and osterix are osteoblast-specific transcription factors essential for the development of osteoblastic cells and bone formation. PTH given intermittently has anabolic effects on bone; however, the exact role remains to be understood completely. The purpose of this study was both to investigate whether PTH regulates Runx2 as well as osterix expression and to identify the signaling used. Using RT-PCR, we confirmed that PTH (1-34) regulated Runx2 and osterix mRNA expression, in rat osteoblast-like cell line UMR 106, in a dose- and time-dependent manner. PTH in low concentrations stimulated both Runx2 and osterix mRNA expression while that in high concentrations did not. Forskolin, an adenylate cyclase activator, also enhanced Runx2 and osterix transcription, and the stimulatory effects of PTH and forskolin were blocked by the pre-treatment of the cells with H-89, a protein kinase A (PKA) inhibitor. In contrast, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) had no effect on Runx2 transcription, but induced an increase in osterix mRNA level at the concentration of 500 nM at 12 h after treatment. Moreover, pre-treatment of the cells with calphostin C, a PKC-specific inhibitor, reduced the increase in osterix transcripts enhanced by PTH and PMA 12 h after treatment. However, these inhibitory effects were not sustained for longer terms. These observations demonstrate that PTH stimulates Runx2 and osterix expression in vitro, at least in part, at transcriptional level. Induction of Runx2 mRNA is mediated through the activation of cAMP/PKA signal transduction. In the case of osterix, although the increase in mRNA level is predominantly mediated via cAMP/PKA signaling, PKC activation might also be involved in this process.

  3. Kisspeptin inhibits a slow afterhyperpolarization current via protein kinase C and reduces spike frequency adaptation in GnRH neurons

    PubMed Central

    Zhang, Chunguang

    2013-01-01

    Kisspeptin signaling via its cognate receptor G protein-coupled receptor 54 (GPR54) in gonadotropin-releasing hormone (GnRH) neurons plays a critical role in regulating pituitary secretion of luteinizing hormone and thus reproductive function. GPR54 is Gq-coupled to activation of phospholipase C and multiple second messenger signaling pathways. Previous studies have shown that kisspeptin potently depolarizes GnRH neurons through the activation of canonical transient receptor potential channels and inhibition of inwardly rectifying K+ channels to generate sustained firing. Since the initial studies showing that kisspeptin has prolonged effects, the question has been why is there very little spike frequency adaption during sustained firing? Presently, we have discovered that kisspeptin reduces spike frequency adaptation and prolongs firing via the inhibition of a calcium-activated slow afterhyperpolarization current (IsAHP). GnRH neurons expressed two distinct IsAHP, a kisspeptin-sensitive and an apamin-sensitive IsAHP. Essentially, kisspeptin inhibited 50% of the IsAHP and apamin inhibited the other 50% of the current. Furthermore, the kisspeptin-mediated inhibition of IsAHP was abrogated by the protein kinase C (PKC) inhibitor calphostin C, and the PKC activator phorbol 12,13-dibutyrate mimicked and occluded any further effects of kisspeptin on IsAHP. The protein kinase A (PKA) inhibitors H-89 and the Rp diastereomer of adenosine 3′,5′-cyclic monophosphorothioate had no effect on the kisspeptin-mediated inhibition but were able to abrogate the inhibitory effects of forskolin on the IsAHP, suggesting that PKA is not involved. Therefore, in addition to increasing the firing rate through an overt depolarization, kisspeptin can also facilitate sustained firing through inhibiting an apamin-insensitive IsAHP in GnRH neurons via a PKC. PMID:23548613

  4. Effect of Hyp delivery system on PKCα activity: What will happen after pkcα gene silencing and Hyp photo-activation?

    NASA Astrophysics Data System (ADS)

    Misuth, Matus; Joniova, Jaroslava; Ferencakova, Michaela; Miskovsky, Pavol; Nadova, Zuzana

    2015-08-01

    Low density lipoproteins (LDL) are considered as suitable natural in vivo delivery system for hydrophobic photosensitizers (pts) such as hypericin (Hyp) and it was shown that over expression of LDL-receptors in tumor cells can be used for specific targeting. Activation of pts by irradiation results in a formation of reactive oxygen species (ROS) at the place of light application and starts destructive mechanism. PKCα plays a key role in the cell survival and its overexpression was observed in glioma cell lines. In the present study we aim to present the effectivity of the pts delivery in the glioma cells and consequences of silencing pkcα gene on cell death/survival after Hyp photo-activation. Pts can be delivered through two pathways: endocytosis - when cells are incubated with LDL/Hyp complex and Hyp transport through cellular membrane without any carrier. Preliminary results show that incubation of cells with or without LDL leads to PKCα activation. Photo-activated Hyp seems to be more effective in terms of apoptosis induction when compared to photo-activated LDL/Hyp complex. We have evaluated the influence of photo-activated Hyp on cell death in non-transfected and transfected (PKCα-) human glioma cells (U87-MG). Level of ROS production and type of cell death was notably affected by silencing pkca gene resulting in significant increase of necrosis after Hyp photo-activation.

  5. β-arrestin-1 mediates the TCR-triggered re-routing of distal receptors to the immunological synapse by a PKC-mediated mechanism

    PubMed Central

    Fernández-Arenas, Elena; Calleja, Enrique; Martínez-Martín, Nadia; Gharbi, Severine I; Navajas, Rosana; García-Medel, Noel; Penela, Petronila; Alcamí, Antonio; Mayor, Federico; Albar, Juan P; Alarcón, Balbino

    2014-01-01

    T-cell receptors (TCR) recognize their antigen ligand at the interface between T cells and antigen-presenting cells, known as the immunological synapse (IS). The IS provides a means of sustaining the TCR signal which requires the continual supply of new TCRs. These are endocytosed and redirected from distal membrane locations to the IS. In our search for novel cytoplasmic effectors, we have identified β-arrestin-1 as a ligand of non-phosphorylated resting TCRs. Using dominant-negative and knockdown approaches we demonstrate that β-arrestin-1 is required for the internalization and downregulation of non-engaged bystander TCRs. Furthermore, TCR triggering provokes the β-arrestin-1-mediated downregulation of the G-protein coupled chemokine receptor CXCR4, but not of other control receptors. We demonstrate that β-arrestin-1 recruitment to the TCR, and bystander TCR and CXCR4 downregulation, are mechanistically mediated by the TCR-triggered PKC-mediated phosphorylation of β-arrestin-1 at Ser163. This mechanism allows the first triggered TCRs to deliver a stop migration signal, and to promote the internalization of distal TCRs and CXCR4 and their translocation to the IS. This receptor crosstalk mechanism is critical to sustain the TCR signal. PMID:24502978

  6. Def-6, a novel regulator of small GTPases in podocytes, acts downstream of atypical protein kinase C (aPKC) λ/ι.

    PubMed

    Worthmann, Kirstin; Leitges, Michael; Teng, Beina; Sestu, Marcello; Tossidou, Irini; Samson, Thomas; Haller, Hermann; Huber, Tobias B; Schiffer, Mario

    2013-12-01

    The atypical protein kinase C (aPKC) isotypes PKCλ/ι and PKCζ are both expressed in podocytes; however, little is known about differences in their function. Previous studies in mice have demonstrated that podocyte-specific loss of PKCλ/ι leads to a severe glomerular phenotype, whereas mice deficient in PKCζ develop no renal phenotype. We analyzed various effects caused by PKCλ/ι and PKCζ deficiency in cultured murine podocytes. In contrast to PKCζ-deficient podocytes, PKCλ/ι-deficient podocytes exhibited a severe actin cytoskeletal phenotype, reduced cell size, decreased number of focal adhesions, and increased activation of small GTPases. Comparative microarray analysis revealed that the guanine nucleotide exchange factor Def-6 was specifically up-regulated in PKCλ/ι-deficient podocytes. In vivo Def-6 expression is significantly increased in podocytes of PKCλ/ι-deficient mice. Cultured PKCλ/ι-deficient podocytes exhibited an enhanced membrane association of Def-6, indicating enhanced activation. Overexpression of aPKCλ/ι in PKCλ/ι-deficient podocytes could reduce the membrane-associated expression of Def-6 and rescue the actin phenotype. In the present study, PKCλ/ι was identified as an important factor for actin cytoskeletal regulation in podocytes and Def-6 as a specific downstream target of PKCλ/ι that regulates the activity of small GTPases and subsequently the actin cytoskeleton of podocytes.

  7. BRO1, a novel gene that interacts with components of the Pkc1p-mitogen-activated protein kinase pathway in Saccharomyces cerevisiae.

    PubMed Central

    Nickas, M E; Yaffe, M P

    1996-01-01

    Yeast cells with mutations in BRO1 display phenotypes similar to those caused by deletion of BCK1, a gene encoding a MEK kinase that functions in a mitogen-activated protein kinase pathway mediating maintenance of cell integrity. bro1 cells exhibit a temperature-sensitive growth defect that is suppressed by the addition of osmotic stabilizers or Ca2+ to the growth medium or by additional copies of the BCK1 gene. At permissive temperatures, bro1 mutants are sensitive to caffeine and respond abnormally to nutrient limitation. A null mutation in BRO1 is synthetically lethal with null mutations in BCK1, MPK1, which encodes a mitogen-activated protein kinase that functions downstream of Bck1p, or PKC1, a gene encoding a protein kinase C homolog that activates Bck1p. Analysis of the isolated BRO1 gene revealed that it encodes a novel, 97-kDa polypeptide which contains a putative SH3 domain-binding motif and is homologous to a protein of unknown function in Caenorhabditis elegans. PMID:8649366

  8. The aPKC/Par3/Par6 Polarity Complex and Membrane Order Are Functionally Interdependent in Epithelia During Vertebrate Organogenesis.

    PubMed

    Abu-Siniyeh, Ahmed; Owen, Dylan M; Benzing, Carola; Rinkwitz, Silke; Becker, Thomas S; Majumdar, Arindam; Gaus, Katharina

    2016-01-01

    The differential distribution of lipids between apical and basolateral membranes is necessary for many epithelial cell functions, but how this characteristic membrane organization is integrated within the polarity network during ductal organ development is poorly understood. Here we quantified membrane order in the gut, kidney and liver ductal epithelia in zebrafish larvae at 3-11 days post fertilization (dpf) with Laurdan 2-photon microscopy. We then applied a combination of Laurdan imaging, antisense knock-down and analysis of polarity markers to understand the relationship between membrane order and apical-basal polarity. We found a reciprocal relationship between membrane order and the cell polarity network. Reducing membrane condensation by exogenously added oxysterol or depletion of cholesterol reduced apical targeting of the polarity protein, aPKC. Conversely, using morpholino knock down in zebrafish, we found that membrane order was dependent upon the Crb3 and Par3 polarity protein expression in ductal epithelia. Hence our data suggest that the biophysical property of membrane lipid packing is a regulatory element in apical basal polarity.

  9. The anticoagulant effect of PGI2S and tPA in transgenic umbilical vein endothelial cells is linked to up-regulation of PKA and PKC.

    PubMed

    Wang, Jian-Hua; Yuan, Lin-Jing; Zhong, Zhi-Min; Wen, Zhe-Sheng; Deng, Jian-Ming; Liang, Rong-Xin; Zheng, Min

    2014-02-19

    The selection of vascular grafts for coronary artery bypass surgery is crucial for a positive outcome. This study aimed to establish a novel line of vascular endothelial cells with a potent anticoagulant effect. A lentiviral vector was used to stably transfect human umbilical vein endothelial cells (HUVECs) with PGI2S alone (HUVEC-PGI2S) or both PGI2S and tPA (HUVEC-PGI2S-tPA). Both HUVEC-PGI2S and HUVEC-PGI2S-tPA cells over-expressing PGI2S and tPA were compared to mock-transfected cells. The enzyme-linked immuno sorbent assay (ELISAs) demonstrated that the anticoagulation components, ATIII and PLG, were up-regulated and coagulation factor FVIII was down-regulated in both cell lines. QRT-PCR and western blotting demonstrated the vasodilation and platelet disaggregation proteins PKA, PKC, and PTGIR were up-regulated in both cell lines, but MAPK expression was not altered in either cell line. However, cell viability and colony formation assays and cell cycle analysis demonstrated that both cell lines had a lower rate of cell growth and induced G1 phase arrest. HUVEC-PGI2S and HUVEC-PGI2S-tPA cells have a potent anticoagulant effect and their use in vascular heterografts may decrease the risk of thrombosis.

  10. Estrogenic compounds inhibit gap junctional intercellular communication in mouse Leydig TM3 cells

    SciTech Connect

    Iwase, Yumiko . E-mail: Iwase.Yumiko@mg.m-pharma.co.jp; Fukata, Hideki . E-mail: fukata@faculty.chiba-u.jp; Mori, Chisato . E-mail: cmori@faculty.chiba-u.jp

    2006-05-01

    Some estrogenic compounds are reported to cause testicular disorders in humans and/or experimental animals by direct action on Leydig cells. In carcinogenesis and normal development, gap junctional intercellular communication (GJIC) plays an essential role in maintaining homeostasis. In this study, we examine the effects of diethylstilbestrol (DES, a synthetic estrogen), 17{beta}-estradiol (E{sub 2}, a natural estrogen), and genistein (GEN, a phytoestrogen) on GJIC between mouse Leydig TM3 cells using Lucifer yellow microinjection. The three compounds tested produced GJIC inhibition in the TM3 cells after 24 h. Gradually, 10 {mu}M DES began to inhibit GJIC for 24 h and this effect was observed until 72 h. On the other hand, both 20 {mu}M E{sub 2} and 25 {mu}M GEN rapidly inhibited GJIC in 6 h and 2 h, respectively. The effects continued until 24 h, but weakened by 72 h. Furthermore, a combined effect at {mu}M level between DES and E{sub 2} on GJIC inhibition was observed, but not between GEN and E{sub 2}. DES and E{sub 2} showed GJIC inhibition at low dose levels (nearly physiological estrogen levels) after 72 h, but GEN did not. DES-induced GJIC inhibition at 10 pM and 10 {mu}M was completely counteracted by ICI 182,780 (ICl), an estrogen receptor antagonist. On the other hand, the inhibitory effects on GJIC with E{sub 2} (10 pM and 20 {mu}M) and GEN (25 {mu}M) were partially blocked by ICI or calphostin C, a protein kinase C (PKC) inhibitor, and were completely blocked by the combination of ICI and calphostin C. These results demonstrate that DES inhibits GJIC between Leydig cells via the estrogen receptor (ER), and that E{sub 2} and GEN inhibit GJIC via ER and PKC. These estrogenic compounds may have different individual nongenotoxic mechanism including PKC pathway on testicular carcinogenesis or development.

  11. [Tyrosine kinase inhibitors].

    PubMed

    Robert, Jacques

    2011-11-01

    Membrane receptors with tyrosine kinase activity and cytoplasmic tyrosine kinases have emerged as important potential targets in oncology. Starting from basic structures such as anilino-quinazoline, numerous compounds have been synthesised, with the help of tyrosine kinase crystallography, which has allowed to optimise protein-ligand interactions. The catalytic domains of all kinases present similar three-dimensional structures, which explains that it may be difficult to identify molecules having a high specificity for a given tyrosine kinase. Some tyrosine kinase inhibitors are relatively specific for epidermal growth factor receptor (EGFR) such as géfitinib and erlotinib; other are mainly active against platelet-derived growth factor receptor (PDGFR) and the receptor KIT, such as imatinib or nilotinib, and other against vascular endothelial growth factor (VEGF) receptors involved in angiogenesis, such as sunitinib and sorafenib. The oral formulation of tyrosine kinase inhibitors is well accepted by the patients but may generate sometimes compliance problems requiring pharmacokinetic monitoring. This chemical family is in full expansion and several dozens of compounds have entered clinical trials.

  12. Cell cycle arrest or survival signaling through αv integrins, activation of PKC and ERK1/2 lead to anoikis resistance of ovarian cancer spheroids.

    PubMed

    Carduner, Ludovic; Picot, Cédric R; Leroy-Dudal, Johanne; Blay, Lyvia; Kellouche, Sabrina; Carreiras, Franck

    2014-01-15

    Ovarian cancer is the most lethal gynecologic cancer mainly due to spheroids organization of cancer cells that disseminate within the peritoneal cavity. We have investigated the molecular mechanisms by which ovarian cancer spheroids resist anoikis, choosing as models the 2 well-characterized human ovarian cancer cell lines IGROV1 and SKOV3. These cell lines have the propensity to float as clusters, and were isolated from tumor tissue and ascites, respectively. To form spheroids, IGROV1 and SKOV3 ovarian adenocarcinoma cells were maintained under anchorage-independent culture conditions, in which both lines survive at least a week. A short apoptotic period prior to a survival signaling commitment was observed for IGROV1 cells whereas SKOV3 cells entered G0/G1 phase of the cell cycle. This difference in behavior was due to different signals. With regard to SKOV3 cells, activation of p38 and an increase in p130/Rb occurred once anchorage-independent culture was established. Analyses of the survival signaling pathway switched on by IGROV1 cells showed that activation of ERK1/2 was required to evade apoptosis, an effect partly dependent on PKC activation and αv integrins. αv-integrin expression is essential for survival through activation of ERK1/2 phosphorylation. The above data indicate that ovarian cancer cells can resist anoikis in the spheroid state by arrest in the cell cycle or through activation of αv-integrin-ERK-mediated survival signals. Such signaling might result in the selection of resistant cells within disseminating spheroids, favoring further relapse in ovarian cancers.

  13. Synthesis of Lysine Methyltransferase Inhibitors

    NASA Astrophysics Data System (ADS)

    Ye, Tao; Hui, Chunngai

    2015-07-01

    Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

  14. Voltage-Independent Inhibition of the Tetrodotoxin-Sensitive Sodium Currents by Oxotremorine and Angiotensin II in Rat Sympathetic Neurons.

    PubMed

    Puente, Erika I; De la Cruz, Lizbeth; Arenas, Isabel; Elias-Viñas, David; Garcia, David E

    2016-04-01

    Tetrodotoxin-sensitive Na(+) currents have been extensively studied because they play a major role in neuronal firing and bursting. In this study, we showed that voltage-dependent Na(+) currents are regulated in a slow manner by oxotremorine (oxo-M) and angiotensin II in rat sympathetic neurons. We found that these currents can be readily inhibited through a signaling pathway mediated by G proteins and phospholipase C (PLC) β1. This inhibition is slowly established, pertussis toxin-insensitive, partially reversed within tens of seconds after oxo-M washout, and not relieved by a strong depolarization, suggesting a voltage-insensitive mechanism of inhibition. Specificity of the M1 receptor was tested by the MT-7 toxin. Activation and inactivation curves showed no shift in the voltage dependency under the inhibition by oxo-M. This inhibition is blocked by a PLC inhibitor (U73122, 1-(6-{[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione), and recovery from inhibition is prevented by wortmannin, a PI3/4 kinase inhibitor. Hence, the pathway involves Gq/11 and is mediated by a diffusible second messenger. Oxo-M inhibition is occluded by screening phosphatidylinositol 4,5-bisphosphate (PIP2)-negative charges with poly-l-lysine and prevented by intracellular dialysis with a PIP2 analog. In addition, bisindolylmaleimide I, a specific ATP-competitive protein kinase C (PKC) inhibitor, rules out that this inhibition may be mediated by this protein kinase. Furthermore, oxo-M-induced suppression of Na(+) currents remains unchanged when neurons are treated with calphostin C, a PKC inhibitor that targets the diacylglycerol-binding site of the kinase. These results support a general mechanism of Na(+) current inhibition that is widely present in excitable cells through modulation of ion channels by specific G protein-coupled receptors.

  15. Sequencing of aromatase inhibitors

    PubMed Central

    Bertelli, G

    2005-01-01

    Since the development of the third-generation aromatase inhibitors (AIs), anastrozole, letrozole and exemestane, these agents have been the subject of intensive research to determine their optimal use in advanced breast cancer. Not only have they replaced progestins in second-line therapy and challenged the role of tamoxifen in first-line, but there is also evidence for a lack of cross-resistance between the steroidal and nonsteroidal AIs, meaning that they may be used in sequence to obtain prolonged clinical benefit. Many questions remain, however, as to the best sequence of the two types of AIs and of the other available agents, including tamoxifen and fulvestrant, in different patient groups. PMID:16100523

  16. Sirtuin activators and inhibitors

    PubMed Central

    Villalba, José M.; Alcaín, Francisco J.

    2012-01-01

    Sirtuins 1-7 (SIRT1-7) belong to the third class of deacetylase enzymes, which are dependent on NAD+ for activity. Sirtuins activity is linked to gene repression, metabolic control, apoptosis and cell survival, DNA repair, development, inflammation, neuroprotection and healthy aging. Because sirtuins modulation could have beneficial effects on human diseases there is a growing interest in the discovery of small molecules modifying their activity. We review here those compounds known to activate or inhibit sirtuins, discussing the data that support the use of sirtuin-based therapies. Almost all sirtuin activators have been described only for SIRT1. Resveratrol is a natural compound which activates SIRT1, and may help in the treatment or prevention of obesity, and in preventing tumorigenesis and the aging-related decline in heart function and neuronal loss. Due to its poor bioavailability, reformulated versions of resveratrol with improved bioavailability have been developed (resVida, Longevinex®, SRT501). Molecules that are structurally unrelated to resveratrol (SRT1720, SRT2104, SRT2379, among others) have been also developed to stimulate sirtuin activities more potently than resveratrol. Sirtuin inhibitors with a wide range of core structures have been identified for SIRT1, SIRT2, SIRT3 and SIRT5 (splitomicin, sirtinol, AGK2, cambinol, suramin, tenovin, salermide, among others). SIRT1 inhibition has been proposed in the treatment of cancer, immunodeficiency virus infections, Fragile X mental retardation syndrome and for preventing or treating parasitic diseases, whereas SIRT2 inhibitors might be useful for the treatment of cancer and neurodegenerative diseases. PMID:22730114

  17. Biological abatement of cellulase inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bio-abatement uses a fungus to metabolize and remove fermentation inhibitors. To determine whether bio-abatement could alleviate enzyme inhibitor effects observed in biomass liquors after pretreatment, corn stover at 10% (w/v) solids was pretreated with either dilute acid or liquid hot water. The ...

  18. Flow regulation of collecting duct endothelin-1 production.

    PubMed

    Lyon-Roberts, Brianna; Strait, Kevin A; van Peursem, Evan; Kittikulsuth, Wararat; Pollock, Jennifer S; Pollock, David M; Kohan, Donald E

    2011-03-01

    Collecting duct (CD) endothelin-1 (ET-1) is an important autocrine inhibitor of CD Na(+) reabsorption. Salt loading is thought to increase CD ET-1 production; however, definitive evidence of this, as well as understanding of the mechanisms transducing this effect, is lacking. Tubule fluid flow increases in response to Na(+) loading; hence, we studied flow modulation of CD ET-1 production. Three days of a high-salt diet increased mouse and rat inner medullary CD (IMCD) ET-1 mRNA expression. Acute furosemide infusion increased urinary ET-1 excretion in anesthetized rats. Primary cultures of mouse or rat IMCD detached in response to flow using a closed perfusion chamber, consequently a CD cell line (mpkCCDcl4) was examined. Flow increased ET-1 mRNA at shear stress rates exceeding 1 dyne/cm(2), with the maximal effect seen between 2 and 10 dyne/cm(2). Induction of ET-1 mRNA was first evident after 1 h, and most apparent after 2 h, of flow. Inhibition of calmodulin or dihydropyridine-sensitive Ca(2+) channels did not alter the flow response; however, chelation of intracellular Ca(2+) or removal of extracellular Ca(2+) largely prevented flow-stimulated ET-1 mRNA accumulation. Downregulation of protein kinase C (PKC) using phorbol 12-myristate 13-acetate, or PKC inhibition with calphostin C, markedly reduced flow-stimulated ET-1 mRNA levels. Flow-stimulated ET-1 mRNA accumulation was abolished by inhibition of phospholipase C (PLC). Taken together, these data indicate that flow increases CD ET-1 production and this is dependent on extracellular and intracellular Ca(2+), PKC, and PLC. These studies suggest a novel pathway for coupling alterations in extracellular fluid volume to CD ET-1 production and ultimately control of CD Na(+) reabsorption.

  19. Cellular pathways involved in the ex vivo expression of bovine leukemia virus.

    PubMed Central

    Kerkhofs, P; Adam, E; Droogmans, L; Portetelle, D; Mammerickx, M; Burny, A; Kettmann, R; Willems, L

    1996-01-01

    Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis. The virus adopts a strategy based on the lack of viral expression in vivo; only very rare BLV-infected B lymphocytes express viral information. When the cells are isolated from animals in persistent lymphocytosis and cultivated ex vivo, a tremendous increase in viral expression occurs. To gain insight into this mechanism, we employed a general approach using chemicals that interfere specifically with cellular pathways involved in signal transduction from the cell membrane to the nucleus. Our data demonstrate that BLV expression is not correlated with the activity of protein kinase A (PKA) and is even inhibited by cyclic AMP (cAMP). The cAMP/PKA pathway is thus apparently not involved in ex vivo viral expression. In contrast, PKC appears to play a key role in this process. Phorbol myristate acetate can directly activate viral expression in B cells (in the absence of T cells). Furthermore, calphostin C, a highly specific inhibitor of PKC, partly decreases ex vivo BLV expression. Our data further demonstrate that calmodulin and calcineurin, a calmodulin-dependent phosphatase, play a key role in the induction of viral expression. The involvement of this calmodulin-dependent pathway could explain the induction of expression that cannot be assigned to PKC. Furthermore, it appears that the activation of viral expression requires a calmodulin but not a PKA-dependent pathway. These data highlight major differences between transient transfection and ex vivo experiments. Finally, despite their homologies, BLV and human T-cell leukemia virus appear to use different signal transduction pathways to induce viral expression. PMID:8642639

  20. Authentic HIV-1 integrase inhibitors

    PubMed Central

    Liao, Chenzhong; Marchand, Christophe; Burke, Terrence R; Pommier, Yves; Nicklaus, Marc C

    2010-01-01

    HIV-1 integrase (IN) is indispensable for HIV-1 replication and has become a validated target for developing anti-AIDS agents. In two decades of development of IN inhibition-based anti-HIV therapeutics, a significant number of compounds were identified as IN inhibitors, but only some of them showed antiviral activity. This article reviews a number of patented HIV-1 IN inhibitors, especially those that possess high selectivity for the strand transfer reaction. These compounds generally have a polar coplanar moiety, which is assumed to chelate two magnesium ions in the binding site. Resistance to those compounds, when given to patients, can develop as a result of IN mutations. We refer to those compounds as authentic IN inhibitors. Continued drug development has so far delivered one authentic IN inhibitor to the market (raltegravir in 2007). Current and future attention will be focused on the development of novel authentic IN inhibitors with the goal of overcoming viral resistance. PMID:21426159

  1. A-Kinase Anchoring Protein 4 (AKAP4) is an ERK1/2 substrate and a switch molecule between cAMP/PKA and PKC/ERK1/2 in human spermatozoa

    PubMed Central

    Rahamim Ben-Navi, Liat; Almog, Tal; Yao, Zhong; Seger, Rony; Naor, Zvi

    2016-01-01

    Mammalian spermatozoa undergo capacitation and acrosome reaction in order to fertilize the egg. The PKC-ERK1/2 pathway plays an important role in human spermatozoa motility, capacitation and the acrosome reaction. Here we demonstrate that ERK1/2 phosphorylates proAKAP4 on Thr265 in human spermatozoa in vitro and in vivo. Cyclic AMP (cAMP) had no effect on ERK1/2 activity in human spermatozoa, but stimulated the MAPK in mouse pituitary LβT2 gonadotrope cells. cAMP via PKA attenuates PKC-dependent ERK1/2 activation only in the presence of proAKAP4. St-HT31, which disrupts PKA-regulatory subunit II (PKA-RII) binding to AKAP abrogates the inhibitory effect of cAMP in human spermatozoa and in HEK293T cells expressing proAKAP4. In transfected HEK293T cells, PMA relocated proAKAP4, but not proAKAP4-T265A to the Golgi in an ERK1/2-dependnet manner. Similarly, AKAP4 is localized to the spermatozoa principal piece and is relocated to the mid-piece and the postacrosomal region by PMA. Furthermore, using capacitated sperm we found that cAMP reduced PMA-induced ERK1/2 activation and acrosome reaction. Thus, the physiological role of the negative crosstalk between the cAMP/PKA/AKAP4 and the PKC/ERK1/2 pathways is to regulate capacitation and acrosome reaction. PMID:27901058

  2. Differential effect of 1{alpha},25-dihydroxyvitamin D{sub 3} on Hsp28 and PKC{beta} gene expression in the phorbol ester-resistant human myeloid HL-525 leukemic cells

    SciTech Connect

    Lee, Yong J.; Galoforo, S.S.; Berns, C.M.

    1997-08-01

    We investigated the effect of 1{alpha},25-dihydroxyvitamin D{sub 3} [1,25-(OH){sub 2}D{sub 3}] on the expression of the 28-kDa heat shock protein gene (hsp28) and the protein kinase C beta gene (PKC{beta}) in the human myeloid HL-60 leukemic cell variant HL-525, which is resistance to phorbol ester-induced macrophage differentiation. Northern and Western blot analysis showed little or no hsp28 gene expression in the HL-60 cell variant, HL-205, which is susceptible to such differentiation, while a relatively high basal level of hps28 gene expression was observed in the HL-525 cells. However, both cell lines demonstrated heat shock-induced expression of this gene. During treatment with 50-300 nM 1,25-(OH){sub 2}D{sub 3}, a marked reduction of hsp28 gene expression was not associated with heat shock transcription factor-heat shock element (HSF-HSE) binding activity. Our results suggest that the differential effect of 1,25-(OH){sub 2}D{sub 3} on hsp28 and PKC{beta} gene expression is due to the different sequence composition of the vitamin D response element in the in the promoter region as well as an accessory factor for each gene or that 1,25-(OH){sub 2}D{sub 3} increases PKC{beta} gene expression, which in turn negatively regulates the expression of the hsp28 gene, or vice versa.

  3. Anti-neuroinflammatory efficacy of the aldose reductase inhibitor FMHM via phospholipase C/protein kinase C-dependent NF-κB and MAPK pathways

    SciTech Connect

    Zeng, Ke-Wu; Li, Jun; Dong, Xin; Wang, Ying-Hong; Ma, Zhi-Zhong; Jiang, Yong; Jin, Hong-Wei; Tu, Peng-Fei

    2013-11-15

    Aldose reductase (AR) has a key role in several inflammatory diseases: diabetes, cancer and cardiovascular diseases. Therefore, AR inhibition seems to be a useful strategy for anti-inflammation therapy. In the central nervous system (CNS), microglial over-activation is considered to be a central event in neuroinflammation. However, the effects of AR inhibition in CNS inflammation and its underlying mechanism of action remain unknown. In the present study, we found that FMHM (a naturally derived AR inhibitor from the roots of Polygala tricornis Gagnep.) showed potent anti-neuroinflammatory effects in vivo and in vitro by inhibiting microglial activation and expression of inflammatory mediators. Mechanistic studies showed that FMHM suppressed the activity of AR-dependent phospholipase C/protein kinase C signaling, which further resulted in downstream inactivation of the IκB kinase/IκB/nuclear factor-kappa B (NF-κB) inflammatory pathway. Therefore, AR inhibition-dependent NF-κB inactivation negatively regulated the transcription and expression of various inflammatory genes. AR inhibition by FMHM exerted neuroprotective effects in lipopolysaccharide-induced neuron–microglia co-cultures. These findings suggested that AR is a potential target for neuroinflammation inhibition and that FMHM could be an effective agent for treating or preventing neuroinflammatory diseases. - Highlights: • FMHM is a natural-derived aldose reductase (AR) inhibitor. • FMHM inhibits various neuroinflammatory mediator productions in vitro and in vivo. • FMHM inhibits neuroinflammation via aldose reductase/PLC/PKC-dependent NF-κB pathway. • FMHM inhibits neuroinflammation via aldose reductase/PLC/PKC-dependent MAPK pathway. • FMHM protects neurons against inflammatory injury in microglia-neuron co-cultures.

  4. Shengmaisan Regulates Pacemaker Potentials in Interstitial Cells of Cajal in Mice

    PubMed Central

    Kim, Byung Joo

    2013-01-01

    Objectives: Shengmaisan (SMS) is a traditional Chinese medicine prescription widely used for the treatment of diverse organs in Korea. The interstitial cells of Cajal (ICCs) are pacemaker cells that play an important role in the generation of coordinated gastrointestinal (GI) motility. We have aimed to investigate the effects of SMS in the ICCs in the mouse small intestine. Methods: To dissociate the ICCs, we used enzymatic digestions from the small intestine in a mouse. After that, the ICCs were identified immunologically by using the anti-c-kit antibody. In the ICCs, the electrophysiological whole-cell patch-clamp configuration was used to record pacemaker potentials in the cultured ICCs. Results: The ICCs generated pacemaker potentials in the mouse small intestine. SMS produced membrane depolarization with concentration-dependent manners in the current clamp mode. Pretreatment with a Ca2+ free solution and thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum, stopped the generation of the pacemaker potentials. In the case of Ca2+-free solutions, SMS induced membrane depolarizations. However, when thapsigargin in a bath solution was applied, the membrane depolarization was not produced by SMS. The membrane depolarizations produced by SMS were inhibited by U-73122, an active phospholipase C (PLC) inhibitors. Furthermore, chelerythrine and calphostin C, a protein kinase C (PKC) inhibitors had no effects on SMS-induced membrane depolarizations. Conclusions: These results suggest that SMS might affect GI motility by modulating the pacemaker activity through an internal Ca2+- and PLC-dependent and PKC-independent pathway in the ICCs. PMID:25780681

  5. The phosphorylation state of neuronal processes determines growth cone formation after neuronal injury.

    PubMed

    Geddis, Matthew S; Rehder, Vincent

    2003-10-15

    Growth cones are essential for neuronal pathfinding during embryonic development and again after injury, when they aid in neuronal regeneration. This study was aimed at investigating the role of kinases in the earliest events in neuronal regeneration, namely, the formation of new growth cones from injured neuronal processes. Neurites of identified snail neurons grown in vitro were severed, and the formation of growth cones was observed from the ends of such transected processes. Under control conditions, all neurites formed a new growth cone within 45 min of transection. In contrast, growth cone formation in the presence of a general kinase inhibitor, K252a, was significantly inhibited. Moreover, decreasing the phosphorylation state of neurites by activating protein phosphatases with C2-ceramide also reduced growth cone formation. Pharmacological analysis with specific kinase inhibitors suggested that targets of protein kinase C (PKC) and tyrosine kinase (PTK) phosphorylation control growth cone formation. Inhibition of PKC with calphostin C and cerebroside completely blocked growth cone formation, whereas the inhibition of PTK with erbstatin analog significantly reduced growth cone formation. In contrast, inhibitors of protein kinase A, protein kinase G, CaM-kinase II, myosin light-chain kinase, Rho kinase, and PI-3 kinase had little or no effect 45 min after transection. These results suggest that the transformation underlying the formation of a growth cone from an injured (transected) neurite stump is highly sensitive to the phosphorylation state of key target proteins. Therefore, injury-induced signaling events will determine the outcome of neuronal regeneration through their action on kinase and phosphatase activities.

  6. Muscle cell depolarization induces a gain in surface GLUT4 via reduced endocytosis independently of AMPK.

    PubMed

    Wijesekara, Nadeeja; Tung, Amanda; Thong, Farah; Klip, Amira

    2006-06-01

    Contracting skeletal muscle increases glucose uptake to sustain energy demand. This is achieved through a gain in GLUT4 at the membrane, but the traffic mechanisms and regulatory signals involved are unknown. Muscle contraction is elicited by membrane depolarization followed by a rise in cytosolic Ca2+ and actomyosin activation, drawing on ATP stores. It is unknown whether one or more of these events triggers the rise in surface GLUT4. Here, we investigate the effect of membrane depolarization on GLUT4 cycling using GLUT4myc-expressing L6 myotubes devoid of sarcomeres and thus unable to contract. K+-induced membrane depolarization elevated surface GLUT4myc, and this effect was additive to that of insulin, was not prevented by inhibiting phosphatidylinositol 3-kinase (PI3K) or actin polymerization, and did not involve Akt activation. Instead, depolarization elevated cytosolic Ca2+, and the surface GLUT4myc elevation was prevented by dantrolene (an inhibitor of Ca2+ release from sarcoplasmic reticulum) and by extracellular Ca2+ chelation. Ca2+-calmodulin-dependent protein kinase-II (CaMKII) was not phosphorylated after 10 min of K+ depolarization, and the CaMK inhibitor KN62 did not prevent the gain in surface GLUT4myc. Interestingly, although 5'-AMP-activated protein kinase (AMPK) was phosphorylated upon depolarization, lowering AMPKalpha via siRNA did not alter the surface GLUT4myc gain. Conversely, the latter response was abolished by the PKC inhibitors bisindolylmaleimide I and calphostin C. Unlike insulin, K+ depolarization caused only a small increase in GLUT4myc exocytosis and a major reduction in its endocytosis. We propose that K+ depolarization reduces GLUT4 internalization through signals and mechanisms distinct from those engaged by insulin. Such a pathway(s) is largely independent of PI3K, Akt, AMPK, and CaMKII but may involve PKC.

  7. Small Molecule Inhibitors of BAF; A Promising Family of Compounds in HIV-1 Latency Reversal.

    PubMed

    Stoszko, Mateusz; De Crignis, Elisa; Rokx, Casper; Khalid, Mir Mubashir; Lungu, Cynthia; Palstra, Robert-Jan; Kan, Tsung Wai; Boucher, Charles; Verbon, Annelies; Dykhuizen, Emily C; Mahmoudi, Tokameh

    2016-01-01

    Persistence of latently infected cells in presence of Anti-Retroviral Therapy presents the main obstacle to HIV-1 eradication. Much effort is thus placed on identification of compounds capable of HIV-1 latency reversal in order to render infected cells susceptible to viral cytopathic effects and immune clearance. We identified the BAF chromatin remodeling complex as a key player required for maintenance of HIV-1 latency, highlighting its potential as a molecular target for inhibition in latency reversal. Here, we screened a recently identified panel of small molecule inhibitors of BAF (BAFi's) for potential to activate latent HIV-1. Latency reversal was strongly induced by BAFi's Caffeic Acid Phenethyl Ester and Pyrimethamine, two molecules previously characterized for clinical application. BAFi's reversed HIV-1 latency in cell line based latency models, in two ex vivo infected primary cell models of latency, as well as in HIV-1 infected patient's CD4 + T cells, without inducing T cell proliferation or activation. BAFi-induced HIV-1 latency reversal was synergistically enhanced upon PKC pathway activation and HDAC-inhibition. Therefore BAFi's constitute a promising family of molecules for inclusion in therapeutic combinatorial HIV-1 latency reversal.

  8. Small Molecule Inhibitors of BAF; A Promising Family of Compounds in HIV-1 Latency Reversal

    PubMed Central

    Stoszko, Mateusz; De Crignis, Elisa; Rokx, Casper; Khalid, Mir Mubashir; Lungu, Cynthia; Palstra, Robert-Jan; Kan, Tsung Wai; Boucher, Charles; Verbon, Annelies; Dykhuizen, Emily C.; Mahmoudi, Tokameh

    2015-01-01

    Persistence of latently infected cells in presence of Anti-Retroviral Therapy presents the main obstacle to HIV-1 eradication. Much effort is thus placed on identification of compounds capable of HIV-1 latency reversal in order to render infected cells susceptible to viral cytopathic effects and immune clearance. We identified the BAF chromatin remodeling complex as a key player required for maintenance of HIV-1 latency, highlighting its potential as a molecular target for inhibition in latency reversal. Here, we screened a recently identified panel of small molecule inhibitors of BAF (BAFi's) for potential to activate latent HIV-1. Latency reversal was strongly induced by BAFi's Caffeic Acid Phenethyl Ester and Pyrimethamine, two molecules previously characterized for clinical application. BAFi's reversed HIV-1 latency in cell line based latency models, in two ex vivo infected primary cell models of latency, as well as in HIV-1 infected patient's CD4 + T cells, without inducing T cell proliferation or activation. BAFi-induced HIV-1 latency reversal was synergistically enhanced upon PKC pathway activation and HDAC-inhibition. Therefore BAFi's constitute a promising family of molecules for inclusion in therapeutic combinatorial HIV-1 latency reversal. PMID:26870822

  9. [ACE inhibitors and the kidney].

    PubMed

    Hörl, W H

    1996-01-01

    Treatment with ACE inhibitors results in kidney protection due to reduction of systemic blood pressure, intraglomerular pressure, an antiproliferative effect, reduction of proteinuria and a lipid-lowering effect in proteinuric patients (secondary due to reduction of protein excretion). Elderly patients with diabetes melitus, coronary heart disease or peripheral vascular occlusion are at risk for deterioration of kidney function due to a high frequency of renal artery stenosis in these patients. In patients with renal insufficiency dose reduction of ACE inhibitors is necessary (exception: fosinopril) but more important is the risk for development of hyperkalemia. Patients at risk for renal artery stenosis and patients pretreated with diuretics should receive a low ACE inhibitor dosage initially ("start low - go slow"). For compliance reasons once daily ACE inhibitor dosage is recommended.

  10. Selective Inhibitors of Protein Methyltransferases

    PubMed Central

    2015-01-01

    Mounting evidence suggests that protein methyltransferases (PMTs), which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and human diseases. In particular, PMTs have been recognized as major players in regulating gene expression and chromatin state. PMTs are divided into two categories: protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs). There has been a steadily growing interest in these enzymes as potential therapeutic targets and therefore discovery of PMT inhibitors has also been pursued increasingly over the past decade. Here, we present a perspective on selective, small-molecule inhibitors of PMTs with an emphasis on their discovery, characterization, and applicability as chemical tools for deciphering the target PMTs’ physiological functions and involvement in human diseases. We highlight the current state of PMT inhibitors and discuss future directions and opportunities for PMT inhibitor discovery. PMID:25406853

  11. Sodium phenylbutyrate enhances astrocytic neurotrophin synthesis via protein kinase C (PKC)-mediated activation of cAMP-response element-binding protein (CREB): implications for Alzheimer disease therapy.

    PubMed

    Corbett, Grant T; Roy, Avik; Pahan, Kalipada

    2013-03-22

    Neurotrophins, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are believed to be genuine molecular mediators of neuronal growth and homeostatic synapse activity. However, levels of these neurotrophic factors decrease in different brain regions of patients with Alzheimer disease (AD). Induction of astrocytic neurotrophin synthesis is a poorly understood phenomenon but represents a plausible therapeutic target because neuronal neurotrophin production is aberrant in AD and other neurodegenerative diseases. Here, we delineate that sodium phenylbutyrate (NaPB), a Food and Drug Administration-approved oral medication for hyperammonemia, induces astrocytic BDNF and NT-3 expression via the protein kinase C (PKC)-cAMP-response element-binding protein (CREB) pathway. NaPB treatment increased the direct association between PKC and CREB followed by phosphorylation of CREB (Ser(133)) and induction of DNA binding and transcriptional activation of CREB. Up-regulation of markers for synaptic function and plasticity in cultured hippocampal neurons by NaPB-treated astroglial supernatants and its abrogation by anti-TrkB blocking antibody suggest that NaPB-induced astroglial neurotrophins are functionally active. Moreover, oral administration of NaPB increased the levels of BDNF and NT-3 in the CNS and improved spatial learning and memory in a mouse model of AD. Our results highlight a novel neurotrophic property of NaPB that may be used to augment neurotrophins in the CNS and improve synaptic function in disease states such as AD.

  12. [Ca{sup 2+}]{sub i} and PKC-{alpha} are involved in the inhibitory effects of Ib, a novel nonpeptide AngiotensinII subtype AT{sub 1} receptor antagonist, on AngiotensinII-induced vascular contraction in vitro

    SciTech Connect

    Wang Yu; Wang Wei; Wang Qiujuan Wu Jinhui; Xu Jinyi; Wu Xiaoming

    2007-12-07

    The vasoactive peptide AngiotensinII (AngII) is an important factor in the cardiovascular system, exerting most of its effects through AngII receptor type 1 (AT{sub 1}). Ib, a new nonpeptide AT{sub 1} receptor antagonist, has been observed to play a positive role in the treatment of hypertension in preclinical tests. In this study, the inhibitory effects of Ib on AngII-induced vascular contraction in vitro were investigated, and its molecular mechanisms were further explored. In endothelium-denuded aortic rings from rabbits, Ib produced a rightward shift in the concentration-response curve for AngII with a decrease in the maximal contractile response and the pD{sub 2}{sup '} was 7.29. In vascular smooth muscle cells (VSMCs), the specific binding of [{sup 125}I]AngII to AT{sub 1} receptors was inhibited by Ib in a concentration-dependent manner with IC{sub 50} value of 0.96 nM. Ib could inhibit both AngII-induced Ca{sup 2+} mobilization from internal stores and Ca{sup 2+} influx. Moreover, the translocation of PKC-{alpha} stimulated by AngII was inhibited by Ib. Thus, the inhibitory effects of Ib might be related with the depression on AngII-induced increase in [Ca{sup 2+}]{sub i} and translocation of PKC-{alpha} through blocking AT{sub 1} receptors.

  13. Inhibitors of pig kidney trehalase.

    PubMed

    Kyosseva, S V; Kyossev, Z N; Elbein, A D

    1995-02-01

    Trehazolin, a new trehalase inhibitor isolated from the culture broth of Micromonospora, was reported to be a highly specific inhibitor for porcine and silk worm trehalases with IC50 values of 5.5 x 10(-9) and 3.7 x 10(-9) M, respectively (O. Ando, H. Satake, K. Itoi, A. Sato, M. Nakajima, S. Takashi, H. Haruyama, Y. Ohkuma, T. Kinoshita, and R. Enokita (1991) J. Antibiot. 44, 1165-1168). We also found that trehazolin is a very powerful and quite specific inhibitor against purified pig kidney trehalase, giving an IC50 value of 1.9 x 10(-8) M. Lineweaver-Burk plots showed that this compound was a competitive inhibitor of the trehalase. However, even at concentrations of 200 micrograms/ml, trehazolin did not inhibit the rat intestinal maltase or sucrase, yeast alpha-glucosidase or almond beta-glucosidase. Validoxylamine A and validamycin A, two other trehalase inhibitors, showed potent competitive inhibition against purified pig kidney trehalase, with IC50 values of 2.4 x 10(-9) and 2.5 x 10(-4) M, respectively. On the other hand, validoxylamine A was almost inactive against rat intestinal sucrase and maltase, with some inhibition being observed at millimolar concentration. A number of other glucosidase inhibitors, such as MDL 25637, castanospermine, and deoxynojirimycin were also tested against the purified trehalase and showed reasonable inhibitory activity.

  14. Gastrin stimulates expression of plasminogen activator inhibitor-1 in gastric epithelial cells.

    PubMed

    Nørsett, Kristin G; Steele, Islay; Duval, Cedric; Sammut, Stephen J; Murugesan, Senthil V M; Kenny, Susan; Rainbow, Lucille; Dimaline, Rod; Dockray, Graham J; Pritchard, D Mark; Varro, Andrea

    2011-09-01

    Plasminogen activator inhibitor (PAI)-1 is associated with cancer progression, fibrosis and thrombosis. It is expressed in the stomach but the mechanisms controlling its expression there, and its biological role, are uncertain. We sought to define the role of gastrin in regulating PAI-1 expression and to determine the relevance for gastrin-stimulated cell migration and invasion. In gastric biopsies from subjects with elevated plasma gastrin, the abundances of PAI-1, urokinase plasminogen activator (uPA), and uPA receptor (uPAR) mRNAs measured by quantitative PCR were increased compared with subjects with plasma concentrations in the reference range. In patients with hypergastrinemia due to autoimmune chronic atrophic gastritis, there was increased abundance of PAI-1, uPA, and uPAR mRNAs that was reduced by octreotide or antrectomy. Immunohistochemistry revealed localization of PAI-1 to parietal cells and enterochromaffin-like cells in micronodular neuroendocrine tumors in hypergastrinemic subjects. Transcriptional mechanisms were studied by using a PAI-1-luciferase promoter-reporter construct transfected into AGS-G(R) cells. There was time- and concentration-dependent increase of PAI-1-luciferase expression in response to gastrin that was reversed by inhibitors of the PKC and MAPK pathways. In Boyden chamber assays, recombinant PAI-1 inhibited gastrin-stimulated AGS-G(R) cell migration and invasion, and small interfering RNA treatment increased responses to gastrin. We conclude that elevated plasma gastrin concentrations are associated with increased expression of gastric PAI-1, which may act to restrain gastrin-stimulated cell migration and invasion.

  15. Novel isoquinolone PDK1 inhibitors discovered through fragment-based lead discovery

    NASA Astrophysics Data System (ADS)

    Johnson, M. Catherine; Hu, Qiyue; Lingardo, Laura; Ferre, Rose Ann; Greasley, Samantha; Yan, Jiangli; Kath, John; Chen, Ping; Ermolieff, Jacques; Alton, Gordon

    2011-07-01

    Phosphoinositide-dependent kinase-1 (PDK1) is a critical enzyme in the PI3K/AKT pathway and to the activation of AGC family protein kinases, including S6K, SGK, and PKC. Dysregulation of this pathway plays a key role in cancer cell growth, survival and tumor angiogenesis. As such, inhibitors of PDK1 offer the promise of a new therapeutic modality for cancer treatment. Fragment based drug screening has recently become a viable entry point for hit identification. In this work, NMR spectroscopy fragment screening of PDK1 afforded novel chemotypes as orthogonal starting points from HTS screening hits. Compounds identified as hits by NMR spectroscopy were tested in a biochemical assay, and fragments with activity in both assays were clustered. The Pfizer compound file was mined via substructure and 2D similarity search, and the chemotypes were prioritized by ligand efficiency (LE), SAR mining, chemical attractiveness, and chemical enablement of promising vectors. From this effort, an isoquinolone fragment hit, 5 (IC50 870 μM, LE = 0.39), was identified as a novel, ligand efficient inhibitor of PDK1 and a suitable scaffold for further optimization. Initially in the absence of crystallographic data, a fragment growing approach efficiently explored four vectors of the isoquinolone scaffold via parallel synthesis to afford a compound with crystallographic data, 16 (IC50 41.4 μM, LE = 0.33). Subsequent lead optimization efforts provided 24 (IC50 1.8 μM, LE = 0.42), with greater than fivefold selectivity against other key pathway kinases.

  16. Engineering trypsin for inhibitor resistance.

    PubMed

    Batt, Anna R; St Germain, Commodore P; Gokey, Trevor; Guliaev, Anton B; Baird, Teaster

    2015-09-01

    The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity. A key step in developing inhibitor resistance is the identification of key residues in protease-inhibitor interaction. Given that majority of the protease therapeutics currently in use are trypsin-fold, trypsin itself serves as an ideal model for studying protease-inhibitor interaction. To test the importance of several trypsin-inhibitor interactions on the prime-side binding interface, we created four trypsin single variants Y39A, Y39F, K60A, and K60V and report biochemical sensitivity against bovine pancreatic trypsin inhibitor (BPTI) and M84R ecotin. All variants retained catalytic activity against small, commercially available peptide substrates [kcat /KM  = (1.2 ± 0.3) × 10(7) M(-1 ) s(-1) . Compared with wild-type, the K60A and K60V variants showed increased sensitivity to BPTI but less sensitivity to ecotin. The Y39A variant was less sensitive to BPTI and ecotin while the Y39F variant was more sensitive to both. The relative binding free energies between BPTI complexes with WT, Y39F, and Y39A were calculated based on 3.5 µs combined explicit solvent molecular dynamics simulations. The BPTI:Y39F complex resulted in the lowest binding energy, while BPTI:Y39A resulted in the highest. Simulations of Y39F revealed increased conformational rearrangement of F39, which allowed formation of a new hydrogen bond between BPTI R17 and H40 of the variant. All together, these data suggest that positions 39 and 60 are key for inhibitor binding to trypsin, and likely more trypsin-fold proteases.

  17. [New anticoagulants - direct thrombin inhibitors].

    PubMed

    Brand, B; Graf, L

    2012-11-01

    Direct thrombin-inhibitors inactivate not only free but also fibrin-bound thrombin. The group of parenteral direct thrombin-inhibitors includes the recombinant hirudins lepirudin and desirudin, the synthetic hirudin bivalirudin, and the small molecule argatroban. All these compounds do not interact with PF4/heparin-antibodies. Therefore, argatroban as well as bivalirudin are currently used to treat heparin-induced thrombocytopenia (HIT). The oral direct thrombin-inhibitor dabigatran etexilate is already licensed in many countries for the treatment of non-valvular atrial fibrillation. Dabigatran etexilate reveals a stable and predictable effect that allows a medication without dose adjustment or monitoring. The substance shows only few interactions with other drugs but strong inhibitors of p-glycoprotein can increase plasma levels of dabigatran substantially. After oral intake, the prodrug dabigatran etexilate is cleaved by esterase-mediated hydrolyses to the active compound dabigatran. Elimination of dabigatran is predominantly renal. Safety and efficacy of dabigatran etexilate were tested in an extensive clinical study program. Non-inferiority compared to current standard treatments was shown for prophylaxis of venous thromboembolic events after total knee and hip replacement, for stroke prevention in atrial fibrillation, and for treatment of acute venous thromboembolism. In daily practice, Dabigatran etexilate competes against the new direct factor Xa-inhibitors. In the absence of direct comparative clinical trials, it is not yet clear if one class of substances has distinct advantages over the other.

  18. Mechanisms of protease-activated receptor 2-evoked hyperexcitability of nociceptive neurons innervating the mouse colon

    PubMed Central

    Kayssi, Ahmed; Amadesi, Silvia; Bautista, Francisco; Bunnett, Nigel W; Vanner, Stephen

    2007-01-01

    Agonists of protease-activated receptor 2 (PAR2) evoke hyperexcitability of dorsal root ganglia (DRG) neurons by unknown mechanisms. We examined the cellular mechanisms underlying PAR2-evoked hyperexcitability of mouse colonic DRG neurons to determine their potential role in pain syndromes such as visceral hyperalgesia. Colonic DRG neurons were identified by injecting Fast Blue and DiI retrograde tracers into the mouse colon. Using immunofluorescence, we found that DiI-labelled neurons contained PAR2 immunoreactivity, confirming the presence of receptors on colonic neurons. Whole-cell current-clamp recordings of acutely dissociated neurons demonstrated that PAR2 activation with a brief application (3 min) of PAR2 agonists, SLIGRL-NH2 and trypsin, evoked sustained depolarizations (up to 60 min) which were associated with increased input resistance and a marked reduction in rheobase (50% at 30 min). In voltage clamp, SLIGRL-NH2 markedly suppressed delayed rectifier IK currents (55% at 10 min), but had no effect on the transient IA current or TTX-resistant Na+ currents. In whole-cell current-clamp recordings, the sustained excitability evoked by PAR2 activation was blocked by the PKC inhibitor, calphostin, and the ERK1/2 inhibitor PD98059. Studies of ERK1/2 phosphorylation using confocal microscopy demonstrated that SLIGRL-NH2 increased levels of immunoreactive pERK1/2 in DRG neurons, particularly in proximity to the plasma membrane. Thus, activation of PAR2 receptors on colonic nociceptive neurons causes sustained hyperexcitability that is related, at least in part, to suppression of delayed rectifier IK currents. Both PKC and ERK1/2 mediate the PAR2-induced hyperexcitability. These studies describe a novel mechanism of sensitization of colonic nociceptive neurons that may be implicated in conditions of visceral hyperalgesia such as irritable bowel syndrome. PMID:17289784

  19. Mastoparan-induced phosphatidylcholine hydrolysis by phospholipase D activation in human astrocytoma cells.

    PubMed Central

    Mizuno, K.; Nakahata, N.; Ohizumi, Y.

    1995-01-01

    1. The effect of mastoparan on phosphatidylcholine hydrolysis was examined in 1321N1 human astrocytoma cells. Mastoparan (3-30 microM) caused an accumulation of diacylglycerol (DG) and phosphatidic acd (PA) accompanied by choline release in a concentration- and time-dependent manner. 2. In the presence of 2% n-butanol, mastoparan (3-100 microM) induced phosphatidylbutanol (PBut) accumulation in a concentration- and time-dependent manner, suggesting that mastoparan activates phospholipase D (PLD). Propranolol (30-300 microM), a phosphatidate phosphohydrolase inhibitor, inhibited DG accumulation induced by mastoparan, supporting this idea. 3. Depletion of extracellular free calcium ion did not alter the effect of mastoparan on PLD activity. 4. A protein kinase C (PKC) inhibitor, calphostin C (1 microM), did not inhibit mastoparan-induce PLD activation but the ability of mastoparan to stimulate phospholipase D activity was decreased in the PKC down regulated cells. 5. PLD activity stimulated by mastoparan was not prevented by pretreatment of the cells with pertussis toxin (PT) or C3 ADP-ribosyltransferase. Furthermore, guanine nucleotides did not affect PLD activity stimulation by mastoparan in membrane preparations. 6. Mastoparan stimulated PLD in several cell lines such as RBL-2H3, RBL-1, HL-60, P388, endothelial cells, as well as 1321N1 human astrocytoma cells. 7. These results suggest that mastoparan induces phosphatidylcholine (PC) hydrolysis by activation of PLD, not by activation of phosphatidylcholine-specific phospholipase C (PC-PLC); mastoparan-induced PLD activation is not mediated by G proteins. PMID:8640350

  20. Differential regulation of redox responsive transcription factors by the nephrocarcinogen 2,3,5-Tris(glutathion-S-yl)hydroquinone.

    PubMed

    Weber, T J; Huang, Q; Monks, T J; Lau, S S

    2001-07-01

    2,3,5-Tris(glutathion-S-yl)hydroquinone [TGHQ] is a potent nephrotoxicant and nephrocarcinogen, and induces a spectrum of mutations in human and bacterial cells consistent with those attributed to reactive oxygen species (ROS). Studies were conducted to determine whether the oxidative stress induced by TGHQ in renal proximal tubule epithelial cells (LLC-PK(1)) modulates transcriptional activities widely implicated in transformation responses, namely 12-O-tetradecanoyl phorbol 13-acetate (TPA) responsive element (TRE)- and nuclear factor kappa B (NF-kappaB)-binding activity. TGHQ increased TRE- and NF-kappaB-binding activity in a concentration- and time-dependent manner. Catalase fully inhibited peak TGHQ-mediated TRE- and NF-kappaB-binding activity. In contrast, although deferoxamine fully inhibited TGHQ-mediated TRE-binding activity, it had only a marginal effect on NF-kappaB-binding activity. Collectively, these data indicate that TGHQ modulates TRE- and NF-kappaB-binding activity in an ROS-dependent fashion. Cycloheximide and actinomycin D fully inhibited TGHQ-mediated TRE-binding activity, but in the absence of TGHQ increased NF-kappaB-binding activity. Although protein kinase C (PKC) is widely implicated in stress response signaling, pretreatment of cells with PKC inhibitors (H-89, calphostin C) did not modulate TGHQ-mediated DNA-binding activities. In contrast, pretreatment of cells with (PD098059), a mitogen activated protein kinase kinase (MEK) inhibitor, markedly reduced TGHQ-mediated TRE-binding activity, but enhanced TGHQ-mediated NF-kappaB-binding activity. We conclude that TGHQ-mediated TRE- and NF-kappaB-binding activities are ROS-dependent. Although there is a common requirement for hydrogen peroxide (H(2)O(2)) in the regulation of these DNA-binding activities, there appears to be divergent regulation after H(2)O(2) generation in renal epithelial cells.

  1. Activation of presynaptic kainate receptors suppresses GABAergic synaptic transmission in the rat globus pallidus

    PubMed Central

    Jin, Xiao-Tao; Smith, Yoland

    2007-01-01

    The globus pallidus (GP) plays a central integrative role in the basal ganglia circuitry. It receives strong GABAergic inputs from the striatum and significant glutamatergic afferents from the subthalamic nucleus (STN). The change in firing rate and pattern of GP neurons is a cardinal feature of Parkinson’s disease pathophysiology. Kainate receptor GluR6/7 subunits immunoreactivity is expressed presynaptically in GABAergic striatopallidal terminals (Kane-Jackson and Smith 2003; Jin et al., 2006), which provides a substrate for regulation of GABAergic transmission in GP. To test this hypothesis, we recorded GABAA-mediated inhibitory postsynaptic currents (IPSCs) in the GP following electrical stimulation of the striatum. Following blockade of AMPA and NMDA receptors with selective antagonists, bath application of kainate (KA) (0.3–3 μM) reduced significantly the amplitude of evoked IPSCs. This inhibition was associated with a significant increase in paired-pulse facilitation ratio and a reduction of the frequency, but not amplitude, of miniature IPSCs (mIPSCs), suggesting a presynaptic site of KA action. The KA effects on striatopallidal GABAergic transmission were blocked by the G-protein inhibitor, N-ethylmaleimide (NEM), or protein kinase C (PKC) inhibitor calphostin C. Our results demonstrate that KAR activation inhibits GABAergic transmission through a presynaptic G protein-coupled, PKC-dependent metabotropic mechanism in the rat GP. These findings open up the possibility for the development of kainate-mediated pharmacotherapies aim at decreasing the excessive and abnormally regulated inhibition of GP neurons in Parkinson’s disease. PMID:17881134

  2. Corrosion inhibitors from expired drugs.

    PubMed

    Vaszilcsin, Nicolae; Ordodi, Valentin; Borza, Alexandra

    2012-07-15

    This paper presents a method of expired or unused drugs valorization as corrosion inhibitors for metals in various media. Cyclic voltammograms were drawn on platinum in order to assess the stability of pharmaceutically active substances from drugs at the metal-corrosive environment interface. Tafel slope method was used to determine corrosion rates of steel in the absence and presence of inhibitors. Expired Carbamazepine and Paracetamol tablets were used to obtain corrosion inhibitors. For the former, the corrosion inhibition of carbon steel in 0.1 mol L(-1) sulfuric acid solution was about 90%, whereas for the latter, the corrosion inhibition efficiency of the same material in the 0.25 mol L(-1) acetic acid-0.25 mol L(-1) sodium acetate buffer solution was about 85%.

  3. Electrochemical studies of corrosion inhibitors

    NASA Technical Reports Server (NTRS)

    Danford, M. D.

    1990-01-01

    The effect of single salts, as well as multicomponent mixtures, on corrosion inhibition was studied for type 1010 steel; for 5052, 1100, and 2219-T87 aluminum alloys; and for copper. Molybdate-containing inhibitors exhibit an immediate, positive effect for steel corrosion, but an incubation period may be required for aluminum before the effect of a given inhibitor can be determined. The absence of oxygen was found to provide a positive effect (smaller corrosion rate) for steel and copper, but a negative effect for aluminum. This is attributed to the two possible mechanisms by which aluminum can oxidize. Corrosion inhibition is generally similar for oxygen-rich and oxygen-free environments. The results show that the electrochemical method is an effective means of screening inhibitors for the corrosion of single metals, with caution to be exercised in the case of aluminum.

  4. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, Joanna S.; MacGregor, Robert R.; Wolf, Alfred P.; Langstrom, Bengt

    1990-01-01

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  5. An environmentally friendly scale inhibitor

    SciTech Connect

    Dobbs, J.B.; Brown, J.M.

    1999-11-01

    This paper describes a method of inhibiting the formation of scales such as barium and strontium sulfate in low pH aqueous systems, and calcium carbonate in systems containing high concentrations of dissolved iron. The solution, chemically, involves treating the aqueous system with an inhibitor designed to replace organic-phosphonates. Typical low pH aqueous systems where the inhibitor is particularly useful are oilfield produced-water, resin bed water softeners that form scale during low pH, acid regeneration operations. Downhole applications are recommended where high concentrations of dissolved iron are present in the produced water. This new approach to inhibition replaces typical organic phosphonates and polymers with a non-toxic, biodegradable scale inhibitor that performs in harsh environments.

  6. Positron emitter labeled enzyme inhibitors

    SciTech Connect

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

    1990-04-03

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  7. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

    1987-05-22

    This invention involved a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide in activators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.

  8. STAT inhibitors for cancer therapy

    PubMed Central

    2013-01-01

    Signal Transducer and Activator of Transcription (STAT) proteins are a family of cytoplasmic transcription factors consisting of 7 members, STAT1 to STAT6, including STAT5a and STAT5b. STAT proteins are thought to be ideal targets for anti-cancer therapy since cancer cells are more dependent on the STAT activity than their normal counterparts. Inhibitors targeting STAT3 and STAT5 have been developed. These included peptidomimetics, small molecule inhibitors and oligonucleotides. This review summarized advances in preclinical and clinical development of these compounds. PMID:24308725

  9. [Kinase inhibitors against hematological malignancies].

    PubMed

    Tojo, Arinobu

    2014-06-01

    Dysregulation of protein phosphorylation, especially on tyrosine residues, plays a crucial role in development and progression of hematological malignancies. Since remarkable success in imatinib therapy of CML and Ph+ALL, extensive efforts have made to explore candidate molecular targets and next breakthrough drugs. Now that next generation ABL kinase inhibitors are available for CML, the therapeutic algorithm has been revolutionized. As for AML and lymphoid malignancies, many kinase inhibitors targeting FLT3, BTK and aurora-A are on early and late clinical trials, and a number of promising drugs including ibrutinib are picked up for further evaluation.

  10. EGFR inhibitors and autophagy in cancer treatment.

    PubMed

    Cui, Jie; Hu, Yun-Feng; Feng, Xie-Min; Tian, Tao; Guo, Ya-Huan; Ma, Jun-Wei; Nan, Ke-Jun; Zhang, Hong-Yi

    2014-12-01

    Epidermal growth factor receptor (EGFR) inhibitor treatment is a strategy for cancer therapy. However, innate and acquired resistance is a major obstacle of the efficacy. Autophagy is a self-digesting process in cells, which is considered to be associated with anti-cancer drug resistance. The activation of EGFR can regulate autophagy through multiple signal pathways. EGFR inhibitors can induce autophagy, but the specific function of the induction of autophagy by EGFR inhibitors remains biphasic. On the one hand, autophagy induced by EGFR inhibitors acts as a cytoprotective response in cancer cells, and autophagy inhibitors can enhance the cytotoxic effects of EGFR inhibitors. On the other hand, a high level of autophagy after treatment of EGFR inhibitors can also result in autophagic cell death lacking features of apoptosis, and the combination of EGFR inhibitors with an autophagy inducer might be beneficial. Thus, autophagy regulation represents a promising approach for improving the efficacy of EGFR inhibitors in the treatment of cancer patients.

  11. Acetylcholinesterase Inhibitors: Pharmacology and Toxicology

    PubMed Central

    Čolović, Mirjana B; Krstić, Danijela Z; Lazarević-Pašti, Tamara D; Bondžić, Aleksandra M; Vasić, Vesna M

    2013-01-01

    Acetylcholinesterase is involved in the termination of impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine in numerous cholinergic pathways in the central and peripheral nervous systems. The enzyme inactivation, induced by various inhibitors, leads to acetylcholine accumulation, hyperstimulation of nicotinic and muscarinic receptors, and disrupted neurotransmission. Hence, acetylcholinesterase inhibitors, interacting with the enzyme as their primary target, are applied as relevant drugs and toxins. This review presents an overview of toxicology and pharmacology of reversible and irreversible acetylcholinesterase inactivating compounds. In the case of reversible inhibitors being commonly applied in neurodegenerative disorders treatment, special attention is paid to currently approved drugs (donepezil, rivastigmine and galantamine) in the pharmacotherapy of Alzheimer’s disease, and toxic carbamates used as pesticides. Subsequently, mechanism of irreversible acetylcholinesterase inhibition induced by organophosphorus compounds (insecticides and nerve agents), and their specific and nonspecific toxic effects are described, as well as irreversible inhibitors having pharmacological implementation. In addition, the pharmacological treatment of intoxication caused by organophosphates is presented, with emphasis on oxime reactivators of the inhibited enzyme activity administering as causal drugs after the poisoning. Besides, organophosphorus and carbamate insecticides can be detoxified in mammals through enzymatic hydrolysis before they reach targets in the nervous system. Carboxylesterases most effectively decompose carbamates, whereas the most successful route of organophosphates detoxification is their degradation by corresponding phosphotriesterases. PMID:24179466

  12. Benzimidazole derivatives as kinase inhibitors.

    PubMed

    Garuti, Laura; Roberti, Marinella; Bottegoni, Giovanni

    2014-01-01

    Benzimidazole is a common kinase inhibitor scaffold and benzimidazole-based compounds interact with enzymes by multiple binding modes. In some cases, the benzimidazole acts as part of the hinge-binding motif, in others it has a scaffolding role without evidence for direct hinge binding. Several of these compounds are ATP-competitive inhibitors and show high selectivity by exploiting unique structural properties that distinguish one kinase from the majority of other kinases. However, the high specificity for a single target is not always sufficient. Thus another approach, called multi-target therapy, has been developed over the last few years. The simultaneous inhibition of various kinases may be useful because the disease is attacked at several relevant targets. Moreover, if a kinase becomes drug-resistant, a multitargeted drug can act on the other kinases. Some benzimidazole derivatives are multi-target inhibitors. In this article benzimidazole inhibitors are reported with their mechanisms of action, structure-activity relationship (SAR) and biological properties.

  13. Biocatalysts with enhanced inhibitor tolerance

    DOEpatents

    Yang, Shihui; Linger, Jeffrey; Franden, Mary Ann; Pienkos, Philip T.; Zhang, Min

    2015-12-08

    Disclosed herein are biocatalysts for the production of biofuels, including microorganisms that contain genetic modifications conferring tolerance to growth and fermentation inhibitors found in many cellulosic feedstocks. Methods of converting cellulose-containing materials to fuels and chemicals, as well as methods of fermenting sugars to fuels and chemicals, using these biocatalysts are also disclosed.

  14. Azidoblebbistatin, a photoreactive myosin inhibitor

    PubMed Central

    Képiró, Miklós; Várkuti, Boglárka H.; Bodor, Andrea; Hegyi, György; Drahos, László; Kovács, Mihály; Málnási-Csizmadia, András

    2012-01-01

    Photoreactive compounds are important tools in life sciences that allow precisely timed coval