Science.gov

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. © 2014 UICC.

  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 Central

    BHAVANASI, DHEERAJ; KOSTYAK, JOHN C.; SWINDLE, JOHN; KILPATRICK, LAURIE E.; KUNAPULI, SATYA P.

    2014-01-01

    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. Pretreatment 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. PMID:24433221

  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. 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.

  15. 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.

  16. Photoexcited calphostin C selectively destroys nuclear lamin B1 in neoplastic human and rat cells - a novel mechanism of action of a photodynamic tumor therapy agent.

    PubMed

    Chiarini, Anna; Whitfield, James F; Pacchiana, Raffaella; Armato, Ubaldo; Dal Pra, Ilaria

    2008-09-01

    Lamin B1, a major component of the nuclear lamina, anchors the nucleus to the cytoskeletal cage, and controls nuclear orientation, chromosome positioning and, alongside several enzymes, fundamental nuclear functions. Exposing polyomavirus-transformed rat pyF111 fibroblasts and human cervical carcinoma (HCC) C4-I cells for 30 min to photoexcited perylenequinone calphostin C, i.e. Cal C(phiE), an established reactive oxygen species (ROS)-generator and protein kinase C (PKC) inhibitor, caused the cells to selectively oxidize and then totally destroy their nuclear lamin B1 by only 60 min after starting the treatment, i.e. when apoptotic caspases' activities had not yet increased. However, while the oxidized lamin B1 was being destroyed, lamins A/C, the lamin A-associated nuclear envelope protein emerin, and the nucleoplasmic protein cyclin E were neither oxidized nor destroyed. The oxidized lamin B was ubiquitinated and demolished in the proteasome probably by an enhanced peptidyl-glutaminase-like activity. Hence, the Cal C(phiE)-induced rapid and selective lamin B1 oxidation and proteasomal destruction ahead of the activation of apoptotic caspases was by itself a most severe molecular lesion impairing vital nuclear functions. Conversely, Cal C directly added to the cells kept in the dark damaged neither nuclear lamin B1 nor cell viability. Thus, our findings reveal a novel cell-damaging mechanism of a photodynamic tumor therapeutic agent.

  17. Pre-treatment with a PKC or PKA inhibitor prevents the development of morphine tolerance but not physical dependence in mice.

    PubMed

    Gabra, Bichoy H; Bailey, Chris P; Kelly, Eamonn; Smith, Forrest L; Henderson, Graeme; Dewey, William L

    2008-06-27

    We previously demonstrated that intracerebroventricular (i.c.v.) administration of protein kinase C (PKC) or protein kinase A (PKA) inhibitors reversed morphine antinociceptive tolerance in 3-day morphine-pelleted mice. The present study aimed at evaluating whether pre-treating mice with a PKC or PKA inhibitor prior to pellet implantation would prevent the development of morphine tolerance and physical dependence. Antinociception was assessed using the warm-water tail immersion test and physical dependence was evaluated by quantifying/scoring naloxone-precipitated withdrawal signs. While drug-naïve mice pelleted with a 75 mg morphine pellet for 3 days developed a 5.8-fold tolerance to morphine antinociception, mice pre-treated i.c.v. with the PKC inhibitors bisindolylmaleimide I, Go-7874 or Go-6976, or with the myristoylated PKA inhibitor, PKI-(14-22)-amide failed to develop any tolerance to morphine antinociception. Experiments were also conducted to determine whether morphine-pelleted mice were physically dependent when pre-treated with PKC or PKA inhibitors. The same inhibitor doses that prevented morphine tolerance were evaluated in other mice injected s.c. with naloxone and tested for precipitated withdrawal. The pre-treatment with PKC or PKA inhibitors failed to attenuate or block the signs of morphine withdrawal including jumping, wet-dog shakes, rearing, forepaw tremor, increased locomotion, grooming, diarrhea, tachypnea and ptosis. These data suggest that elevations in the activity of PKC and PKA in the brain are critical to the development of morphine tolerance. However, it appears that tolerance can be dissociated from physical dependence, indicating a role for PKC and PKA to affect antinociception but not those signs mediated through the complex physiological processes of withdrawal.

  18. 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.

  19. Ca2+/Calmodulin-dependent Protein Kinase II Inhibitors Disrupt AKAP79-dependent PKC Signaling to GluA1 AMPA Receptors*

    PubMed Central

    Brooks, Ian M.; Tavalin, Steven J.

    2011-01-01

    GluA1 (formerly GluR1) AMPA receptor subunit phosphorylation at Ser-831 is an early biochemical marker for long-term potentiation and learning. This site is a substrate for Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) and protein kinase C (PKC). By directing PKC to GluA1, A-kinase anchoring protein 79 (AKAP79) facilitates Ser-831 phosphorylation and makes PKC a more potent regulator of GluA1 than CaMKII. PKC and CaM bind to residues 31–52 of AKAP79 in a competitive manner. Here, we demonstrate that common CaMKII inhibitors alter PKC and CaM interactions with AKAP79(31–52). Most notably, the classical CaMKII inhibitors KN-93 and KN-62 potently enhanced the association of CaM to AKAP79(31–52) in the absence (apoCaM) but not the presence of Ca2+. In contrast, apoCaM association to AKAP79(31–52) was unaffected by the control compound KN-92 or a mechanistically distinct CaMKII inhibitor (CaMKIINtide). In vitro studies demonstrated that KN-62 and KN-93, but not the other compounds, led to apoCaM-dependent displacement of PKC from AKAP79(31–52). In the absence of CaMKII activation, complementary cellular studies revealed that KN-62 and KN-93, but not KN-92 or CaMKIINtide, inhibited PKC-mediated phosphorylation of GluA1 in hippocampal neurons as well as AKAP79-dependent PKC-mediated augmentation of recombinant GluA1 currents. Buffering cellular CaM attenuated the ability of KN-62 and KN-93 to inhibit AKAP79-anchored PKC regulation of GluA1. Therefore, by favoring apoCaM binding to AKAP79, KN-62 and KN-93 derail the ability of AKAP79 to efficiently recruit PKC for regulation of GluA1. Thus, AKAP79 endows PKC with a pharmacological profile that overlaps with CaMKII. PMID:21156788

  20. 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.

  1. Proto-oncogene c-erbB2 initiates rat primordial follicle growth via PKC and MAPK pathways

    PubMed Central

    2010-01-01

    Background c-erbB2, a proto-oncogene coding epidermal growth factor receptor-like receptor, also as a chemosensitivity/prognosis marker for gynecologic cancer, may be involved in initiation of growth of rat primordial follicles. The aim of the present study is to investigate the role and signal pathway of c-erbB2 in onset of rat primordial follicle development. Methods The expression of c-erbB2 mRNA and protein in neonatal ovaries cultured 4 and 8 days with/without epidermal growth factor (EGF) were examined by in situ hybridization, RT-PCR and western blot. The function of c-erbB2 in the primordial folliculogenesis was abolished by small interfering RNA transfection. Furthermore, MAPK inhibitor PD98059 and PKC inhibitor calphostin were used to explore the possible signaling pathway of c-erbB2 in primordial folliculogenesis. Results The results showed that c-erbB2 mRNA was expressed in ooplasm and the expression of c-erbB2 decreased after transfection with c-erbB2 siRNA. Treatment with EGF at 50 ng/ml significantly increased c-erbB2 expression and primary and secondary follicle formation in ovaries. However, this augmenting effect was remarkably inhibited by c-erbB2 siRNA transfection. Furthermore, folliculogenesis offset was blocked by calphostin (5 × 10(-4) mmol/L) and PD98059 (5 × 10(-2) mmol/L), but both did not down-regulate c-erbB2 expression. In contrast, the expressions of p-ERK and p-PKC were decreased obviously by c-erbB2 siRNA transfection. Conclusions c-erbB2 initiates rat primordial follicle growth via PKC and MAPK pathways, suggesting an important role of c-erbB2 in rat primordial follicle initiation and development. PMID:20565902

  2. Calphostin C, a remarkable multimodal photodynamic killer of neoplastic cells by selective nuclear lamin B1 destruction and apoptogenesis (Review).

    PubMed

    Chiarini, Anna; Whitfield, James F; Pacchiana, Raffaella; Marconi, Maddalena; Armato, Ubaldo; Dal Prà, Ilaria

    2010-04-01

    Perylenequinones that generate reactive oxygen species (ROS) when illuminated with visible light have been recommended as photodynamic chemotherapeutic agents. One of these is calphostin C (CalC), the action of the photo-activated derivative of which, CalCphiE, has been ascribed to its ability to selectively and irreversibly inhibit protein kinase Cs (PKCs). But recent results of experiments with neoplastic rat fibroblasts and human breast and uterine cervix cancer cells have revealed that the action of CalCphiE involves more than PKC inhibition. Besides suppressing PKC activity, CalCphiE rapidly causes endoplasmic reticulum (ER) stress in breast cancer cells and the selective complete oxidation and proteasomal destruction of the functionally essential nuclear envelope protein lamin B1, in human cervical carcinoma (HCC) cells and neoplastic rat fibroblasts. When these lamin B1-lacking cells are placed in the dark, cytoplasmic membrane-linked PKC activities suddenly rebound and apoptogenesis is initiated as indicated by the immediate release of cytochrome c from mitochondria and later on the activation of caspases. Hence, CalCphiE is a photodynamic cytocidal agent attacking multiple targets in cancer cells and it would be worth determining, even for their best applicative use, whether other perylenequinones also share the so far unexpectedly complex deadly properties of the CalCphiE.

  3. 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.

  4. VP-16 (etoposide) and calphostin C trigger different nuclear but akin cytoplasmic patterns of changes in the distribution and activity of protein kinase C-betaI in polyomavirus-transformed pyF111 rat fibroblasts.

    PubMed

    Chiarini, Anna; Whitfield, James F; Armato, Ubaldo; Dal Pra, Ilaria

    2006-01-01

    Protein kinase C (PKC) isoforms regulate cell proliferation and apoptosis. Since the PKC isoenzyme complement varies considerably from cell type to cell type, a PKC's responsiveness to an apoptogenic challenge must be defined for both the type of apoptogen and the type of cell. We have already reported that the changes in the distribution and activity of PKC-delta in apoptosing polyomavirus-infected/transformed Fischer rat embryo pyF111 fibroblasts depend on the type of apoptogen. Here, we show that this is also true for PKC-betaI in pyF111 cells treated with the slow DNA-damaging VP-16 (etoposide) or the fast-acting (in the cytoplasm) calphostin C. These apoptogens caused quite different shifts of the PKC-betaI level and activity in the nuclear membrane (NM) and nucleoplasm (NP), but corresponding changes in the cytosol (CS) and cytoplasmic particulate (CP) fractions. The hefty translocation of PKC-betaI onto the CP fraction and its increased activity there suggest the possible triggering of a cytochrome c/caspase-mediated apoptosis-inducing mechanism common to both agents. The present results are a necessary lead-up to functional proteomic analyses aimed at identifying the molecules forming the local PKC-betaI signalling modules under different conditions.

  5. The proto-oncogene c-src is involved in primordial follicle activation through the PI3K, PKC and MAPK signaling pathways

    PubMed Central

    2012-01-01

    Background C-src is an evolutionarily conserved proto-oncogene that regulates cell proliferation, differentiation and apoptosis. In our previous studies, we have reported that another proto-oncogene, c-erbB2, plays an important role in primordial follicle activation and development. We also found that c-src was expressed in mammalian ovaries, but its functions in primordial follicle activation remain unclear. The objective of this study is to investigate the role and mechanism of c-src during the growth of primordial follicles. Methods Ovaries from 2-day-old rats were cultured in vitro for 8 days. Three c-src-targeting and one negative control siRNA were designed and used in the present study. PCR, Western blotting and primordial follicle development were assessed for the silencing efficiency of the lentivirus c-src siRNA and its effect on primordial follicle onset. The expression of c-src mRNA and protein in primordial follicle growth were examined using the PCR method and immunohistochemical staining. Furthermore, the MAPK inhibitor PD98059, the PKC inhibitor Calphostin and the PI3K inhibitor LY294002 were used to explore the possible signaling pathways of c-src in primordial folliculogenesis. Results The results showed that Src protein was distributed in the ooplasmic membrane and the granulosa cell membrane in the primordial follicles, and c-src expression level increased with the growth of primordial follicle. The c-src -targeting lentivirus siRNAs had a silencing effect on c-src mRNA and protein expression. Eight days after transfection of rat ovaries with c-src siRNA, the GFP fluorescence in frozen ovarian sections was clearly discernible under a fluorescence microscope, and its relative expression level was 5-fold higher than that in the control group. Furthermore, the c-src-targeting lentivirus siRNAs lowered its relative expression level 1.96 times. We also found that the development of cultured primordial follicles was completely arrested after c-src si

  6. The proto-oncogene c-src is involved in primordial follicle activation through the PI3K, PKC and MAPK signaling pathways.

    PubMed

    Du, Xiao-Yu; Huang, Jian; Xu, Liang-Quan; Tang, Dan-Feng; Wu, Lei; Zhang, Li-Xia; Pan, Xiao-Ling; Chen, Wei-Yun; Zheng, Li-Ping; Zheng, Yue-Hui

    2012-08-20

    C-src is an evolutionarily conserved proto-oncogene that regulates cell proliferation, differentiation and apoptosis. In our previous studies, we have reported that another proto-oncogene, c-erbB2, plays an important role in primordial follicle activation and development. We also found that c-src was expressed in mammalian ovaries, but its functions in primordial follicle activation remain unclear. The objective of this study is to investigate the role and mechanism of c-src during the growth of primordial follicles. Ovaries from 2-day-old rats were cultured in vitro for 8 days. Three c-src-targeting and one negative control siRNA were designed and used in the present study. PCR, Western blotting and primordial follicle development were assessed for the silencing efficiency of the lentivirus c-src siRNA and its effect on primordial follicle onset. The expression of c-src mRNA and protein in primordial follicle growth were examined using the PCR method and immunohistochemical staining. Furthermore, the MAPK inhibitor PD98059, the PKC inhibitor Calphostin and the PI3K inhibitor LY294002 were used to explore the possible signaling pathways of c-src in primordial folliculogenesis. The results showed that Src protein was distributed in the ooplasmic membrane and the granulosa cell membrane in the primordial follicles, and c-src expression level increased with the growth of primordial follicle. The c-src -targeting lentivirus siRNAs had a silencing effect on c-src mRNA and protein expression. Eight days after transfection of rat ovaries with c-src siRNA, the GFP fluorescence in frozen ovarian sections was clearly discernible under a fluorescence microscope, and its relative expression level was 5-fold higher than that in the control group. Furthermore, the c-src-targeting lentivirus siRNAs lowered its relative expression level 1.96 times. We also found that the development of cultured primordial follicles was completely arrested after c-src siRNA knockdown of c

  7. Aldose reductase inhibitor improves insulin-mediated glucose uptake and prevents migration of human coronary artery smooth muscle cells induced by high glucose.

    PubMed

    Yasunari, K; Kohno, M; Kano, H; Minami, M; Yoshikawa, J

    2000-05-01

    We examined involvement of the polyol pathway in high glucose-induced human coronary artery smooth muscle cell (SMC) migration using Boyden's chamber method. Chronic glucose treatment for 72 hours potentiated, in a concentration-dependent manner (5.6 to 22.2 mol/L), platelet-derived growth factor (PDGF) BB-mediated SMC migration. This potentiation was accompanied by an increase in PDGF BB binding, because of an increased number of PDGF-beta receptors, and this potentiation was blocked by the aldose reductase inhibitor epalrestat. Epalrestat at concentrations of 10 and 100 nmol/L inhibited high glucose-potentiated (22.2 mmol/L), PDGF BB-mediated migration. Epalrestat at 100 nmol/L inhibited a high glucose-induced increase in the reduced/oxidized nicotinamide adenine dinucleotide ratio and membrane-bound protein kinase C (PKC) activity in SMCs. PKC inhibitors calphostin C (100 nmol/L) and chelerythrine (1 micromol/L) each inhibited high glucose-induced, PDGF BB-mediated SMC migration. High glucose-induced suppression of insulin-mediated [(3)H]-deoxyglucose uptake, which was blocked by both calphostin C (100 nmol/L) and chelerythrine (1 micromol/L), was decreased by epalrestat (100 nmol/L). Chronic high glucose treatment for 72 hours increased intracellular oxidative stress, which was directly measured by flow cytometry using carboxydichlorofluorescein diacetate bis-acetoxymethyl ester, and this increase was significantly suppressed by epalrestat (100 nmol/L). Antisense oligonucleotide to PKC-beta isoform inhibited high glucose-mediated changes in SMC migration, insulin-mediated [(3)H]-deoxyglucose uptake, and oxidative stress. These findings suggest that high glucose concentrations potentiate SMC migration in coronary artery and that the aldose reductase inhibitor epalrestat inhibits high glucose-potentiated, PDGF BB-induced SMC migration, possibly through suppression of PKC (PKC-beta), impaired insulin-mediated glucose uptake, and oxidative stress.

  8. 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.

  9. Involvement of HDAC1 and the PI3K/PKC signaling pathways in NF-{kappa}B activation by the HDAC inhibitor apicidin

    SciTech Connect

    Kim, Yong Kee . E-mail: yksnbk@kwandong.ac.kr; Seo, Dong-Wan; Kang, Dong-Won; Lee, Hoi Young; Han, Jeung-Whan; Kim, Su-Nam . E-mail: snkim@kist.re.kr

    2006-09-08

    Histone deacetylase (HDAC) inhibitors are appreciated as one of promising anticancer drugs, but they exert differential responses depending on the cell type. We recently reported the critical role of NF-{kappa}B as a modulator in determining cell fate for apoptosis in response to an HDAC inhibitor. In this study, we investigate a possible signaling pathway required for NF-{kappa}B activation in response to the HDAC inhibitor apicidin. Treatment of HeLa cells with apicidin leads to an increase in transcriptional activity of NF-{kappa}B and the expression of its target genes, IL-8 and TNF-{alpha}. TNF-{alpha} expression by apicidin is induced at earlier time points than NF-{kappa}B activation or IL-8 expression. In addition, our data show that the early expression of TNF-{alpha} does not lead to activation of NF-{kappa}B, because disruption of TNF-{alpha} activity by a neutralizing antibody does not affect nuclear translocation of NF-{kappa}B, I{kappa}B{alpha} degradation or reporter gene activation by apicidin. However, this activation of NF-{kappa}B requires the PI3K and PKC signaling pathways, but not ERK or JNK. Furthermore, apicidin activation of NF-{kappa}B seems to result from HDAC1 inhibition, as evidenced by the observation that overexpression of HDAC1, but not HDAC2, 3 or 4, dramatically inhibits NF-{kappa}B reporter gene activity. Collectively, our results suggest that activation of NF-{kappa}B signaling by apicidin requires both the PI3K/PKC signaling pathways and HDAC1, and functions as a critical modulator in determining the cellular effect of apicidin.

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. DNA repair contributes to the drug-resistant phenotype of primary acute myeloid leukaemia cells with FLT3 internal tandem duplications and is reversed by the FLT3 inhibitor PKC412.

    PubMed

    Seedhouse, C H; Hunter, H M; Lloyd-Lewis, B; Massip, A-M; Pallis, M; Carter, G I; Grundy, M; Shang, S; Russell, N H

    2006-12-01

    The presence of internal tandem duplications (ITD) mutations in the FMS-like tyrosine kinase 3 (FLT3) receptor influences the risk of relapse in acute myeloid leukaemia (AML). We have investigated DNA repair in FLT3-ITD and wild-type (WT) cells. Using the comet assay, we have demonstrated that the FLT3 inhibitor PKC412 significantly inhibits repair of DNA damage in the MV4-11-FLT3-ITD cell line and FLT3-ITD patient samples but not in the HL-60-FLT3-WT cell line or FLT3-WT patient samples. Following the discovery that transcript levels of the DNA repair gene RAD51 are significantly correlated with FLT3 transcript levels in FLT3-ITD patients, we further investigated the role of RAD51 in FLT3-ITD-AML. The reduction in DNA repair in PKC412-treated FLT3-ITD cells was shown to be associated with downregulation of RAD51 mRNA and protein expression and correlates with the maintenance of phosphorylated H2AX levels, implying that PKC412 inhibits the homologous recombination double-strand break repair pathway in FLT3-ITD cells. Using FLT3-short interfering RNA (siRNA), we also demonstrated that genetic silencing of FLT3 results in RAD51 downregulation in FLT3-ITD cells but not in FLT3-WT cells. This work suggests that the use of FLT3 inhibitors such as PKC412 may reverse the drug-resistant phenotype of FLT3-ITD-AML cells by inhibiting repair of chemotherapy-induced genotoxic damage and thereby reduce the risk of disease relapse.

  15. PGE2 upregulates renin through E-prostanoid receptor 1 via PKC/cAMP/CREB pathway in M-1 Cells.

    PubMed

    Gonzalez, Alexis A; Salinas-Parra, Nicolas; Leach, Dan; Navar, L Gabriel; Prieto, Minolfa C

    2017-07-12

    During the early phase of angiotensin (ANG) II-dependent hypertension tubular prostaglandin E2 (PGE2) is increased. Renin synthesis and secretion in the collecting duct (CD) is upregulated by ANGII contributing to further intratubular ANGII formation. However, what happens first and whether the triggering mechanism is independent of tubular ANGII, remain unknown. PGE2 stimulates renin synthesis in juxtaglomerular (JG) cells via E-prostanoid (EP) receptors through cAMP/CREB pathway. EP receptors are also expressed in the CD. Here, we tested the hypothesis that renin is upregulated by PGE2 in CD cells. M-1 CD cell line expressed EP1, EP3 and EP4 but not EP2. Dose response experiments in the presence of AT1 receptor blockade with candesartan demonstrated that 10-6 M PGE2 maximally increases renin mRNA (~4 fold) and prorenin/renin protein levels (~2 fold). This response was prevented by micromolar doses of SC-19220 (EP1 antagonist), attenuated by the EP4 antagonist, L-161,982, and exacerbated by the highly selective EP3 antagonist, L-798106 (~10 fold increase). To further evaluate the signaling pathway involved we used the PKC inhibitor calphostin C and transfections with PKCα dominant negative (DN). Both strategies blunted the PGE2-induced increases in cAMP levels, CREB phosphorylation and augmentation of renin. Knockdown of EP1 receptor and CREB also prevented renin upregulation. These results indicate that PGE2 increases CD renin expression through EP1 receptor via PKC/cAMP/CREB pathway. Therefore, we conclude that during early stages of ANGII-dependent hypertension, there is augmentation of PGE2 that stimulates renin in the CD, resulting in increased tubular ANGII formation and further stimulation of renin. Copyright © 2017, American Journal of Physiology-Renal Physiology.

  16. Protein kinase C (PKC) dependent induction of tissue factor (TF) by mesangial cells in response to inflammatory mediators and release during apoptosis

    PubMed Central

    Lang, Detlef; Terstesse, Martin; Dohle, Frank; Bangen, Philip; Banas, Bernhard; Pauels, Hans-Gerd; Heidenreich, Stefan

    2002-01-01

    In inflammatory kidney diseases procoagulatory activity (PCA) becomes evident. Glomerular fibrin deposits and capillary microthrombi are histopathological hallmarks in most forms of glomerulonephritis. Therefore in this study the expression of tissue factor (TF) as the main inducer of thrombogenesis was examined in cultured human mesangial cells (MC) in response to proinflammatory stimuli such as interleukin-1 (IL-1β), tumour necrosis factor alpha (TNF-α) and lipopolysaccharide (LPS). Also main signalling pathways were investigated. IL-1β, TNF-α and LPS induced TF in MC in a time and dose dependent manner on mRNA and protein levels. Highest activity was found after 12 h of stimulation. Induction of TF was completely blockable by BAPTA-AM, a chelator of intracellular [Ca2+]i as well as calphostin, a protein kinase C (PKC) inhibitor. Activation of the protein kinase A (PKA) pathway had no influence on basal TF expression, but down-regulated cytokine-induced TF. The PKA blocker, KT5720, increased TF formation significantly. Since TF exerts its activity primarily on the surface of cells and after release of encrypted receptors we further tested TF activity in MC supernatants. IL-1β did not significantly increase TF activity in supernatants of intact cells. However, when MC were rendered apoptotic by oxidative metabolites, IL-1β treated MC released highly stimulated TF activity into the supernatants, suggesting that a paracrine activation of the coagulatory cascade can take place under such conditions. Inflammatory mediators up-regulate TF expression in MC by a PKC dependent pathway whereas PKA can serve as a negative feed-back link. Apoptosis of inflammatory MC may trigger to spread PCA. PMID:12429585

  17. 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.

  18. ATP competitive protein kinase C inhibitors demonstrate distinct state-dependent inhibition.

    PubMed

    Smith, Ida M; Hoshi, Naoto

    2011-01-01

    We previously reported that some ATP competitive protein kinase C (PKC) inhibitors are either competitive or uncompetitive inhibitors with respect to substrate peptides. In this report, we demonstrate how the interactions between PKC and inhibitors change PKC activation kinetics. A substrate competitive inhibitor, bisindolylmaleimide I, targets activated PKC and stabilizes PKC in the activated conformation. This leads to transient activation and prolonged deactivation of PKC in the presence of bisindolylmaleimide I. In contrast, an uncompetitive substrate inhibitor, bisindolylmaleimide IV, targets quiescent PKC and stabilizes PKC in the quiescent conformation, which generates slower activation and suppressed translocation upon activation of PKC.

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

    PubMed Central

    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

  20. 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.

  1. 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.

  2. 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

  3. 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…

  4. 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…

  5. 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.

  6. 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

  7. Protein kinase C (PKC) participates in acetaminophen hepatotoxicity through JNK dependent and independent signaling pathways

    PubMed Central

    Johnson, Heather S.; Gaarde, William A.; Han, Derick; Kaplowitz, Neil

    2013-01-01

    This study examines the role of protein kinase C (PKC) and AMP-activated kinase (AMPK) in acetaminophen (APAP) hepatotoxicity. Treatment of primary mouse hepatocytes with broad-spectrum PKC inhibitors (Ro-31-8245, Go6983), protected against APAP cytotoxicity despite sustained JNK activation. Broad-spectrum PKC inhibitor treatment enhanced p-AMPK levels and AMPK regulated survival-energy pathways including autophagy. AMPK inhibition by compound C or activation using an AMPK activator oppositely modulated APAP cytotoxicity, suggesting p-AMPK and AMPK regulated energy survival pathways particularly autophagy play a critical role in APAP cytotoxicity. Ro-31-8245 treatment in mice upregulated p-AMPK levels, increased autophagy (i.e. increased LC3-II formation, p62 degradation) and protected against APAP-induced liver injury, even in the presence of sustained JNK activation and translocation to mitochondria. In contrast, treatment of hepatocytes with classical PKC inhibitor (Go6976) protected against APAP by inhibiting JNK activation. Knockdown of PKC-α using antisense (ASO) in mice also protected against APAP-induced liver injury by inhibiting JNK activation. APAP treatment resulted in PKC-α translocation to mitochondria and phosphorylation of mitochondrial PKC substrates. JNK 1 and 2 silencing in vivo decreased APAP-induced PKC-α translocation to mitochondria, suggesting PKC-α and JNK interplay in a feed-forward mechanism to mediate APAP-induced liver injury. Conclusion: PKC-α and other PKC(s) regulate death (JNK) and survival (AMPK) proteins, to modulate APAP-induced liver injury. PMID:23873604

  8. 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

    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

    2016-05-05

    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.

  9. 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

  10. 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.

  11. 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.

  12. Isoform-specific translocation of PKC isoforms in NIH3T3 cells by TPA

    SciTech Connect

    Kazi, Julhash U.; Soh, Jae-Won

    2007-12-14

    Protein kinase C (PKC), a multi-gene family of enzymes, plays key roles in the pathways of signal transduction, growth control and tumorigenesis. Variations in the intracellular localization of the individual isoforms are thought to be an important mechanism for the isoform-specific regulation of enzyme activity and substrate specificity. To provide a dynamic method of analyzing the localization of the specific isoforms of PKC in living cells, we generated fluorescent fusion proteins of the various PKC isoforms by using the green fluorescent protein (GFP) as a fluorescent marker at the carboxyl termini of these enzymes. The intracellular localization of the specific PKC isoforms was then examined by fluorescence microscopy after transient transfection of the respective PKC-GFP expression vector into NIH3T3 cells and subsequent TPA stimulation. We found that the specific isoforms of PKC display distinct localization patterns in untreated NIH3T3 cells. For example, PKC{alpha} is localized mainly in the cytoplasm while PKC{epsilon} is localized mainly in the Golgi apparatus. We also observed that PKC{alpha}, {beta}1, {beta}2, {gamma}, {delta}, {epsilon}, and {eta} translocate to the plasma membrane within 10 min of the start of TPA treatment, while the cellular localizations of PKC{zeta} and {iota} were not affected by TPA. Using a protein kinase inhibitor, we also showed that the kinase activity was not important for the translocation of PKC. These results suggest that specific PKC isoforms exert spatially distinct biological effects by virtue of their directed translocation to different intracellular sites.

  13. Combined PKC and MEK inhibition for treating metastatic uveal melanoma.

    PubMed

    Sagoo, M S; Harbour, J W; Stebbing, J; Bowcock, A M

    2014-09-25

    Uveal melanoma (UM) is the most common primary intraocular malignancy and the second most common form of melanoma. UM has a strong tendency for metastatic disease, and no effective treatments have yet been identified. Activating oncogenic mutations are commonly found in GNAQ and GNA11 in UM, and inhibiting key downstream effectors of the GNAQ/11 signaling pathway represents a rational therapeutic approach for treating metastatic UM. Chen et al., doi:10.1038/onc.2013.418, now confirm activation of the MAPK and PKC pathways as a result of GNAQ and GNA11 activating mutations in melanocytes, and they demonstrate that MAPK activation occurs downstream of PKC activation. PKC inhibitors disrupt MAPK signaling and block proliferation of GNAQ/11 mutant UM cell lines and slow the in vivo growth of xenografted UM tumors without inducing their shrinkage. However, a combination of PKC and MEK inhibition led to sustained MAPK pathway inhibition and tumor regression in vivo. Hence, the authors concluded that MEK and PKC inhibition is synergistic, with superior efficacy to treatment of GNAQ/GNA11 mutant UMs with either drug alone.

  14. Ca2+ oscillation induced by P2Y2 receptor activation and its regulation by a neuron-specific subtype of PKC (gammaPKC).

    PubMed

    Ashida, Noriaki; Ueyama, Takehiko; Rikitake, Kyoko; Shirai, Yasuhito; Eto, Mika; Kondoh, Takeshi; Kohmura, Eiji; Saito, Naoaki

    2008-12-03

    We found that stimulation of P2Y2 receptor (P2Y2R), which is endogenously expressed in CHO-K1 cells, induced intracellular calcium ([Ca2+]i) oscillation with a low frequency of 11.4 +/- 2.7 mHz. When CHO-K1 cells expressing GFP-tagged kinase-negative gammaPKC (gammaPKC-KN-GFP), which is a neuron-specific subtype of PKC, were stimulated with UDP, gammaPKC-KN-GFP, but not wild-type gammaPKC (gammaPKC-GFP) showed an oscillatory translocation. The oscillatory translocation of gammaPKC-KN-GFP corresponded with [Ca2+]i oscillation, which was not observed in the cells expressing gammaPKC-GFP. We examined the mechanism ofP2Y2R-induced [Ca2+]i oscillation pharmacologically. gammaPKC-KN-GFP oscillation was stopped by an extracellular Ca2+ chelator, EGTA, an antagonist of P2Y2R, Suramin, and store-operated calcium channel (SOC) inhibitors, SKF96365 and 2-ABP. Taken together, P2Y2R-induced [Ca2+]i oscillation in CHO-K1 cells is related with Ca2+ influx through SOC, whose function may be negatively regulated by gammaPKC. This [Ca2]i oscillation was distinct from that induced by metabotropic glutamate receptor 5 (mGluR5) stimulation in the frequency (72.3 +/- 5.3 mHz) and in the regulatory mechanism.

  15. 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.

  16. PKC in rat dorsal raphe nucleus plays a key role in sleep-wake regulation.

    PubMed

    Li, Sheng-Jie; Cui, Su-Ying; Zhang, Xue-Qiong; Yu, Bin; Sheng, Zhao-Fu; Huang, Yuan-Li; Cao, Qing; Xu, Ya-Ping; Lin, Zhi-Ge; Yang, Guang; Cui, Xiang-Yu; Zhang, Yong-He

    2015-12-03

    Studies suggest a tight relationship between protein kinase C (PKC) and circadian clock. However, the role of PKC in sleep-wake regulation remains unclear. The present study was conducted to investigate the role of PKC signaling in sleep-wake regulation in the rat. Our results showed that the phosphorylation level of PKC in dorsal raphe nucleus (DRN) was decreased after 6h sleep deprivation, while no alterations were found in ventrolateral preoptic nucleus (VLPO) or locus coeruleus (LC). Microinjection of a pan-PKC inhibitor, chelerythrine chloride (CHEL, 5 or 10nmol), into DRN of freely moving rats promoted non rapid eye movement sleep (NREMS) without influences on rapid eye movement sleep (REMS). Especially, CHEL application at 5nmol increased light sleep (LS) time while CHEL application at 10nmol increased slow wave sleep (SWS) time and percentage. On the other hand, microinjection of CaCl2 into DRN not only increased the phosphorylation level of PKC, but also reduced NREMS time, especially SWS time and percentage. While CHEL abolished the inhibitory effect of CaCl2 on NREMS and SWS. These data provide the first direct evidence that inhibition of intracellular PKC signaling in DRN could increase NREMS time including SWS time and percentage, while activation of PKC could suppress NREMS and reduce SWS time and percentage. These novel findings further our understanding of the basic cellular and molecular mechanisms of sleep-wake regulation. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Role of Calcium and PKC in Salivary Mucous Cell Exocrine Secretion

    PubMed Central

    Culp, D.J.; Zhang, Z.; Evans, R.L.

    2011-01-01

    Fluid and exocrine secretion of mucins by salivary mucous glands is regulated predominantly by parasympathetic activation of muscarinic receptors. A direct role for subsequent putative signaling steps, phospholipase C (PLC), increased intracellular calcium ([Ca2+]i), and isoforms of protein kinase C (PKC) in mediating muscarinic exocrine secretion has not been elucidated, and these are potential therapeutic targets to enhance mucin secretion in hyposalivary patients. We found that muscarinic-induced mucin secretion by rat sublingual tubulo-acini was dependent upon PLC activation and the subsequent increase in [Ca2+]i, and further identified a transient PKC-independent component of secretion dependent upon Ca2+ release from intracellular stores, whereas sustained secretion required entry of extracellular Ca2+. Interactions among carbachol, PKC inhibitors, phorbol 12-myristate 13-acetate, and thapsigargin to modulate [Ca2+]i implicated conventional PKC isoforms in mediating sustained secretion. With increasing times during carbachol perfusion of glands, in situ, PKC-α redistributed across glandular membrane compartments and underwent a rapid and persistent accumulation near the luminal borders of mucous cells. PKC-β1 displayed transient localization near luminal borders, whereas the novel PKCs, PKC-δ or PKC-ϵ, displayed little or no redistribution in mucous cells. Collective results implicate synergistic interactions between diacylglycerol (DAG) and increasing [Ca2+]i levels to activate cPKCs in mediating sustained muscarinic-induced secretion. PMID:21933938

  18. 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

  19. 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(-).

  20. Combined PKC and MEK inhibition in uveal melanoma with GNAQ and GNA11 mutations

    PubMed Central

    Chen, Xu; Wu, Qiuxia; Tan, Lujian; Porter, Dale; Jager, Martine J.; Emery, Caroline; Bastian, Boris C.

    2015-01-01

    Uveal melanoma (UM) is a genetically and biologically distinct type of melanoma, and once metastatic there is no effective treatment currently available. 80% of UMs harbor mutations in the Gαq family members GNAQ and GNA11. Understanding the effector pathways downstream of these oncoproteins is important to identify opportunities for targeted therapy. We report consistent activation of the protein kinase C (PKC) and MAPK pathways as a consequence of GNAQ or GNA11 mutation. PKC inhibition with AEB071 or AHT956 suppressed PKC and MAPK signalling and induced G1 arrest selectively in melanoma cell lines carrying GNAQ or GNA11 mutations. In contrast, treatment with two different MEK inhibitors, PD0325901 and MEK162, inhibited the proliferation of melanoma cell lines irrespective of their mutation status, indicating that in the context of GNAQ or GNA11 mutation, MAPK activation can be attributed to activated PKC. AEB071 significantly slowed the growth of tumors in an allograft model of GNAQQ209L transduced melanocytes, but did not induce tumor shrinkage. In vivo and in vitro studies showed that PKC inhibitors alone were unable to induce sustained suppression of MAP-kinase signaling. However, combinations of PKC and MEK inhibition, using either PD0325901 or MEK162, led to sustained MAP-kinase pathway inhibition and showed a strong synergistic effect in halting proliferation and in inducing apoptosis in vitro. Furthermore, combining PKC and MEK inhibition was efficacious in vivo, causing marked tumor regression in a uveal melanoma xenograft model. Our data identifies PKC as a rational therapeutic target for melanoma patients with GNAQ or GNA11 mutations, and demonstrates combined MEK and PKC inhibition is synergistic, with superior efficacy compared to treatment with either approach alone. PMID:24141786

  1. Combined PKC and MEK inhibition in uveal melanoma with GNAQ and GNA11 mutations.

    PubMed

    Chen, X; Wu, Q; Tan, L; Porter, D; Jager, M J; Emery, C; Bastian, B C

    2014-09-25

    Uveal melanoma (UM) is a genetically and biologically distinct type of melanoma, and once metastatic there is no effective treatment currently available. Eighty percent of UMs harbor mutations in the Gαq family members GNAQ and GNA11. Understanding the effector pathways downstream of these oncoproteins is important to identify opportunities for targeted therapy. We report consistent activation of the protein kinase C (PKC) and MAPK pathways as a consequence of GNAQ or GNA11 mutation. PKC inhibition with AEB071 or AHT956 suppressed PKC and MAPK signalling and induced G1 arrest selectively in melanoma cell lines carrying GNAQ or GNA11 mutations. In contrast, treatment with two different MEK inhibitors, PD0325901 and MEK162, inhibited the proliferation of melanoma cell lines irrespective of their mutation status, indicating that in the context of GNAQ or GNA11 mutation MAPK activation can be attributed to activated PKC. AEB071 significantly slowed the growth of tumors in an allograft model of GNAQ(Q209L)-transduced melanocytes, but did not induce tumor shrinkage. In vivo and in vitro studies showed that PKC inhibitors alone were unable to induce sustained suppression of MAP-kinase signaling. However, combinations of PKC and MEK inhibition, using either PD0325901or MEK162, led to sustained MAP-kinase pathway inhibition and showed a strong synergistic effect in halting proliferation and in inducing apoptosis in vitro. Furthermore, combining PKC and MEK inhibition was efficacious in vivo, causing marked tumor regression in a UM xenograft model. Our data identify PKC as a rational therapeutic target for melanoma patients with GNAQ or GNA11 mutations and demonstrate that combined MEK and PKC inhibition is synergistic, with superior efficacy compared to treatment with either approach alone.

  2. 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

  3. 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.

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

    PubMed

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

    2011-06-01

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

  5. Interaction with AKAP79 modifies the cellular pharmacology of PKC.

    PubMed

    Hoshi, Naoto; Langeberg, Lorene K; Gould, Christine M; Newton, Alexandra C; Scott, John D

    2010-02-26

    A-kinase anchoring proteins (AKAPs) coordinate cell signaling events. AKAP79 brings together different combinations of enzyme binding partners to customize the regulation of effector proteins. In neurons, muscarinic agonists mobilize an AKAP79-anchored pool of PKC that phosphorylates the KCNQ2 subunit of the M channel. This inhibits potassium permeability to enhance neuronal excitability. Using a dual fluorescent imaging/patch-clamp technique, we visualized AKAP79-anchored PKC phosphorylation of the kinase activity reporter CKAR concurrently with electrophysiological changes in KCNQ2 channels to show that AKAP79 synchronizes both signaling events to optimize the attenuation of M currents. AKAP79 also protects PKC from certain ATP-competitive inhibitors. Related studies suggest that context-dependent protein-protein interactions alter the susceptibility of another protein kinase, PDK1, to ATP analog inhibitors. This implies that intracellular binding partners not only couple individual molecular events in a cell signaling process but can also change the pharmacological profile of certain protein kinases. Copyright 2010 Elsevier Inc. All rights reserved.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Phosphoproteomics profiling suggests a role for nuclear βΙPKC in transcription processes of undifferentiated murine embryonic stem cells.

    PubMed

    Costa-Junior, Helio Miranda; Garavello, Nicole Milaré; Duarte, Mariana Lemos; Berti, Denise Aparecida; Glaser, Talita; de Andrade, Alexander; Labate, Carlos A; Ferreira, André Teixeira da Silva; Perales, Jonas Enrique Aguilar; Xavier-Neto, José; Krieger, José Eduardo; Schechtman, Deborah

    2010-12-03

    Protein kinase C (PKC) plays a key role in embryonic stem cell (ESC) proliferation, self-renewal, and differentiation. However, the function of specific PKC isoenzymes have yet to be determined. Of the PKCs expressed in undifferentiated ESCs, βIPKC was the only isoenzyme abundantly expressed in the nuclei. To investigate the role of βΙPKC in these cells, we employed a phosphoproteomics strategy and used two classical (cPKC) peptide modulators and one βIPKC-specific inhibitor peptide. We identified 13 nuclear proteins that are direct or indirect βΙPKC substrates in undifferentiated ESCs. These proteins are known to be involved in regulating transcription, splicing, and chromatin remodeling during proliferation and differentiation. Inhibiting βΙPKC had no effect on DNA synthesis in undifferentiated ESCs. However, upon differentiation, many cells seized to express βΙPKC and βΙPKC was frequently found in the cytoplasm. Taken together, our results suggest that βIPKC takes part in the processes that maintain ESCs in their undifferentiated state.

  12. 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.

  13. Hypertensive encephalopathy and the blood-brain barrier: is deltaPKC a gatekeeper?

    PubMed

    Chou, Wen-Hai; Messing, Robert O

    2008-01-01

    Hypertensive encephalopathy is a life-threatening condition due to elevation of cerebral perfusion pressure beyond the limits of autoregulation. Breakdown of the blood-brain barrier (BBB) leads to cerebral edema and reduced blood flow. In this issue of the JCI, Mochly-Rosen and colleagues demonstrate a novel molecular strategy for preserving the BBB in a model of hypertension-induced encephalopathy (see the related article beginning on page 173). Using a rationally designed peptide inhibitor of deltaPKC, they stabilized the BBB and improved mortality in hypertensive rats. This study highlights the therapeutic potential of deltaPKC inhibitors in hypertensive encephalopathy and provides incentive to elucidate deltaPKC signaling pathways that mediate BBB dysfunction in other disease states.

  14. 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.

  15. 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

  16. Protein kinase C (PKC) participates in acetaminophen hepatotoxicity through c-jun-N-terminal kinase (JNK)-dependent and -independent signaling pathways.

    PubMed

    Saberi, Behnam; Ybanez, Maria D; Johnson, Heather S; Gaarde, William A; Han, Derick; Kaplowitz, Neil

    2014-04-01

    This study examines the role of protein kinase C (PKC) and AMP-activated kinase (AMPK) in acetaminophen (APAP) hepatotoxicity. Treatment of primary mouse hepatocytes with broad-spectrum PKC inhibitors (Ro-31-8245, Go6983), protected against APAP cytotoxicity despite sustained c-jun-N-terminal kinase (JNK) activation. Broad-spectrum PKC inhibitor treatment enhanced p-AMPK levels and AMPK regulated survival-energy pathways including autophagy. AMPK inhibition by compound C or activation using an AMPK activator oppositely modulated APAP cytotoxicity, suggesting that p-AMPK and AMPK regulated energy survival pathways, particularly autophagy, play a critical role in APAP cytotoxicity. Ro-31-8245 treatment in mice up-regulated p-AMPK levels, increased autophagy (i.e., increased LC3-II formation, p62 degradation), and protected against APAP-induced liver injury, even in the presence of sustained JNK activation and translocation to mitochondria. In contrast, treatment of hepatocytes with a classical PKC inhibitor (Go6976) protected against APAP by inhibiting JNK activation. Knockdown of PKC-α using antisense (ASO) in mice also protected against APAP-induced liver injury by inhibiting JNK activation. APAP treatment resulted in PKC-α translocation to mitochondria and phosphorylation of mitochondrial PKC substrates. JNK 1 and 2 silencing in vivo decreased APAP-induced PKC-α translocation to mitochondria, suggesting PKC-α and JNK interplay in a feed-forward mechanism to mediate APAP-induced liver injury. PKC-α and other PKC(s) regulate death (JNK) and survival (AMPK) proteins, to modulate APAP-induced liver injury. Copyright © 2014 by the American Association for the Study of Liver Diseases.

  17. PKC?-dependent activation of the ubiquitin proteasome system is responsible for high glucose-induced human breast cancer MCF-7 cell proliferation, migration and invasion.

    PubMed

    Zhu, Shan; Yao, Feng; Li, Wen-Huan; Wan, Jin-Nan; Zhang, Yi-Min; Tang, Zhao; Khan, Shahzad; Wang, Chang-Hua; Sun, Sheng-Rong

    2013-01-01

    Type 2 diabetes mellitus (T2DM) has contributed to advanced breast cancer development over the past decades. However, the mechanism underlying this contribution is poorly understood. In this study, we determined that high glucose enhanced proteasome activity was accompanied by enhanced proliferation, migration and invasion, as well as suppressed apoptosis, in human breast cancer MCF-7 cells. Proteasome inhibitor bortezomib (BZM) pretreatment mitigated high glucose-induced MCF-7 cell growth and invasion. Furthermore, high glucose increased protein kinase C delta (PKC?)-phosphorylation. Administration of the specific PKC? inhibitor rottlerin attenuated high glucose-stimulated cancer cell growth and invasion. In addition, PKC? inhibition by both rottlerin and PKC? shRNA significantly suppressed high glucose-induced proteasome activity. Our results suggest that PKC?-dependent ubiquitin proteasome system activation plays an important role in high glucose- induced breast cancer cell growth and metastasis.

  18. 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.

  19. Lysophosphatidic acid upregulates connective tissue growth factor expression in osteoblasts through the GPCR/PKC and PKA pathways.

    PubMed

    Yu, Zi-Li; Li, Dian-Qi; Huang, Xiang-Yu; Xing, Xin; Yu, Ru-Qing; Li, Zhi; Li, Zu-Bing

    2016-02-01

    Lysophosphatidic acid (LPA) is an efficient, bioactive phospholipid involved in various biological processes. In this study, LPA-induced connective tissue growth factor (CTGF/CCN2) expression and the underlying mechanisms were investigated using the MC3T3-E1 cell line. The MC3T3-E1 cells were stimulated with an inhibitor of LPA receptors, an activator and inhibitor of protein kinase C (PKC) and protein kinase A (PKA) for indicated periods of time. RT-qPCR and western blot analyses were used to measure the expression levels of CCN2. Immunofluorescence staining was used to observe the translocation of PKC. The mRNA expression level of CCN2 was increased following stimulation of the cells with LPA; LPA transiently induced the mRNA expression of CCN2; maximum expression levels were observed 2 h following stimulation with LPA. This increase was accompanied by CCN2 protein synthesis. LPA receptor1/3 was inhibited by Ki16425, a specific inhibitor of LPA1/3; as a result, the LPA-induced increase in CCN2 expression was abrogated. LPA also induced the membrane translocation of PKC and enhanced PKC activity in the osteoblasts. Pre-treatment of the osteoblasts with staurosporine prevented the increase in CCN2 expression by induced by LPA, and the activation of PKC by phorbol 12-myristate 13-acetate (PMA) enhanced CCN2 expression, indicating that the PKC pathway is involved in the LPA-induced increase in CCN2 expression. The interference of PKA signaling also led to the induction of CCN2 expresion by LPA. These data indicate that LPA increases CCN2 expression through the activation of PKC and PKA. Thus, the regulatory functions of the PKC and PKA pathways are implicated in the LPA-induced increase in CCN2 expression.

  20. 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.

  1. 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

  2. Enhancement of potency and efficacy of NADA by PKC-mediated phosphorylation of vanilloid receptor.

    PubMed

    Premkumar, Louis S; Qi, Zhan-Heng; Van Buren, Jeremy; Raisinghani, Manish

    2004-03-01

    The search for an endogenous ligand for the vanilloid receptor (VR or TRPV1) has led to the identification of N-arachidonyl dopamine (NADA). This study investigates the role of protein kinase C (PKC)-mediated phosphorylation on NADA-induced membrane currents in Xenopus oocytes heterologously expressing TRPV1 and in dorsal root ganglion (DRG) neurons. In basal state, current induced by 10 microM NADA is 5-10% of the current induced by 1 microM capsaicin or protons at pH 5. However, PKC activator, phorbol 12,13-dibutyrate (PDBu) strongly potentiated ( approximately 15-fold) the NADA-induced current. Repeated application of NADA at short intervals potentiated its own response approximately fivefold in a PKC-dependent manner. PKC inhibitor, bisindolylmaleimide (BIM, 500 nM), a mutant TRPV1 (S800A/S502A), and maximal activation of PKC abolished the potentiation induced by repeated application of NADA. As a further confirmation that NADA could stimulate PKC, pretreatment with NADA potentiated the response of protons at pH 5 (approximately 20 fold), which was dramatically reduced in the mutant TRPV1. In DRG neurons, capsaicin (100 nM) induced a approximately 15 mV depolarization and initiated a train of action potentials compared with 1 microM NADA that produced a approximately 5 mV response. Pretreatment with PDBu induced significantly larger depolarization and potentiated NADA-induced current. Furthermore, exposure of NADA to the intracellular surface of the membrane-induced larger currents suggesting inaccessibility to the intracellular binding site might contribute to its weaker action. These results indicate that NADA is a potent agonist of VR when the receptor is in the PKC-mediated phosphorylation state.

  3. 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.

  4. Targeting aPKC disables oncogenic signaling by both the EGFR and the proinflammatory cytokine TNFα in glioblastoma

    PubMed Central

    Perry, Anthony S.; Rushing, Elisabeth J.; Mandell, Edward K.; Dietrich, Justin D.; Errasti, Andrea E.; Gibbs, Daniel; Berens, Michael E.; Loftus, Joseph C.; Hulme, Christopher; Yang, Weiwei; Lu, Zhimin; Aldape, Kenneth; Sanai, Nader; Rothlin, Carla V.; Ghosh, Sourav

    2015-01-01

    Grade IV glioblastoma is characterized by increased kinase activity of epidermal growth factor receptor (EGFR); however, EGFR kinase inhibitors have failed to improve survival in individuals with this cancer because resistance to these drugs often develops. We showed that tumor necrosis factor–α (TNFα) produced in the glioblastoma microenvironment activated atypical protein kinase C (aPKC), thereby producing resistance to EGFR kinase inhibitors. Additionally, we identified that aPKC was required both for paracrine TNFα-dependent activation of the transcription factor nuclear factor κB (NF-κB) and for tumor cell–intrinsic receptor tyrosine kinase signaling. Targeting aPKC decreased tumor growth in mouse models of glioblastoma, including models of EGFR kinase inhibitor–resistant glioblastoma. Furthermore, aPKC abundance and activity were increased in human glioblastoma tumor cells, and high aPKC abundance correlated with poor prognosis. Thus, targeting aPKC might provide an improved molecular approach for glioblastoma therapy. PMID:25118327

  5. Tauroursodeoxycholic acid exerts anticholestatic effects by a cooperative cPKC alpha-/PKA-dependent mechanism in rat liver.

    PubMed

    Wimmer, R; Hohenester, S; Pusl, T; Denk, G U; Rust, C; Beuers, U

    2008-10-01

    Ursodeoxycholic acid (UDCA) exerts anticholestatic effects in part by protein kinase C (PKC)-dependent mechanisms. Its taurine conjugate, TUDCA, is a cPKC alpha agonist. We tested whether protein kinase A (PKA) might contribute to the anticholestatic action of TUDCA via cooperative cPKC alpha-/PKA-dependent mechanisms in taurolithocholic acid (TLCA)-induced cholestasis. In perfused rat liver, bile flow was determined gravimetrically, organic anion secretion spectrophotometrically, lactate dehydrogenase (LDH) release enzymatically, cAMP response-element binding protein (CREB) phosphorylation by immunoblotting, and cAMP by immunoassay. PKC/PKA inhibitors were tested radiochemically. In vitro phosphorylation of the conjugate export pump, Mrp2/Abcc2, was studied in rat hepatocytes and human Hep-G2 hepatoma cells. In livers treated with TLCA (10 micromol/l)+TUDCA (25 micromol/l), combined inhibition of cPKC by the cPKC-selective inhibitor Gö6976 (100 nmol/l) or the non-selective PKC inhibitor staurosporine (10 nmol/l) and of PKA by H89 (100 nmol/l) reduced bile flow by 36% (p<0.05) and 48% (p<0.01), and secretion of the Mrp2/Abcc2 substrate, 2,4-dinitrophenyl-S-glutathione, by 31% (p<0.05) and 41% (p<0.01), respectively; bile flow was unaffected in control livers or livers treated with TUDCA only or TLCA+taurocholic acid. Inhibition of cPKC or PKA alone did not affect the anticholestatic action of TUDCA. Hepatic cAMP levels and CREB phosphorylation as readout of PKA activity were unaffected by the bile acids tested, suggesting a permissive effect of PKA for the anticholestatic action of TUDCA. Rat and human hepatocellular Mrp2 were phosphorylated by phorbol ester pretreatment and recombinant cPKC alpha, nPKC epsilon, and PKA, respectively, in a staurosporine-sensitive manner. UDCA conjugates exert their anticholestatic action in bile acid-induced cholestasis in part via cooperative post-translational cPKC alpha-/PKA-dependent mechanisms. Hepatocellular Mrp2 may be one

  6. PKC-mediated cerebral vasoconstriction: Role of myosin light chain phosphorylation versus actin cytoskeleton reorganization.

    PubMed

    El-Yazbi, Ahmed F; Abd-Elrahman, Khaled S; Moreno-Dominguez, Alejandro

    2015-06-15

    Defective protein kinase C (PKC) signaling has been suggested to contribute to abnormal vascular contraction in disease conditions including hypertension and diabetes. Our previous work on agonist and pressure-induced cerebral vasoconstriction implicated PKC as a major contributor to force production in a myosin light chain (LC20) phosphorylation-independent manner. Here, we used phorbol dibutyrate to selectively induce a PKC-dependent constriction in rat middle cerebral arteries and delineate the relative contribution of different contractile mechanisms involved. Specifically, we employed an ultra-sensitive 3-step western blotting approach to detect changes in the content of phosphoproteins that regulate myosin light chain phosphatase (MLCP) activity, thin filament activation, and actin cytoskeleton reorganization. Data indicate that PKC activation evoked a greater constriction at a similar level of LC20 phosphorylation achieved by 5-HT. PDBu-evoked constriction persisted in the presence of Gö6976, a selective inhibitor of Ca(2+)-dependent PKC, and in the absence of extracellular Ca(2+). Biochemical evidence indicates that either + or - extracellular Ca(2+), PDBu (i) inhibits MLCP activity via the phosphorylation of myosin targeting subunit of myosin phosphatase (MYPT1) and C-kinase potentiated protein phosphatase-1 inhibitor (CPI-17), (ii) increases the phosphorylation of paxillin and heat shock protein 27 (HSP27), and reduces G-actin content, and (iii) does not change the phospho-content of the thin filament proteins, calponin and caldesmon. PDBu-induced constriction was more sensitive to disruption of actin cytoskeleton compared to inhibition of cross-bridge cycling. In conclusion, this study provided evidence for the pivotal contribution of cytoskeletal actin polymerization in force generation following PKC activation in cerebral resistance arteries. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. 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.

  8. Interleukin-1β-induced barrier dysfunction is signaled through PKC-θ in human brain microvascular endothelium.

    PubMed

    Rigor, Robert R; Beard, Richard S; Litovka, Olesya P; Yuan, Sarah Y

    2012-05-15

    Blood-brain barrier dysfunction is a serious consequence of inflammatory brain diseases, cerebral infections, and trauma. The proinflammatory cytokine interleukin (IL)-1β is central to neuroinflammation and contributes to brain microvascular leakage and edema formation. Although it is well known that IL-1β exposure directly induces hyperpermeability in brain microvascular endothelium, the molecular mechanisms mediating this response are not completely understood. In the present study, we found that exposure of the human brain microvascular endothelium to IL-1β triggered activation of novel PKC isoforms δ, μ, and θ, followed by decreased transendothelial electrical resistance (TER). The IL-1β-induced decrease in TER was prevented by small hairpin RNA silencing of PKC-θ or by treatment with the isoform-selective PKC inhibitor Gö6976 but not by PKC inhibitors that are selective for all PKC isoforms other than PKC-θ. Decreased TER coincided with increased phosphorylation of regulatory myosin light chain and with increased proapoptotic signaling indicated by decreased uptake of mitotracker red in response to IL-1β treatment. However, neither of these observed effects were prevented by Gö6976 treatment, indicating lack of causality with respect to decreased TER. Instead, our data indicated that the mechanism of decreased TER involves PKC-θ-dependent phosphorylation of the tight junction protein zona occludens (ZO)-1. Because IL-1β is a central inflammatory mediator, our interpretation is that inhibition of PKC-θ or inhibition of ZO-1 phosphorylation could be viable strategies for preventing blood-brain barrier dysfunction under a variety of neuroinflammatory conditions.

  9. PKC isotype functions in T lymphocytes.

    PubMed

    Baier, G

    2007-01-01

    The main function of mature T cells is to recognize and respond to foreign antigens by a complex activation process involving differentiation of the resting cell to a proliferating lymphoblast actively secreting immunoregulatory lymphokines or displaying targeted cytotoxicity, ultimately leading to recruitment of other cell types and initiation of an effective immune response. In order to understand the physiology and pathophysiology of T lymphocytes, it is necessary to decode the biochemical processes that integrate signals from antigen, cytokine, integrin and death receptors. The principal upon which our work is based is to explore and identify gene products of distinct members of the AGC family of protein serine/threonine kinases as key players mediating cell growth regulation. Given the established important role of PKC theta as regulator of T cell fate and knowing that several other PKC isotypes are also expressed in T cells at a high level, we now summarize the physiological and non-redundant functions of PKC alpha, beta, delta, epsilon, zeta and theta isotypes in T cells. This review describes the current knowledge of the physiological and non-redundant functions of the PKC gene products in T cells.

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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.

  15. Amoebic PI3K and PKC Is Required for Jurkat T Cell Death Induced by Entamoeba histolytica

    PubMed Central

    Lee, Young Ah; Kim, Kyeong Ah; Min, Arim

    2014-01-01

    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. PMID:25246714

  16. The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC.

    PubMed

    Ren, L; Hong, S H; Cassavaugh, J; Osborne, T; Chou, A J; Kim, S Y; Gorlick, R; Hewitt, S M; Khanna, C

    2009-02-12

    Ezrin is a member of the ERM (ezrin, radixin, moesin) protein family and links F-actin to the cell membrane following phosphorylation. Ezrin has been associated with tumor progression and metastasis in several cancers including the pediatric solid tumors, osteosarcoma and rhabdomyosarcoma. In this study, we were surprised to find that ezrin was not constitutively phosphorylated but rather was dynamically regulated during metastatic progression in osteosarcoma. Metastatic osteosarcoma cells expressed phosphorylated ERM early after their arrival in the lung, and then late in progression, only at the invasive front of larger metastatic lesions. To pursue mechanisms for this regulation, we found that inhibitors of PKC (protein kinase C) blocked phosphorylation of ezrin, and that ezrin coimmunoprecipitated in cells with PKCalpha, PKCiota and PKCgamma. Furthermore, phosphorylated forms of ezrin and PKC had identical expression patterns at the invasive front of pulmonary metastatic lesions in murine and human patient samples. Finally, we showed that the promigratory effects of PKC were linked to ezrin phosphorylation. These data are the first to suggest a dynamic regulation of ezrin phosphorylation during metastasis and to connect the PKC family members with this regulation.

  17. 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.

  18. 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

  19. Effect of Protein Kinase C delta (PKC-δ) Inhibition on the Transcriptome of Normal and Systemic Sclerosis Human Dermal Fibroblasts In Vitro

    PubMed Central

    Wermuth, Peter J.; Addya, Sankar; Jimenez, Sergio A.

    2011-01-01

    Previous studies demonstrated that protein kinase C- δ (PKC-δ) inhibition with the selective inhibitor, rottlerin, resulted in potent downregulation of type I collagen expression and production in normal human dermal fibroblasts and abrogated the exaggerated type I collagen production and expression in fibroblasts cultured from affected skin from patients with the fibrosing disorder systemic sclerosis (SSc). To elucidate the mechanisms involved in the ability of PKC-δ to regulate collagen production in fibroblasts, we examined the effects of PKC-δ inhibition on the transcriptome of normal and SSc human dermal fibroblasts. Normal and SSc human dermal fibroblasts were incubated with rottlerin (5 µM), and their gene expression was analyzed by microarrays. Pathway analysis and gene ontology analysis of differentially expressed genes in each comparison were performed. Identification of significantly overrepresented transcriptional regulatory elements (TREs) was performed using the Promoter Analysis and Interaction Network Toolset (PAINT) program. PKC-δ activity was also inhibited using RNA interference (siRNA) and by treating fibroblasts with a specific PKC-δ inhibitory cell permeable peptide. Differential gene expression of 20 genes was confirmed using real time PCR. PKC-δ inhibition caused a profound change in the transcriptome of normal and SSc human dermal fibroblasts in vitro. Pathway and gene ontology analysis identified multiple cellular and organismal pathways affected by PKC-δ inhibition. Furthermore, both pathway and PAINT analyses indicated that the transcription factor NFκB played an important role in the transcriptome changes induced by PKC-δ inhibition. Multiple genes involved in the degradation of the extracellular matrix components were significantly reduced in SSc fibroblasts and their expression was increased by PKC-δ inhibition. These results indicate that isoform-specific inhibition of PKC-δ profibrotic effects may represent a novel

  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. PKC412 normalizes mutation-related keratin filament disruption and hepatic injury in mice by promoting keratin-myosin binding.

    PubMed

    Kwan, Raymond; Chen, Lu; Looi, Koksun; Tao, Guo-Zhong; Weerasinghe, Sujith V; Snider, Natasha T; Conti, Mary Anne; Adelstein, Robert S; Xie, Qing; Omary, M Bishr

    2015-12-01

    Keratins, among other cytoskeletal intermediate filament proteins, are mutated at a highly conserved arginine with consequent severe disease phenotypes due to disruption of keratin filament organization. We screened a kinase inhibitor library, using A549 cells that are transduced with a lentivirus keratin 18 (K18) construct, to identify compounds that normalize filament disruption due to K18 Arg90Cys mutation at the conserved arginine. High-throughput screening showed that PKC412, a multikinase inhibitor, ameliorated K18 Arg90Cys-mediated keratin filament disruption in cells and in the livers of previously described transgenic mice that overexpress K18 Arg90Cys. Furthermore, PKC412 protected cultured A549 cells that express mutant or wild-type K18 and mouse livers of the K18 Arg90Cys-overexpressing transgenic mice from Fas-induced apoptosis. Proteomic analysis of proteins that associated with keratins after exposure of K18-expressing A549 cells to PKC412 showed that nonmuscle myosin heavy chain-IIA (NMHC-IIA) partitions with the keratin fraction. The nonmuscle myosin-IIA (NM-IIA) association with keratins was confirmed by immune staining and by coimmunoprecipitation. The keratin-myosin association is myosin dephosphorylation-dependent; occurs with K8, the obligate K18 partner; is enhanced by PKC412 in cells and mouse liver; and is blocked by hyperphosphorylation conditions in cultured cells and mouse liver. Furthermore, NMHC-IIA knockdown inhibits PKC412-mediated normalization of K18 R90C filaments. The inhibitor PKC412 normalizes K18 Arg90Cys mutation-induced filament disruption and disorganization by enhancing keratin association with NM-IIA in a myosin dephosphorylation-regulated manner. Targeting of intermediate filament disorganization by compounds that alter keratin interaction with their associated proteins offers a potential novel therapeutic approach for keratin and possibly other intermediate filament protein-associated diseases. © 2015 by the American

  3. 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…

  4. 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…

  5. 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

  6. Bryostatin modulates latent HIV-1 infection via PKC and AMPK signaling but inhibits acute infection in a receptor independent manner.

    PubMed

    Mehla, Rajeev; Bivalkar-Mehla, Shalmali; Zhang, Ruonan; Handy, Indhira; Albrecht, Helmut; Giri, Shailendra; Nagarkatti, Prakash; Nagarkatti, Mitzi; Chauhan, Ashok

    2010-06-16

    HIV's ability to establish long-lived latent infection is mainly due to transcriptional silencing in resting memory T lymphocytes and other non dividing cells including monocytes. Despite an undetectable viral load in patients treated with potent antiretrovirals, current therapy is unable to purge the virus from these latent reservoirs. In order to broaden the inhibitory range and effectiveness of current antiretrovirals, the potential of bryostatin was investigated as an HIV inhibitor and latent activator. Bryostatin revealed antiviral activity against R5- and X4-tropic viruses in receptor independent and partly via transient decrease in CD4/CXCR4 expression. Further, bryostatin at low nanomolar concentrations robustly reactivated latent viral infection in monocytic and lymphocytic cells via activation of Protein Kinase C (PKC) -alpha and -delta, because PKC inhibitors rottlerin and GF109203X abrogated the bryostatin effect. Bryostatin specifically modulated novel PKC (nPKC) involving stress induced AMP Kinase (AMPK) inasmuch as an inhibitor of AMPK, compound C partially ablated the viral reactivation effect. Above all, bryostatin was non-toxic in vitro and was unable to provoke T-cell activation. The dual role of bryostatin on HIV life cycle may be a beneficial adjunct to the treatment of HIV especially by purging latent virus from different cellular reservoirs such as brain and lymphoid organs.

  7. Bryostatin Modulates Latent HIV-1 Infection via PKC and AMPK Signaling but Inhibits Acute Infection in a Receptor Independent Manner

    PubMed Central

    Zhang, Ruonan; Handy, Indhira; Albrecht, Helmut; Giri, Shailendra; Nagarkatti, Prakash; Nagarkatti, Mitzi; Chauhan, Ashok

    2010-01-01

    HIV's ability to establish long-lived latent infection is mainly due to transcriptional silencing in resting memory T lymphocytes and other non dividing cells including monocytes. Despite an undetectable viral load in patients treated with potent antiretrovirals, current therapy is unable to purge the virus from these latent reservoirs. In order to broaden the inhibitory range and effectiveness of current antiretrovirals, the potential of bryostatin was investigated as an HIV inhibitor and latent activator. Bryostatin revealed antiviral activity against R5- and X4-tropic viruses in receptor independent and partly via transient decrease in CD4/CXCR4 expression. Further, bryostatin at low nanomolar concentrations robustly reactivated latent viral infection in monocytic and lymphocytic cells via activation of Protein Kinase C (PKC) -α and -δ, because PKC inhibitors rottlerin and GF109203X abrogated the bryostatin effect. Bryostatin specifically modulated novel PKC (nPKC) involving stress induced AMP Kinase (AMPK) inasmuch as an inhibitor of AMPK, compound C partially ablated the viral reactivation effect. Above all, bryostatin was non-toxic in vitro and was unable to provoke T-cell activation. The dual role of bryostatin on HIV life cycle may be a beneficial adjunct to the treatment of HIV especially by purging latent virus from different cellular reservoirs such as brain and lymphoid organs. PMID:20585398

  8. 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.

  9. 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

  10. In Vitro Neutrophil Migration Requires Protein Kinase C-Delta (δ-PKC)-Mediated Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Phosphorylation.

    PubMed

    Sheats, Mary K; 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 myristoylated alanine-rich C-kinase substrate (MARCKS), a well-known actin binding protein and protein kinase C (PKC) substrate, 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 f-Met-Leu-Phe (fMLF)-induced MARCKS phosphorylation (IC50=5.709 μM), adhesion (IC50=8.4 μM), and migration (IC50=6.7 μM), 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.

  11. PKC412 normalizes mutation-related keratin filament disruption and hepatic injury in mice by promoting keratin-myosin binding

    PubMed Central

    Kwan, Raymond; Chen, Lu; Looi, Koksun; Tao, Guo-Zhong; Weerasinghe, Sujith V; Snider, Natasha T; Conti, Mary Anne; Adelstein, Robert S; Xie, Qing; Omary, M Bishr

    2015-01-01

    Keratins, among other cytoskeletal intermediate filament proteins, are mutated at a highly conserved arginine with consequent severe disease phenotypes due to disruption of keratin filament organization. We screened a kinase inhibitor library, using A549 cells that are transduced with a lentivirus keratin 18 (K18) construct, to identify compounds that normalize filament disruption due to K18 Arg90Cys mutation at the conserved arginine. The high throughput screening showed that PKC412, a multi-kinase inhibitor, ameliorated K18 Arg90Cys-mediated keratin filament disruption in cells and in the livers of previously described transgenic mice that overexpress K18 Arg90Cys. Furthermore, PKC412 protected cultured A549 cells that express mutant or wild-type K18 and mouse livers of the K18 Arg90Cys-overexpressing transgenic mice from Fas-induced apoptosis. Proteomic analysis of proteins that associated with keratins after exposure of K18- expressing A549 cells to PKC412 showed that non-muscle myosin heavy chain-IIA (NMHC-IIA) partitions with the keratin fraction. NMHC-IIA association with keratins was confirmed by immune staining and by coimmunoprecipitation. The keratin-myosin association is myosin dephosphorylation-dependent and occurs with K8, the obligate K18 partner, and is enhanced by PKC412 in cells and mouse liver and blocked by hyperphosphorylation conditions in cultured cells and mouse liver. Furthermore, NMHC-IIA knockdown inhibits PKC412-mediated normalization of K18 R90C filaments. Conclusion PKC412 normalizes K18 Arg90Cys mutation-induced filament disruption and disorganization by enhancing keratin association with NMHC-IIA in a myosin dephosphorylation regulated manner. Targeting of intermediate filament disorganization by compounds that alters keratin interaction with their associated proteins offers a potential novel therapeutic approach for keratin and possibly other IF-associated diseases. PMID:26126491

  12. 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

  13. 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.

  14. The induction of CXCR4 expression in human osteoblast-like cells (MG63) by CoCr particles is regulated by the PLC-DAG-PKC pathway.

    PubMed

    Drynda, Andreas; Ren, Qiang; Buchhorn, Gottfried H; Lohmann, Christoph H

    2016-08-09

    Osteolysis which leads to aseptic loosening of implants is a fundamental problem in joint replacement surgery (arthroplasty) and the leading cause for implant failure and revision surgery. Metal (CoCr) particles separated from implants by wear cause osteolysis and the failure of orthopedic implants, but the molecular mechanism is not clear. The chemokine receptor CXCR4 has been shown to play a pivotal role in periprosthetic osteolysis. The aim of this study was to determine which signal transduction pathway (PLC-DAG-PKC or MAPK/ERK) induces CXCR4 expression in osteoblast-like cells (MG63) cells. MG63 and Jurkat cells were stimulated with different amounts of particles (10(7) , 10(6) , and 10(5) ) for different time periods (30 min to 24 h), in the presence and absence of specific inhibitors (chelerythrine for the PLC-DAG-PKC pathway and PD98059 for the MAPK/ERK pathway). The expression of CXCR4-specific mRNA was determined by real-time polymerase chain reaction (PCR), and the PKC activity was measured by Western Blot using an antibody specific for PKC-related phosphorylation. Real-time PCR data showed that CXCR4 mRNA expression in MG63 cells induced by CoCr particles was significantly diminished by the PKC-specific inhibitor chelerythrine. This effect was not observed with the MAPK/ERK inhibitor PD98059. The involvement of PKC was also confirmed by an intensified phosphorylation pattern after stimulation with CoCr particles. In Jurkat cells, none of the inhibitors exhibited any effect. The induction of CXCR4-specific mRNA expression in MG63 cells after stimulation with CoCr particles is regulated by the PLC-DAG-PKC pathway and not by the MAPK/ERK pathway. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2016. © 2016 Wiley Periodicals, Inc.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. A novel effect of MARCKS phosphorylation by activated PKC: the dephosphorylation of its serine 25 in chick neuroblasts.

    PubMed

    Toledo, Andrea; Zolessi, Flavio R; Arruti, Cristina

    2013-01-01

    MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) is a peripheral membrane protein, especially abundant in the nervous system, and functionally related to actin organization and Ca-calmodulin regulation depending on its phosphorylation by PKC. However, MARCKS is susceptible to be phosphorylated by several different kinases and the possible interactions between these phosphorylations have not been fully studied in intact cells. In differentiating neuroblasts, as well as some neurons, there is at least one cell-type specific phosphorylation site: serine 25 (S25) in the chick. We demonstrate here that S25 is included in a highly conserved protein sequence which is a Cdk phosphorylatable region, located far away from the PKC phosphorylation domain. S25 phosphorylation was inhibited by olomoucine and roscovitine in neuroblasts undergoing various states of cell differentiation in vitro. These results, considered in the known context of Cdks activity in neuroblasts, suggest that Cdk5 is the enzyme responsible for this phosphorylation. We find that the phosphorylation by PKC at the effector domain does not occur in the same molecules that are phosphorylated at serine 25. The in situ analysis of the subcellular distribution of these two phosphorylated MARCKS variants revealed that they are also segregated in different protein clusters. In addition, we find that a sustained stimulation of PKC by phorbol-12-myristate-13-acetate (PMA) provokes the progressive disappearance of phosphorylation at serine 25. Cells treated with PMA, but in the presence of several Ser/Thr phosphatase (PP1, PP2A and PP2B) inhibitors indicated that this dephosphorylation is achieved via a phosphatase 2A (PP2A) form. These results provide new evidence regarding the existence of a novel consequence of PKC stimulation upon the phosphorylated state of MARCKS in neural cells, and propose a link between PKC and PP2A activity on MARCKS.

  2. A Novel Effect of MARCKS Phosphorylation by Activated PKC: The Dephosphorylation of Its Serine 25 in Chick Neuroblasts

    PubMed Central

    Toledo, Andrea; Zolessi, Flavio R.; Arruti, Cristina

    2013-01-01

    MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) is a peripheral membrane protein, especially abundant in the nervous system, and functionally related to actin organization and Ca-calmodulin regulation depending on its phosphorylation by PKC. However, MARCKS is susceptible to be phosphorylated by several different kinases and the possible interactions between these phosphorylations have not been fully studied in intact cells. In differentiating neuroblasts, as well as some neurons, there is at least one cell-type specific phosphorylation site: serine 25 (S25) in the chick. We demonstrate here that S25 is included in a highly conserved protein sequence which is a Cdk phosphorylatable region, located far away from the PKC phosphorylation domain. S25 phosphorylation was inhibited by olomoucine and roscovitine in neuroblasts undergoing various states of cell differentiation in vitro. These results, considered in the known context of Cdks activity in neuroblasts, suggest that Cdk5 is the enzyme responsible for this phosphorylation. We find that the phosphorylation by PKC at the effector domain does not occur in the same molecules that are phosphorylated at serine 25. The in situ analysis of the subcellular distribution of these two phosphorylated MARCKS variants revealed that they are also segregated in different protein clusters. In addition, we find that a sustained stimulation of PKC by phorbol-12-myristate-13-acetate (PMA) provokes the progressive disappearance of phosphorylation at serine 25. Cells treated with PMA, but in the presence of several Ser/Thr phosphatase (PP1, PP2A and PP2B) inhibitors indicated that this dephosphorylation is achieved via a phosphatase 2A (PP2A) form. These results provide new evidence regarding the existence of a novel consequence of PKC stimulation upon the phosphorylated state of MARCKS in neural cells, and propose a link between PKC and PP2A activity on MARCKS. PMID:23634231

  3. 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

  4. 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.

  5. Taurochenodeoxycholic acid induces NR8383 cells apoptosis via PKC/JNK-dependent pathway.

    PubMed

    Wang, Xu; Zhang, Ziying; He, Xiuling; Mao, Wei; Zhou, Lei; Li, Peifeng

    2016-09-05

    Our former studies have suggested that taurochenodeoxycholic acid (TCDCA) as a signaling molecule shows obvious anti-inflammatory and immune regulation properties. In this research, we tentatively explored the potential effects and the possible mechanism that involve in the apoptotic process in NR8383 cells induced by TCDCA. Using flow cytometry analysis, we evaluated the apoptosis rate. Gene expression levels were determined by qPCR. The expressions of protein kinase C (PKC), Jun N-terminal kinase (JNK) and their phosphorylation were measured by Western Blot. We observed the activities of caspase-3 and caspase-8 with Caspase-Glo® regent. The results demonstrated that TCDCA dramatically improved the apoptosis rate of NR8383 cells in a concentration-dependent manner. In the meantime, PKC mRNA levels and activities were significantly augmented by TCDCA treatments. In addition, JNK, caspase-3 and caspase-8 mRNA expression levels and activities were increased by TCDCA, while they were markedly decreased by specific inhibitors. We conclude that TCDCA contributes to the apoptosis through the activation of the caspase cascade in NR8383 cells, and the PKC/JNK signaling pathway may be involved in this process. These results indicate that TCDCA may be a latent effective pharmaceutical product for apoptosis-related diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. 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.

  7. 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.

  8. A Calcium- and Diacylglycerol-Stimulated Protein Kinase C (PKC), Caenorhabditis elegans PKC-2, Links Thermal Signals to Learned Behavior by Acting in Sensory Neurons and Intestinal Cells.

    PubMed

    Land, Marianne; Rubin, Charles S

    2017-10-01

    Ca(2+)- and diacylglycerol (DAG)-activated protein kinase C (cPKC) promotes learning and behavioral plasticity. However, knowledge of in vivo regulation and exact functions of cPKCs that affect behavior is limited. We show that PKC-2, a Caenorhabditis elegans cPKC, is essential for a complex behavior, thermotaxis. C. elegans memorizes a nutrient-associated cultivation temperature (Tc ) and migrates along the Tc within a 17 to 25°C gradient. pkc-2 gene disruption abrogated thermotaxis; a PKC-2 transgene, driven by endogenous pkc-2 promoters, restored thermotaxis behavior in pkc-2(-/-) animals. Cell-specific manipulation of PKC-2 activity revealed that thermotaxis is controlled by cooperative PKC-2-mediated signaling in both AFD sensory neurons and intestinal cells. Cold-directed migration (cryophilic drive) precedes Tc tracking during thermotaxis. Analysis of temperature-directed behaviors elicited by persistent PKC-2 activation or inhibition in AFD (or intestine) disclosed that PKC-2 regulates initiation and duration of cryophilic drive. In AFD neurons, PKC-2 is a Ca(2+) sensor and signal amplifier that operates downstream from cyclic GMP-gated cation channels and distal guanylate cyclases. UNC-18, which regulates neurotransmitter and neuropeptide release from synaptic vesicles, is a critical PKC-2 effector in AFD. UNC-18 variants, created by mutating Ser(311) or Ser(322), disrupt thermotaxis and suppress PKC-2-dependent cryophilic migration. Copyright © 2017 American Society for Microbiology.

  9. 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.

  10. Comparative analysis of the anti-chikungunya virus activity of novel bryostatin analogs confirms the existence of a PKC-independent mechanism.

    PubMed

    Abdelnabi, Rana; Staveness, Daryl; Near, Katherine E; Wender, Paul A; Delang, Leen; Neyts, Johan; Leyssen, Pieter

    2016-11-15

    Previously, we reported that salicylate-based analogs of bryostatin protect cells from chikungunya virus (CHIKV)-induced cell death. Interestingly, 'capping' the hydroxyl group at C26 of a lead bryostatin analog, a position known to be crucial for binding to and modulation of protein kinase C (PKC), did not abrogate the anti-CHIKV activity of the scaffold, putatively indicating the involvement of a pathway independent of PKC. The work detailed in this study demonstrates that salicylate-derived analog 1 and two capped analogs (2 and 3) are not merely cytoprotective compounds, but act as selective and specific inhibitors of CHIKV replication. Further, a detailed comparative analysis of the effect of the non-capped versus the two capped analogs revealed that compound 1 acts both at early and late stages in the chikungunya virus replication cycle, while the capped analogs only interfere with a later stage process. Co-dosing with the PKC inhibitors sotrastaurin and Gö6976 counteracts the antiviral activity of compound 1 without affecting that of capped analogs 2 and 3, providing further evidence that the latter elicit their anti-CHIKV activity independently of PKC. Remarkably, treatment of CHIKV-infected cells with a combination of compound 1 and a capped analog resulted in a pronounced synergistic antiviral effect. Thus, these salicylate-based bryostatin analogs can inhibit CHIKV replication through a novel, yet still elusive, non-PKC dependent pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Arabinosylated lipoarabinomannan (Ara-LAM) mediated intracellular mechanisms against tuberculosis infection: involvement of protein kinase C (PKC) mediated signaling.

    PubMed

    Das, Shibali; Bhattacharjee, Oindrila; Goswami, Avranil; Pal, Nishith K; Majumdar, Subrata

    2015-03-01

    Tuberculosis causes severe immunosuppression thereby ensuring the loss of the host protective immune responses. During Mycobacterium tuberculosis infection, the pathogen modulates TLR-2 receptor down-stream signaling, indicating the possible involvement of TLR-2 in the regulation of the host immune response. Moreover, different PKC isoforms are also involved in the course of infection. Arabinosylated lipoarabinomannan (Ara-LAM) possesses immuno-modulatory properties which induce the pro-inflammatory responses via induction of TLR-2-mediated signaling. Here, we found that pretreatment of M. tuberculosis-infected macrophages with Ara-LAM caused a significant increase in the conventional PKC expression along with their active association with TLR-2. This association activated the TLR-2 -mediated downstream signaling, facilitating the activation of MAP kinase P38. All these events culminated in the up-regulation of proinflammatory response, which was abrogated by treatment with PKC-α and P38 inhibitors. Moreover, pretreatment of macrophages with Ara-LAM abrogated the IL-10 production while restored MHC-II expression in the infected macrophages. This study demonstrates that Ara-LAM confers protection against tuberculosis via TLR-2/PKC signaling crosstalk which is responsible for the induction of host protective immune response against tuberculosis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. 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.

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

    PubMed

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

    2016-04-01

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

  14. Gabapentin Effects on PKC-ERK1/2 Signaling in the Spinal Cord of Rats with Formalin-Induced Visceral Inflammatory Pain.

    PubMed

    Zhang, Yan-Bo; Guo, Zheng-Dong; Li, Mei-Yi; Fong, Peter; Zhang, Ji-Guo; Zhang, Can-Wen; Gong, Ke-Rui; Yang, Ming-Feng; Niu, Jing-Zhong; Ji, Xun-Ming; Lv, Guo-Wei

    2015-01-01

    Currently, the clinical management of visceral pain remains unsatisfactory for many patients suffering from this disease. While preliminary animal studies have suggested the effectiveness of gabapentin in successfully treating visceral pain, the mechanism underlying its analgesic effect remains unclear. Evidence from other studies has demonstrated the involvement of protein kinase C (PKC) and extracellular signal-regulated kinase1/2 (ERK1/2) in the pathogenesis of visceral inflammatory pain. In this study, we tested the hypothesis that gabapentin produces analgesia for visceral inflammatory pain through its inhibitory effect on the PKC-ERK1/2 signaling pathway. Intracolonic injections of formalin were performed in rats to produce colitis pain. Our results showed that visceral pain behaviors in these rats decreased after intraperitoneal injection of gabapentin. These behaviors were also reduced by intrathecal injections of the PKC inhibitor, H-7, and the ERK1/2 inhibitor, PD98059. Neuronal firing of wide dynamic range neurons in L6-S1 of the rat spinal cord dorsal horn were significantly increased after intracolonic injection of formalin. This increased firing rate was inhibited by intraperitoneal injection of gabapentin and both the individual and combined intrathecal application of H-7 and PD98059. Western blot analysis also revealed that PKC membrane translocation and ERK1/2 phosphorylation increased significantly following formalin injection, confirming the recruitment of PKC and ERK1/2 during visceral inflammatory pain. These effects were also significantly reduced by intraperitoneal injection of gabapentin. Therefore, we concluded that the analgesic effect of gabapentin on visceral inflammatory pain is mediated through suppression of PKC and ERK1/2 signaling pathways. Furthermore, we found that the PKC inhibitor, H-7, significantly diminished ERK1/2 phosphorylation levels, implicating the involvement of PKC and ERK1/2 in the same signaling pathway. Thus, our

  15. Gabapentin Effects on PKC-ERK1/2 Signaling in the Spinal Cord of Rats with Formalin-Induced Visceral Inflammatory Pain

    PubMed Central

    Li, Mei-yi; Fong, Peter; Zhang, Ji-guo; Zhang, Can-wen; Gong, Ke-rui; Yang, Ming-feng; Niu, Jing-zhong; Ji, Xun-ming; Lv, Guo-wei

    2015-01-01

    Currently, the clinical management of visceral pain remains unsatisfactory for many patients suffering from this disease. While preliminary animal studies have suggested the effectiveness of gabapentin in successfully treating visceral pain, the mechanism underlying its analgesic effect remains unclear. Evidence from other studies has demonstrated the involvement of protein kinase C (PKC) and extracellular signal-regulated kinase1/2 (ERK1/2) in the pathogenesis of visceral inflammatory pain. In this study, we tested the hypothesis that gabapentin produces analgesia for visceral inflammatory pain through its inhibitory effect on the PKC-ERK1/2 signaling pathway. Intracolonic injections of formalin were performed in rats to produce colitis pain. Our results showed that visceral pain behaviors in these rats decreased after intraperitoneal injection of gabapentin. These behaviors were also reduced by intrathecal injections of the PKC inhibitor, H-7, and the ERK1/2 inhibitor, PD98059. Neuronal firing of wide dynamic range neurons in L6–S1 of the rat spinal cord dorsal horn were significantly increased after intracolonic injection of formalin. This increased firing rate was inhibited by intraperitoneal injection of gabapentin and both the individual and combined intrathecal application of H-7 and PD98059. Western blot analysis also revealed that PKC membrane translocation and ERK1/2 phosphorylation increased significantly following formalin injection, confirming the recruitment of PKC and ERK1/2 during visceral inflammatory pain. These effects were also significantly reduced by intraperitoneal injection of gabapentin. Therefore, we concluded that the analgesic effect of gabapentin on visceral inflammatory pain is mediated through suppression of PKC and ERK1/2 signaling pathways. Furthermore, we found that the PKC inhibitor, H-7, significantly diminished ERK1/2 phosphorylation levels, implicating the involvement of PKC and ERK1/2 in the same signaling pathway. Thus, our

  16. 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

  17. Map kinase and PKC signaling pathways modulate NGF-mediated apoE transcription.

    PubMed

    Strachan-Whaley, Megan R; Reilly, Kate; Dobson, James; Kalisch, Bettina E

    2015-05-19

    The present study assessed the mechanisms by which nerve growth factor (NGF) increased the level of apolipoprotein E (apoE) in PC12 cells. NGF (50ng/mL) significantly increased apoE protein levels following 72h of treatment. Similarly NGF increased luciferase activity in cells transfected with a luciferase reporter construct containing a 500bp fragment of the apoE promoter, indicating NGF-induced apoE expression is regulated, at least in part, at the level of transcription. The non-selective nitric oxide synthase (NOS) inhibitor N(ɷ)-nitro-L-arginine methylester (L-NAME; 20mM) did not attenuate the NGF-mediated increase in luciferase activity, while the inducible NOS inhibitor s-methylisothiourea (S-MIU; 2mM) partially attenuated this action of NGF. Inhibition of MAP kinase activation with 50μM U0126 or pre-treatment with the PKC inhibitor bisindolylmaleimide 1 (BIS-1; 10μM) prevented the NGF-mediated activation of the apoE promoter. Pre-treatment with the phospholipase C (PLC) inhibitor U73122 (5μM) partially inhibited the NGF-induced increase in luciferase activity while the Akt inhibitor LY294002 (10μM) had no effect. These data suggest NGF-induced apoE transcription requires MAP kinase and PKC activation and that these TrkA signaling pathways may be modulated by NO. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  18. 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

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

    PubMed Central

    Amin, Ruhul; Sharma, Sapna; Ratakonda, Sireesha

    2013-01-01

    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. PMID:23596171

  20. 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.

  1. 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.

  2. Heterologous, PKC-Mediated Desensitization of Human Histamine H3 Receptors Expressed in CHO-K1 Cells.

    PubMed

    Montejo-López, Wilber; Rivera-Ramírez, Nayeli; Escamilla-Sánchez, Juan; García-Hernández, Ubaldo; Arias-Montaño, José-Antonio

    2016-09-01

    Desensitization is a major mechanism to regulate the functional response of G protein-coupled receptors. In this work we studied whether the human histamine H3 receptor of 445 amino acids (hH3R445) experiences heterologous desensitization mediated by PKC activation. Bioinformatic analysis indicated the presence of Serine and Threonine residues susceptible of PKC-mediated phosphorylation on the third intracellular loop and the carboxyl terminus of the hH3R445. In CHO-K1 cells stably transfected with the hH3R445 direct PKC activation by phorbol 12-myristate 13-acetate (TPA, 200 nM) abolished H3R-mediated inhibition of forskolin-stimulated cAMP accumulation. Activation of endogenous purinergic receptors by ATP (adenosine 5'-triphosphate, 10 μM) increased the free calcium intracellular concentration ([Ca(2+)]i) confirming their coupling to phospholipase C stimulation. Incubation with ATP also abolished H3R-mediated inhibition of forskolin-induced cAMP accumulation, and this effect was prevented by the PKC inhibitors Ro-31-8220 and Gö-6976. Pre-incubation with TPA or ATP reduced H3R-mediated stimulation of [(35)S]-GTPγS binding to membranes from CHO-K1-hH3R445 cells by 39.7 and 54.2 %, respectively, with no change in the agonist potency, and the effect was prevented by either Ro-31-8220 or Gö-6976. Exposure to ATP or TPA also resulted in the loss of cell surface H3Rs (-30.4 and -45.1 %) as evaluated by [(3)H]-NMHA binding to intact cells. These results indicate that the hH3R445 undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation.

  3. 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 Central

    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.

    2015-01-01

    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. PMID:26008233

  4. 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.

  5. Sustained pharmacological inhibition of δPKC protects against hypertensive encephalopathy through prevention of blood-brain barrier breakdown in rats

    PubMed Central

    Qi, Xin; Inagaki, Koichi; Sobel, Raymond A.; Mochly-Rosen, Daria

    2007-01-01

    Hypertensive encephalopathy is a potentially fatal condition associated with cerebral edema and the breakdown of the blood-brain barrier (BBB). The molecular pathways leading to this condition, however, are unknown. We determined the role of δPKC, which is thought to regulate microvascular permeability, in the development of hypertensive encephalopathy using δV1-1 — a selective peptide inhibitor of δPKC. As a model of hypertensive encephalopathy, Dahl salt-sensitive rats were fed an 8% high-salt diet from 6 weeks of age and then were infused s.c. with saline, control TAT peptide, or δV1-1 using osmotic minipumps. The mortality rate and the behavioral symptoms of hypertensive encephalopathy decreased significantly in the δV1-1–treated group relative to the control-treated group, and BBB permeability was reduced by more than 60%. Treatment with δV1-1 was also associated with decreased δPKC accumulation in capillary endothelial cells and in the endfeet of capillary astrocytes, which suggests decreased microvasculature disruption. Treatment with δV1-1 prevented hypertension-induced tight junction disruption associated with BBB breakdown, which suggests that δPKC may specifically act to dysregulate tight junction components. Together, these results suggest that δPKC plays a role in the development of hypertension-induced encephalopathy and may be a therapeutic target for the prevention of BBB disruption. PMID:18097471

  6. Sustained pharmacological inhibition of deltaPKC protects against hypertensive encephalopathy through prevention of blood-brain barrier breakdown in rats.

    PubMed

    Qi, Xin; Inagaki, Koichi; Sobel, Raymond A; Mochly-Rosen, Daria

    2008-01-01

    Hypertensive encephalopathy is a potentially fatal condition associated with cerebral edema and the breakdown of the blood-brain barrier (BBB). The molecular pathways leading to this condition, however, are unknown. We determined the role of deltaPKC, which is thought to regulate microvascular permeability, in the development of hypertensive encephalopathy using deltaV1-1 - a selective peptide inhibitor of deltaPKC. As a model of hypertensive encephalopathy, Dahl salt-sensitive rats were fed an 8% high-salt diet from 6 weeks of age and then were infused s.c. with saline, control TAT peptide, or deltaV1-1 using osmotic minipumps. The mortality rate and the behavioral symptoms of hypertensive encephalopathy decreased significantly in the deltaV1-1-treated group relative to the control-treated group, and BBB permeability was reduced by more than 60%. Treatment with deltaV1-1 was also associated with decreased deltaPKC accumulation in capillary endothelial cells and in the endfeet of capillary astrocytes, which suggests decreased microvasculature disruption. Treatment with deltaV1-1 prevented hypertension-induced tight junction disruption associated with BBB breakdown, which suggests that deltaPKC may specifically act to dysregulate tight junction components. Together, these results suggest that deltaPKC plays a role in the development of hypertension-induced encephalopathy and may be a therapeutic target for the prevention of BBB disruption.

  7. 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

  8. 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

  9. 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.

  10. Therapeutic effects of PKC activators in Alzheimer's disease transgenic mice

    PubMed Central

    Etcheberrigaray, René; Tan, Mathew; Dewachter, Ilse; Kuipéri, Cuno; Van der Auwera, Ingrid; Wera, Stefaan; Qiao, Lixin; Bank, Barry; Nelson, Thomas J.; Kozikowski, Alan P.; Van Leuven, Fred; Alkon, Daniel L.

    2004-01-01

    Alzheimer's disease (AD) characteristically presents with early memory loss. Regulation of K+ channels, calcium homeostasis, and protein kinase C (PKC) activation are molecular events that have been implicated during associative memory which are also altered or defective in AD. PKC is also involved in the processing of the amyloid precursor protein (APP), a central element in AD pathophysiology. In previous studies, we demonstrated that benzolactam (BL), a novel PKC activator, reversed K+ channels defects and enhanced secretion of APPα in AD cells. In this study we present data showing that another PKC activator, bryostatin 1, at subnanomolar concentrations dramatically enhances the secretion of the α-secretase product sAPPα in fibroblasts from AD patients. We also show that BL significantly increased the amount of sAPPα and reduced Aβ40 in the brains of APP[V717I] transgenic mice. In a more recently developed AD double-transgenic mouse, bryostatin was effective in reducing both brain Aβ40 and Aβ42. In addition, bryostatin ameliorated the rate of premature death and improved behavioral outcomes. Collectively, these data corroborate PKC and its activation as a potentially important means of ameliorating AD pathophysiology and perhaps cognitive impairment, thus offering a promising target for drug development. Because bryostatin 1 is devoid of tumor-promoting activity and is undergoing numerous clinical studies for cancer treatment in humans, it might be readily tested in patients as a potential therapeutic agent for Alzheimer's disease. PMID:15263077

  11. Tamoxifen in combination with temozolomide induce a synergistic inhibition of PKC-pan in GBM cell lines.

    PubMed

    Balça-Silva, Joana; Matias, Diana; do Carmo, Anália; Girão, Henrique; Moura-Neto, Vivaldo; Sarmento-Ribeiro, Ana Bela; Lopes, Maria Celeste

    2015-04-01

    Glioblastoma (GBM) is a highly proliferative, angiogenic grade IV astrocytoma that develops resistance to the alkylating agents used in chemotherapy, such as temozolomide (TMZ), which is considered the gold standard. The mean survival time for GBM patients is approximately 12 months, increasing to 14.6 months after TMZ treatment. The resistance of GBM to chemotherapy seems to be associated to genetic alterations and to the constitutive activation of several signaling pathways. Therefore, the combination of different drugs with different mechanisms of action may contribute to circumvent the chemoresistance of glioma cells. Here we describe the potential synergistic behavior of the therapeutic combination of tamoxifen (TMX), a known inhibitor of PKC, and TMZ in GBM. We used two GBM cell lines incubated in absence and presence of TMX and/or TMZ and measured cell viability, proliferation, apoptosis, cell cycle, migration ability, cytoskeletal organization and the phosphorylated amount of the p-PKC-pan. The combination of low doses of TMX with increasing doses of TMZ shows an increased antiproliferative and apoptotic effect compared to the effect with TMX alone. The combination of TMX and TMZ seems to potentiate the effect of each other. These alterations seem to be associated to a decrease in the phosphorylation status of PKC. We emphasize that TMX is an inhibitor of the p-PKC-pan and that these combination is more effective in the reduction of proliferation and in the increase of apoptosis than each drug alone, which presents a new therapeutic strategy in GBM treatment. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. 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

  13. Rottlerin induces autophagy and apoptotic cell death through a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells: the protective role of autophagy in apoptosis.

    PubMed

    Song, Kyoung-Sub; Kim, Jong-Seok; Yun, Eun-Jin; Kim, Young-Rae; Seo, Kang-Sik; Park, Ji-Hoon; Jung, Yeon-Joo; Park, Jong-Il; Kweon, Gi-Ryang; Yoon, Wan-Hee; Lim, Kyu; Hwang, Byung-Doo

    2008-07-01

    Rottlerin is widely used as a protein kinase C-delta inhibitor. Recently, several reports have shown the possible apoptosis-inducing effect of rottlerin in some cancer cell lines. Here we report that rottlerin induces not only apoptosis but also autophagy via a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells. Rottlerin treatment induced a dose- and time-dependent inhibition of cell growth, and cytoplasmic vacuolations were markedly shown. These vacuoles were identified as acidic autolysosomes by electron microscopy, acidic vesicular organelle (AVO) staining and transfection of green fluorescent protein-LC3. The LC3-II protein level also increased after treatment with rottlerin. Prolonged exposure to rottlerin eventually caused apoptosis via loss of mitochondrial membrane potential and translocation of AIF from mitochondria to the nucleus. However, the activities of caspase-3, -8 and -9 were not changed, and PARP did not show signs of cleavage. Interestingly, the pretreatment of cells with a specific inhibitor of autophagy (3-methyladenine) accelerated rottlerin-induced apoptosis as revealed by an analysis of the subdiploid fraction and TUNEL assay. Nevertheless, the knockdown of PKC-delta by RNA interference neither affected cell growth nor acidic vacuole formation. Similarly, rottlerin-induced cell death was not prevented by PKC-delta overexpression. Taken together, these findings suggest that rottlerin induces early autophagy and late apoptosis in a PKC-delta-independent manner, and the rottlerin-induced early autophagy may act as a survival mechanism against late apoptosis in HT1080 human fibrosarcoma cells.

  14. Duodenal GLP-1 signaling regulates hepatic glucose production through a PKC-δ-dependent neurocircuitry

    PubMed Central

    Yang, Mengliu; Wang, Jinzhi; Wu, Shaobo; Yuan, Lei; Zhao, Xiaodong; Liu, Chaohong; Xie, Jing; Jia, Yanjun; Lai, Yerui; Zhao, Allan Zijian; Boden, Guenther; Li, Ling; Yang, Gangyi

    2017-01-01

    Intestinal glucagon-like peptide-1 (GLP-1) is a hormone that stimulates insulin secretion and acts as a neuropeptide to control glucose homeostasis, but little is known whether intestinal GLP-1 has any effect in the control of hepatic glucose production (HGP). Here we found that intraduodenal infusion of GLP-1 activated duodenal PKC-δ, lowered HGP and was accompanied by a decrease in hepatic expression of gluconeogenic enzymes and an increase in hepatic insulin signaling in rats. However, gut co-infusion of either the GLP-1 receptor antagonist Ex-9, or the PKCinhibitor rottlerin with GLP-1, negated the ability of gut GLP-1 to lower HGP and to increase hepatic insulin signaling during clamps. The metabolic and molecular signal effects of duodenal GLP-1 were also negated by co-infusion with tetracaine, pharmacologic inhibition of N-methyl-d-aspartate receptors within the dorsalvagal complex, or hepatic vagotomy in rats. In summary, we identified a neural glucoregulatory function of gut GLP-1 signaling. PMID:28182013

  15. (-)-Epigallocatechin gallate suppresses proliferation of vascular smooth muscle cells induced by high glucose by inhibition of PKC and ERK1/2 signalings.

    PubMed

    Yang, Jian; Han, Yu; Sun, Hailan; Chen, Caiyu; He, Duofen; Guo, Jing; Yu, Changqing; Jiang, Baoquan; Zhou, Lin; Zeng, Chunyu

    2011-11-09

    Proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development and progression of diabetes-related vascular complications. (-)-Epigallocatechin gallate (EGCG), the major catechin derived from green tea, is able to exert antidiabetes effects in animal models. However, it is not known whether or not EGCG inhibits VSMC proliferation induced by high glucose. This study tested the hypothesis that EGCG might have an inhibitory effect on VSMC proliferation induced by high glucose. VSMC proliferation was determined by [(3)H]-thymidine incorporation and uptake of 3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide (MTT). Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was determined by immunoblotting, and ERK 1/2 activity was detected by measuring the ability to phosphorylate its substrate Elk-1. Glucose increased VSMC proliferation in a concentration-dependent manner, which was reduced in the presence of EGCG. VSMC proliferation mediated by high glucose (30 mM) was involved in protein kinase C (PKC) and ERK1/2 signalings, because its effect was blocked by PKC inhibitor (PKC inhibitor 19-31) and ERK1/2 inhibitor (PD98059). Pretreatment of VSMCs with EGCG significantly inhibited the stimulatory effect of high glucose on PKC and ERK1/2 activation, followed by attenuation of its downstream transcription factor Elk-1 phosphorylation. Taken together, these results suggest that EGCG could suppress VSMC proliferation induced by high glucose by inhibition of PKC and ERK1/2 signalings in VSMCs, which indicates that EGCG might be a possible medicine to reduce vascular complications in diabetes.

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

    PubMed

    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 [Ca(2+)]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 Ca(2+)-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.

  17. 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

  18. cPKC regulates interphase nuclear size during Xenopus development

    PubMed Central

    Edens, Lisa J.

    2014-01-01

    Dramatic changes in cell and nuclear size occur during development and differentiation, and aberrant nuclear size is associated with many disease states. However, the mechanisms that regulate nuclear size are largely unknown. A robust system for investigating nuclear size is early Xenopus laevis development, during which reductions in nuclear size occur without changes in DNA content. To identify cellular factors that regulate nuclear size during development, we developed a novel nuclear resizing assay wherein nuclei assembled in Xenopus egg extract become smaller in the presence of cytoplasmic interphase extract isolated from post-gastrula Xenopus embryos. We show that nuclear shrinkage depends on conventional protein kinase C (cPKC). Increased nuclear cPKC localization and activity and decreased nuclear association of lamins mediate nuclear size reductions during development, and manipulating cPKC activity in vivo during interphase alters nuclear size in the embryo. We propose a model of steady-state nuclear size regulation whereby nuclear expansion is balanced by an active cPKC-dependent mechanism that reduces nuclear size. PMID:25135933

  19. 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.

  20. Alpha-1 adrenergic receptor agonists modulate ductal secretion of BDL rats via Ca(2+)- and PKC-dependent stimulation of cAMP.

    PubMed

    LeSage, Gene D; Alvaro, Domenico; Glaser, Shannon; Francis, Heather; Marucci, Luca; Roskams, Tania; Phinizy, Jo Lynne; Marzioni, Marco; Benedetti, Antonio; Taffetani, Silvia; Barbaro, Barbara; Fava, Giammarco; Ueno, Yoshiyuki; Alpini, Gianfranco

    2004-11-01

    Acetylcholine potentiates secretin-stimulated ductal secretion by Ca(2+)-calcineurin-mediated modulation of adenylyl cyclase. D2 dopaminergic receptor agonists inhibit secretin-stimulated ductal secretion via activation of protein kinase C (PKC)-gamma. No information exists regarding the effect of adrenergic receptor agonists on ductal secretion in a model of cholestasis induced by bile duct ligation (BDL). We evaluated the expression of alpha-1A/1C, -1beta and beta-1 adrenergic receptors in liver sections and cholangiocytes from normal and BDL rats. We evaluated the effects of the alpha-1 and beta-1 adrenergic receptor agonists (phenylephrine and dobutamine, respectively) on bile and bicarbonate secretion and cholangiocyte IP(3) and Ca(2+) levels in normal and BDL rats. We measured the effect of phenylephrine on lumen expansion in intrahepatic bile duct units (IBDUs) and cyclic adenosine monophosphate (cAMP) levels in cholangiocytes from BDL rats in the absence or presence of BAPTA/AM and Gö6976 (a PKC-alpha inhibitor). We evaluated if the effects of phenylephrine on ductal secretion were associated with translocation of PKC isoforms leading to increased protein kinase A activity. Alpha-1 and beta-1 adrenergic receptors were present mostly in the basolateral domain of cholangiocytes and, following BDL, their expression increased. Phenylephrine, but not dobutamine, increased secretin-stimulated choleresis in BDL rats. Phenylephrine did not alter basal but increased secretin-stimulated IBDU lumen expansion and cAMP levels, which were blocked by BAPTA/AM and Go6976. Phenylephrine increased IP(3) and Ca(2+) levels and activated PKC-alpha and PKC-beta-II. In conclusion, coordinated regulation of ductal secretion by secretin (through cAMP) and adrenergic receptor agonist activation (through Ca(2+)/PKC) induces maximal ductal bicarbonate secretion in liver diseases. (Supplementary material for this article can be found on the HEPATOLOGY website (http

  1. Cocaine-seeking is associated with PKC-dependent reduction of excitatory signaling in accumbens shell D2 dopamine receptor-expressing neurons

    PubMed Central

    Ortinski, Pavel I.; Briand, Lisa A.; Pierce, R. Christopher; Schmidt, Heath D.

    2015-01-01

    Stimulation of D1-like dopamine receptors (D1DRs) or D2-like dopamine receptors (D2DRs) in the nucleus accumbens (NAc) shell reinstates cocaine seeking in rats, an animal model of relapse. D2DRs and D1DRs activate protein kinase C (PKC) and recent studies indicate that activation of PKC in the NAc plays an important role in the reinstatement of drug seeking induced by a systemic cocaine priming injection. In the present study, pharmacological inhibition of PKC in the NAc shell attenuated cocaine seeking induced by intra-accumbens shell microinjection of a D2DR agonist, but not a D1DR agonist. D1DRs and D2DRs are primarily expressed on different accumbens medium spiny (MSN) neurons. Neuronal signaling and activity were assessed in these two populations of NAc neurons with transgenic mice expressing fluorescent labels under the control of D1DR and D2DR promoters. Following the extinction of cocaine self-administration, bath application of a PKC inhibitor produced similar effects on single evoked excitatory and inhibitory post-synaptic currents in D1DR- and D2DR-positive MSNs in the NAc shell. However, inhibition of PKC preferentially improved the ability of excitatory, but not inhibitory, synapses to sustain responding to brief train of stimuli specifically in D2DR-positive MSNs. This effect did not appear to involve modulation of presynaptic release mechanisms. Taken together, these findings indicate that the reinstatement of cocaine seeking is at least partially due to D2DR-dependent increases in PKC signaling in the NAc shell, which reduce excitatory synaptic efficacy in D2DR-expressing MSNs. PMID:25596492

  2. Cocaine-seeking is associated with PKC-dependent reduction of excitatory signaling in accumbens shell D2 dopamine receptor-expressing neurons.

    PubMed

    Ortinski, Pavel I; Briand, Lisa A; Pierce, R Christopher; Schmidt, Heath D

    2015-05-01

    Stimulation of D1-like dopamine receptors (D1DRs) or D2-like dopamine receptors (D2DRs) in the nucleus accumbens (NAc) shell reinstates cocaine seeking in rats, an animal model of relapse. D2DRs and D1DRs activate protein kinase C (PKC) and recent studies indicate that activation of PKC in the NAc plays an important role in the reinstatement of drug seeking induced by a systemic cocaine priming injection. In the present study, pharmacological inhibition of PKC in the NAc shell attenuated cocaine seeking induced by intra-accumbens shell microinjection of a D2DR agonist, but not a D1DR agonist. D1DRs and D2DRs are primarily expressed on different accumbens medium spiny (MSN) neurons. Neuronal signaling and activity were assessed in these two populations of NAc neurons with transgenic mice expressing fluorescent labels under the control of D1DR and D2DR promoters. Following the extinction of cocaine self-administration, bath application of a PKC inhibitor produced similar effects on single evoked excitatory and inhibitory post-synaptic currents in D1DR- and D2DR-positive MSNs in the NAc shell. However, inhibition of PKC preferentially improved the ability of excitatory, but not inhibitory, synapses to sustain responding to brief train of stimuli specifically in D2DR-positive MSNs. This effect did not appear to involve modulation of presynaptic release mechanisms. Taken together, these findings indicate that the reinstatement of cocaine seeking is at least partially due to D2DR-dependent increases in PKC signaling in the NAc shell, which reduce excitatory synaptic efficacy in D2DR-expressing MSNs. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. 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.

  4. Nociceptor Beta II, Delta, and Epsilon Isoforms of PKC Differentially Mediate Paclitaxel-Induced Spontaneous and Evoked Pain

    PubMed Central

    He, Ying

    2015-01-01

    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. PMID:25788678

  5. 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.

  6. Janus kinase inhibition suppresses PKC-induced cytokine release without affecting HIV-1 latency reversal ex vivo.

    PubMed

    Spivak, Adam M; Larragoite, Erin T; Coletti, McKenna L; Macedo, Amanda B; Martins, Laura J; Bosque, Alberto; Planelles, Vicente

    2016-12-20

    Despite the durable viral suppression afforded by antiretroviral therapy, HIV-1 eradication will require strategies to target latently infected cells that persist in infected individuals. Protein kinase C (PKC) activation is a promising strategy to reactivate latent proviruses and allow for subsequent recognition and clearance of infected cells by the immune system. Ingenol derivatives are PKC agonists that induce latency reversal but also lead to T cell activation and the release of pro-inflammatory cytokines, which would be undesirable in vivo. In this work, we sought to identify compounds that would suppress pro-inflammatory cytokine production in the context of PKC activation. We performed an in vitro screen to identify compounds that could dampen pro-inflammatory cytokine release associated with T cell activation, using IL-6 as a model cytokine. We then tested the ability of the most promising screening hit, the FDA-approved Janus Kinase (JAK) inhibitor ruxolitinib, to diminish release of multiple cytokines and its effect on latency reversal using cells from HIV-1-positive, aviremic participants. We demonstrate that co-administration of ruxolitinib with ingenol-3,20-dibenzoate significantly reduces pro-inflammatory cytokine release without impairing latency reversal ex vivo. The combination of ingenol compounds and JAK inhibition represents a novel strategy for HIV-1 eradication.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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,

  15. Excretory-secretory products from Paragonimus westermani increase nitric oxide production in microglia in PKC-dependent and -independent manners.

    PubMed

    Jin, Youngnam; Choi, In Young; Kim, Chunsook; Hong, Suyoung; Kim, Won-Ki

    2009-10-01

    Excretory-secretory products (ESP) from helminthic parasites may play pivotal roles in the immune regulation in hosts. Previously, we reported that ESP produced from Paragonimus westermani induced morphological activation of microglial cells and markedly stimulated nitric oxide (NO) production via activation of mitogen-activated protein kinases (MAPKs). In the present study, we investigated the role of protein kinase C and protein kinase A in MAPKs-dependent NO production by ESP. We found that treatment with protein kinase C inhibitor Go6976 strongly inhibited the phosphorylation of p38 and JNK, but not ERK, of MAPKs and decreased the production of NO in ESP-stimulated microglial cells. Inhibition of ERK, p38 or PKC decreased the ESP-induced activation of NF-kappaB, an important transcription factor for iNOS expression. Furthermore, ESP increased the level of p-CREB in microglial cells. However, adenylyl cyclase activator (forskolin), adenylyl cyclase inhibitor (SQ22536), cAMP analogue (db-cAMP) or protein kinase A inhibitor (H89) was not able to change iNOS expression and NO production in ESP-treated microglial cells. It implies that the cAMP-PKA-CREB pathway is not implicated in the ESP-evoked NO production in microglial cells. Thus, our results indicate that ESP stimulates microglial expression of iNOS via both PKC-dependent and -independent MAPKs phosphorylation and NF-kappaB activation.

  16. Adenoprotection of the heart involves phospholipase C-induced activation and translocation of PKC-ɛ to RACK2 in adult rat and mouse

    PubMed Central

    Fenton, Richard A.; Komatsu, Satoshi; Ikebe, Mitsuo; Shea, Lynne G.; Dobson, James G.

    2009-01-01

    Adenosine protects the heart from adrenergic overstimulation. This adenoprotection includes the direct anti-adrenergic action via adenosine A1 receptors (A1R) on the adrenergic signaling pathway. An indirect A1R-induced attenuation of adrenergic responsiveness involves the translocation of PKC-ɛ to t-tubules and Z-line of cardiomyocytes. We investigated with sarcomere imaging, immunocytochemistry imaging, and coimmunoprecipitation (co-IP) whether A1R activation of PKC-ɛ induces the kinase translocation to receptor for activated C kinase 2 (RACK2) in isolated rat and mouse hearts and whether phospholipase C (PLC) is involved. Rat cardiomyocytes were treated with the A1R agonist chlorocyclopentyladenosine (CCPA) and exposed to primary PKC-ɛ and RACK2 antibodies with secondaries conjugated to Cy3 and Cy5 (indodicarbocyanine), respectively. Scanning confocal microscopy showed that CCPA caused PKC-ɛ to reversibly colocalize with RACK2 within 3 min. Additionally, rat and mouse hearts were perfused and stimulated with CCPA or phenylisopropyladenosine to activate A1R, or with phorbol 12-myristate 13-acetate to activate PKC. RACK2 was immunoprecipitated from heart extracts and resolved with SDS-PAGE. Western blotting showed that CCPA, phenylisopropyladenosine, and phorbol 12-myristate 13-acetate in the rat heart increased the PKC-ɛ co-IP with RACK2 by 186, 49, and >1,000%, respectively. The A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine prevented the CCPA-induced co-IP with RACK2. In mouse hearts, CCPA increased the co-IP of PKC-ɛ with RACK2 by 61%. With rat cardiomyocytes, the β-adrenergic agonist isoproterenol increased sarcomere shortening by 177%. CCPA reduced this response by 47%, an action inhibited by the PLC inhibitor U-73122 and 8-cyclopentyl-1,3-dipropylxanthine. In conclusion, A1R stimulation of the heart is associated with PLC-initiated PKC-ɛ translocation and association with RACK2. PMID:19525381

  17. Ethanol and diolein stimulate PKC (protein kinase C) translocation in astroglial cells

    SciTech Connect

    Skwish, S. ); Shain, W. New York State Department of Health, Albany )

    1990-01-01

    Ethanol exposure stimulates taurine release from astroglial cells. To determine if ethanol mediates this release using protein kinase C (PKC), PKC activity was measured using LRM55 astroglial cells. When ethanol or diolein was applied to cells for 30 seconds, PKC activity was observed to decrease in the cytosol and increase in the membrane fraction of the cell while the whole cell activity remained unchanged. The membrane-associated activity increased by almost 100%. When ethanol and diolein were applied simultaneously, membrane-associated activity increased to become 3-5 times greater than when either PKC activator was applied alone. These changes in PKC activity parallel changes in taurine release observed when cells are exposed to ethanol and the PKC activator diolein. Ethanol-stimulated release may be associated with the translocation of PKC activity from the cytosol to the membrane.

  18. 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

  19. 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.

  20. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. 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.

  2. DOR activation inhibits anoxic/ischemic Na+ influx through Na+ channels via PKC mechanisms in the cortex

    PubMed Central

    Chao, Dongman; He, Xiaozhou; Yang, Yilin; Bazzy-Asaad, Alia; Lazarus, Lawrence H.; Balboni, Gianfranco; Kim, Dong H.; Xia, Ying

    2012-01-01

    Activation of delta-opioid receptors (DOR) is neuroprotective against hypoxic/ischemic injury in the cortex, which is at least partially related to its action against hypoxic/ischemic disruption of ionic homeostasis that triggers neuronal injury. Na+ influx through TTX-sensitive voltage-gated Na+ channels may be a main mechanism for hypoxia-induced disruption of K+ homeostasis, with DOR activation attenuating the disruption of ionic homeostasis by targeting voltage-gated Na+ channels. In the present study we examined the role of DOR in the regulation of Na+ influx in anoxia and simulated ischemia (oxygen-glucose deprivation) as well as the effect of DOR activation on the Na+ influx induced by a Na+ channel opener without anoxic/ischemic stress and explored a potential PKC mechanism underlying the DOR action. We directly measured extracellular Na+ activity in mouse cortical slices with Na+ selective electrodes and found that (1) anoxia-induced Na+ influx occurred mainly through TTX-sensitive Na+ channels; (2) DOR activation inhibited the anoxia/ischemia-induced Na+ influx; (3) veratridine, a Na+ channel opener, enhanced the anoxia-induced Na+ influx; this could be attenuated by DOR activation; (4) DOR activation did not reduce the anoxia-induced Na+ influx in the presence of chelerythrine, a broad-spectrum PKC blocker; and (5) DOR effects were blocked by PKCβII peptide inhibitor, and PKCθ pseudosubstrate inhibitor, respectively. We conclude that DOR activation inhibits anoxia-induced Na+ influx through Na+ channels via PKC (especially PKCβII and PKCθ isoforms) dependent mechanisms in the cortex. PMID:22609332

  3. Active site inhibitors protect protein kinase C from dephosphorylation and stabilize its mature form.

    PubMed

    Gould, Christine M; Antal, Corina E; Reyes, Gloria; Kunkel, Maya T; Adams, Ryan A; Ziyar, Ahdad; Riveros, Tania; Newton, Alexandra C

    2011-08-19

    Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C.

  4. Differential and Conditional Activation of PKC-Isoforms Dictates Cardiac Adaptation during Physiological to Pathological Hypertrophy

    PubMed Central

    Naskar, Shaon; Datta, Kaberi; Mitra, Arkadeep; Pathak, Kanchan; Datta, Ritwik; Bansal, Trisha; Sarkar, Sagartirtha

    2014-01-01

    A cardiac hypertrophy is defined as an increase in heart mass which may either be beneficial (physiological hypertrophy) or detrimental (pathological hypertrophy). This study was undertaken to establish the role of different protein kinase-C (PKC) isoforms in the regulation of cardiac adaptation during two types of cardiac hypertrophy. Phosphorylation of specific PKC-isoforms and expression of their downstream proteins were studied during physiological and pathological hypertrophy in 24 week male Balb/c mice (Mus musculus) models, by reverse transcriptase-PCR, western blot analysis and M-mode echocardiography for cardiac function analysis. PKC-δ was significantly induced during pathological hypertrophy while PKC-α was exclusively activated during physiological hypertrophy in our study. PKC-δ activation during pathological hypertrophy resulted in cardiomyocyte apoptosis leading to compromised cardiac function and on the other hand, activation of PKC-α during physiological hypertrophy promoted cardiomyocyte growth but down regulated cellular apoptotic load resulting in improved cardiac function. Reversal in PKC-isoform with induced activation of PKC-δ and simultaneous inhibition of phospho-PKC-α resulted in an efficient myocardium to deteriorate considerably resulting in compromised cardiac function during physiological hypertrophy via augmentation of apoptotic and fibrotic load. This is the first report where PKC-α and -δ have been shown to play crucial role in cardiac adaptation during physiological and pathological hypertrophy respectively thereby rendering compromised cardiac function to an otherwise efficient heart by conditional reversal of their activation. PMID:25116170

  5. Differential and conditional activation of PKC-isoforms dictates cardiac adaptation during physiological to pathological hypertrophy.

    PubMed

    Naskar, Shaon; Datta, Kaberi; Mitra, Arkadeep; Pathak, Kanchan; Datta, Ritwik; Bansal, Trisha; Sarkar, Sagartirtha

    2014-01-01

    A cardiac hypertrophy is defined as an increase in heart mass which may either be beneficial (physiological hypertrophy) or detrimental (pathological hypertrophy). This study was undertaken to establish the role of different protein kinase-C (PKC) isoforms in the regulation of cardiac adaptation during two types of cardiac hypertrophy. Phosphorylation of specific PKC-isoforms and expression of their downstream proteins were studied during physiological and pathological hypertrophy in 24 week male Balb/c mice (Mus musculus) models, by reverse transcriptase-PCR, western blot analysis and M-mode echocardiography for cardiac function analysis. PKC-δ was significantly induced during pathological hypertrophy while PKC-α was exclusively activated during physiological hypertrophy in our study. PKC-δ activation during pathological hypertrophy resulted in cardiomyocyte apoptosis leading to compromised cardiac function and on the other hand, activation of PKC-α during physiological hypertrophy promoted cardiomyocyte growth but down regulated cellular apoptotic load resulting in improved cardiac function. Reversal in PKC-isoform with induced activation of PKC-δ and simultaneous inhibition of phospho-PKC-α resulted in an efficient myocardium to deteriorate considerably resulting in compromised cardiac function during physiological hypertrophy via augmentation of apoptotic and fibrotic load. This is the first report where PKC-α and -δ have been shown to play crucial role in cardiac adaptation during physiological and pathological hypertrophy respectively thereby rendering compromised cardiac function to an otherwise efficient heart by conditional reversal of their activation.

  6. 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

  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. 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

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

    PubMed

    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 Ca(2+). 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.

  10. 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.

  11. 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

  12. Bryostatin activates HIV-1 latent expression in human astrocytes through a PKC and NF-ĸB-dependent mechanism.

    PubMed

    Díaz, Laura; Martínez-Bonet, Marta; Sánchez, Javier; Fernández-Pineda, Alejandra; Jiménez, José Luis; Muñoz, Eduardo; Moreno, Santiago; Álvarez, Susana; Muñoz-Fernández, Ma Ángeles

    2015-07-22

    Multiple studies have shown that HIV-1 patients may develop virus reservoirs that impede eradication; these reservoirs include the central nervous system (CNS). Despite an undetectable viral load in patients treated with potent antiretrovirals, current therapy is unable to purge the virus from these latent reservoirs. To broaden the inhibitory range and effectiveness of current antiretrovirals, the potential of bryostatin was investigated as a latent HIV-1 activator. We used primary astrocytes, NHA cells, and astrocytoma cells U-87. Infected cells with HIV-1(NL4.3) were treated with bryostatin alone or in combination with different inhibitors. HIV-1 production was quantified by using ELISA. Transcriptional activity was measured using luciferase reporter gene assays by using lipofectin. We performed cotransfection experiments of the LTR promoter with the active NF-κB member p65/relA. To confirm the NF-κB role, Western blot and confocal microscopy were performed. Bryostatin reactivates latent viral infection in the NHA and U87 cells via activation of protein kinase C (PKC)-alpha and -delta, because the PKC inhibitors rottlerin and GF109203X abrogated the bryostatin effect. No alteration in cell proliferation was found. Moreover, bryostatin strongly stimulated LTR transcription by activating the transcription factor NF-κB. Bryostatin could be a beneficial adjunct to the treatment of HIV-1 brain infection.

  13. Bryostatin activates HIV-1 latent expression in human astrocytes through a PKC and NF-ĸB-dependent mechanism

    PubMed Central

    Díaz, Laura; Martínez-Bonet, Marta; Sánchez, Javier; Fernández-Pineda, Alejandra; Jiménez, José Luis; Muñoz, Eduardo; Moreno, Santiago; Álvarez, Susana; Muñoz-Fernández, Mª Ángeles

    2015-01-01

    Multiple studies have shown that HIV-1 patients may develop virus reservoirs that impede eradication; these reservoirs include the central nervous system (CNS). Despite an undetectable viral load in patients treated with potent antiretrovirals, current therapy is unable to purge the virus from these latent reservoirs. To broaden the inhibitory range and effectiveness of current antiretrovirals, the potential of bryostatin was investigated as a latent HIV-1 activator. We used primary astrocytes, NHA cells, and astrocytoma cells U-87. Infected cells with HIV-1NL4.3 were treated with bryostatin alone or in combination with different inhibitors. HIV-1 production was quantified by using ELISA. Transcriptional activity was measured using luciferase reporter gene assays by using lipofectin. We performed cotransfection experiments of the LTR promoter with the active NF-κB member p65/relA. To confirm the NF-κB role, Western blot and confocal microscopy were performed. Bryostatin reactivates latent viral infection in the NHA and U87 cells via activation of protein kinase C (PKC)-alpha and -delta, because the PKC inhibitors rottlerin and GF109203X abrogated the bryostatin effect. No alteration in cell proliferation was found. Moreover, bryostatin strongly stimulated LTR transcription by activating the transcription factor NF-κB. Bryostatin could be a beneficial adjunct to the treatment of HIV-1 brain infection. PMID:26199173

  14. 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.

  15. 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

  16. 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

  17. 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).

  18. Pkc-Mediated Stimulation of Amphibian Cftr Depends on a Single Phosphorylation Consensus Site. Insertion of This Site Confers Pkc Sensitivity to Human Cftr

    PubMed Central

    Button, Brian; Reuss, Luis; Altenberg, Guillermo A.

    2001-01-01

    Mutations of the CFTR, a phosphorylation-regulated Cl− channel, cause cystic fibrosis. Activation of CFTR by PKA stimulation appears to be mediated by a complex interaction between several consensus phosphorylation sites in the regulatory domain (R domain). None of these sites has a critical role in this process. Here, we show that although endogenous phosphorylation by PKC is required for the effect of PKA on CFTR, stimulation of PKC by itself has only a minor effect on human CFTR. In contrast, CFTR from the amphibians Necturus maculosus and Xenopus laevis (XCFTR) can be activated to similar degrees by stimulation of either PKA or PKC. Furthermore, the activation of XCFTR by PKC is independent of the net charge of the R domain, and mutagenesis experiments indicate that a single site (Thr665) is required for the activation of XCFTR. Human CFTR lacks the PKC phosphorylation consensus site that includes Thr665, but insertion of an equivalent site results in a large activation upon PKC stimulation. These observations establish the presence of a novel mechanism of activation of CFTR by phosphorylation of the R domain, i.e., activation by PKC requires a single consensus phosphorylation site and is unrelated to the net charge of the R domain. PMID:11331356

  19. 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

  20. DaPKC-dependent phosphorylation of Crumbs is required for epithelial cell polarity in Drosophila

    PubMed Central

    Sotillos, Sol; Díaz-Meco, María Teresa; Caminero, Eva; Moscat, Jorge; Campuzano, Sonsoles

    2004-01-01

    Both in Drosophila and vertebrate epithelial cells, the establishment of apicobasal polarity requires the apically localized, membrane-associated Par-3–Par-6–aPKC protein complex. In Drosophila, this complex colocalizes with the Crumbs–Stardust (Sdt)–Pals1-associated TJ protein (Patj) complex. Genetic and molecular analyses suggest a functional relationship between them. We show, by overexpression of a kinase-dead Drosophila atypical PKC (DaPKC), the requirement for the kinase activity of DaPKC to maintain the position of apical determinants and to restrict the localization of basolateral ones. We demonstrate a novel physical interaction between the apical complexes, via direct binding of DaPKC to both Crb and Patj, and identify Crumbs as a phosphorylation target of DaPKC. This phosphorylation of Crumbs is functionally significant. Thus, a nonphosphorylatable Crumbs protein behaves in vivo as a dominant negative. Moreover, the phenotypic effect of overexpressing wild-type Crumbs is suppressed by reducing DaPKC activity. These results provide a mechanistic framework for the functional interaction between the Par-3–Par-6–aPKC and Crumbs–Sdt–Patj complexes based in the posttranslational modification of Crb by DaPKC. PMID:15302858

  1. 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.

  2. 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.

  3. 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

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

    PubMed Central

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

    2010-01-01

    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 Ca2+ 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 Ca2+ 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

  5. Involvement of PKA, PKC, CAMK-II and MEK1/2 in the acute antidepressant-like effect of creatine in mice.

    PubMed

    Cunha, Mauricio P; Budni, Josiane; Pazini, Francis L; Oliveira, Ágatha; Rosa, Julia M; Lopes, Mark W; Leal, Rodrigo B; Rodrigues, Ana Lúcia S

    2014-08-01

    The aim of this study was to investigate the involvement of signaling pathways on the creatine antidepressant-like effect in the tail suspension test (TST) in mice. The TST was used to assess the antidepressant-like properties of creatine. The anti-immobility effect of creatine (1mg/kg, p.o.) in the TST was blocked by i.c.v. pretreatment with H-89 (1μg/site, PKA inhibitor), KN-62 (1μg/site, CAMK-II inhibitor), chelerythrine (1μg/site, PKC inhibitor), U0126 (5μg/site, MEK1/2 inhibitor) or PD09058 (5μg/site, MEK1/2 inhibitor). These results suggest that the antidepressant-like effect of creatine is dependent on PKA, CaMK-II, PKC and MEK 1/2 activation. Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. 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

  11. 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

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

    PubMed

    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

    2014-10-07

    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.

  13. 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

  14. 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.

  15. Bioengineered internal anal sphincter derived from isolated human internal anal sphincter smooth muscle cells.

    PubMed

    Somara, Sita; Gilmont, Robert R; Dennis, Robert G; Bitar, Khalil N

    2009-07-01

    The internal anal sphincter (IAS) is a specialized circular smooth muscle that maintains rectoanal continence. In vitro models are needed to study the pathophysiology of human IAS disorders. We bioengineered sphincteric rings from human IAS smooth muscle cells (SMC) and investigated their response to cholinergic stimulation as well as investigated whether protein kinase C (PKC) and Rho kinase signaling pathways remain functional. 3-Dimensional bioengineered ring (3DBR) model of the human IAS was constructed from isolated human IAS SMC obtained from surgery. Contractile properties and force generation in response to acetylcholine, PKC inhibitor calphostin-C, Rho/ROCK inhibitor Y-27632, permeable Rho/ROCK inhibitor c3-exoenzyme, and PKC activator PdBU was measured. The human IAS 3DBR has the essential characteristics of physiologically functional IAS; it generated a spontaneous myogenic basal tone, and the constructs were able to relax in response to relaxants and contract in response to contractile agents. The constructs generated dose-dependent force in response to acetylcholine. Basal tone was significantly reduced by calphostin-C but not with Y-27632. Acetylcholine-induced force generation was also significantly reduced by calphostin-C but not with Y-27632. PdBU generated force that was equal in magnitude to acetylcholine. Thus, calphostin-C inhibited PdBU-induced force generation, whereas Y-27632 and c3 exoenzyme did not. These data indicate that basal tone and acetylcholine-induced force generation depend on signaling through the PKC pathway in human IAS; PKC-mediated force generation is independent of the Rho/ROCK pathway. This human IAS 3DBR model can be used to study the pathophysiology associated with IAS malfunctions.

  16. 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

  17. Biochemical and Genetic Evidence for a SAP-PKC-θ Interaction Contributing to IL-4 Regulation

    PubMed Central

    Cannons, Jennifer L.; Wu, Julie Z.; Gomez-Rodriguez, Julio; Zhang, Jinyi; Dong, Baoxia; Liu, Yin; Shaw, Stephen; Siminovitch, Katherine A.; Schwartzberg, Pamela L.

    2012-01-01

    SAP, an adaptor molecule that recruits Fyn to the SLAM-family of immunomodulatory receptors, is mutated in X-linked lymphoproliferative disease. CD4+ T cells from SAP-deficient mice have defective TCR-induced IL-4 production and impaired T cell-mediated help for germinal center formation; however, the downstream intermediates contributing to these defects remain unclear. We previously found that SAP-deficient CD4+ T cells exhibit decreased PKC-θ recruitment upon TCR stimulation. We demonstrate here using GST-pulldowns and co-immunoprecipitation studies that SAP constitutively associates with PKC-θ in T cells. SAP-PKC-θ interactions required R78 of SAP, a residue previously implicated in Fyn recruitment, yet SAP’s interactions with PKC-θ occurred independent of phosphotyrosine binding and Fyn. Overexpression of SAP in T cells increased and sustained PKC-θ recruitment to the immune synapse and elevated IL-4 production in response to TCR plus SLAM-mediated stimulation. Moreover, PKC-θ, like SAP, was required for SLAM-mediated increases in IL-4 production and conversely, membrane-targeted PKC-θ mutants rescued IL-4 expression in SAP−/− CD4+ T cells, providing genetic evidence that PKC-θ is a critical component of SLAM/SAP-mediated pathways that influence TCR-driven IL-4 production. PMID:20668219

  18. 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

  19. 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.

  20. [The correlation between the concentrations of VEGF and PEDF and Ca2+-PKC signaling pathways in human retinal pigment epithelial cells cultured in vitro after exposuring to blue light].

    PubMed

    Wang, Limin; Cai, Shanjun; Wu, Zhipeng; Gong, Xin; Lyu, Jianping; Su, Gang; Wang, Lili

    2015-11-01

    To investigate the concentrations of vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), inositol triphosphate (IP3) and diacylglycerol (DAG) in human retinal pigment epithelium (RPE) cells after exposuring to blue light, and to explore the relationship with Ca2+-PKC signaling pathways, to evaluate the role of Ca2+-PKC signaling pathways of blue-light irradiation induced apoptosis in RPE cells. The fourth generation human RPE cells in vitro were exposured to blue light (2000±500 lux) for 6 hours, 24 hours prolongation of post-exposure culture. The concentrations of VEGF, PEDF, IP3 and DAG were assayed by enzyme linked immunosorbent assay (ELISA). Cells were randomly divided into 6 groups, group A (control), group B (exposure to blue light), group C (exposure to blue light+PMA), group D (exposure to blue light+Calphostin C), group E (exposure to blue light+Nifedipine), group F (exposure to blue light+Calphostin C+Nifedipine). Flow cytometry was used to detect the apoptosis rate of human RPE cells in A, B and F group. Comparing with group A (584.38±10.66), the concentration of VEGF in group B (700.70±5.88), group C (698.21±6.66) and group E (648.30±4.91) was higher, the difference was statistically significant (P=0.002, 0.002, 0.016). Comparing with group B (700.70±5.88), the concentration of VEGF in Group D (623.87±3.12) and E (648.30±4.91) was lower (P=0.001, 0.002). Comparing with group A (75.96±1.70), the concentration of PEDF in Group B (71.82±1.67) and C (72.43±0.58) was lower (P=0.004, 0.011), but the concentration of PEDF in Group D (86.31±1.35) and E (93.72±1.24) was higher (P=0.000, 0.000). Comparing with group B (71.82±1.67), the concentration of PEDF in Group D (86.31±1.35) and E (93.72±1.24) was higher (P=0.000, 0.000). Comparing with group A (7.70±0.29), the ratio of VEGF to PEDF in Group B (9.85±0.34) and Croup C (9.64±0.02) was higher (P=0.008, 0.027) Comparing with group B, The ratio of VEGF to PEDF

  1. Reversible and irreversible acetylcholinesterase inhibitors cause changes in neuronal amyloid precursor protein processing and protein kinase C level in vitro.

    PubMed

    Pakaski, M; Rakonczay, Z; Kasa, P

    2001-03-01

    The alternative routes of cleavage of the amyloid precursor protein (APP) result in the generation and secretion of both soluble APP and beta-amyloid, the latter being the main component of the amyloid deposits in the brains of individuals with Alzheimer's disease (AD). This study examined the question of whether acetylcholinesterase (AChE) inhibitors can alter the processing of APP and the level of protein kinase C (PKC) in primary rat basal forebrain cultures. Western blotting was used to test two AChE inhibitors (reversible and irreversible) for their ability to enhance the release of APP and PKC content. These inhibitors were ambenonium (AMB) and metrifonate (MTF), at different concentrations. A significant increase was found in the cell-associated APP level in a basal forebrain neuronal culture, and there was an elevation of the APP release into the medium. Increases were similarly observed in the PKC levels after AMB or MTF treatment. The results suggest that these AChE inhibitors promote the non-amyloidogenic route of APP processing, which may be due to their stimulatory effects on PKC. The PKC activation may enhance the alpha-secretase activity and consequently the production of the N-terminal APP. Since both a decreased level of APP secretion and a low activity and level of PKC may be involved in the pathogenesis of AD, it is concluded that the administration of AChE inhibitors to AD patients may facilitate the memory processes and exert a neuroprotective effect.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Defining the Contribution of AMP-activated Protein Kinase (AMPK) and Protein Kinase C (PKC) in Regulation of Glucose Uptake by Metformin in Skeletal Muscle Cells*

    PubMed Central

    Turban, Sophie; Stretton, Clare; Drouin, Olivier; Green, Charlotte J.; Watson, Maria L.; Gray, Alexander; Ross, Fiona; Lantier, Louise; Viollet, Benoit; Hardie, D. Grahame; Marette, Andre; Hundal, Harinder S.

    2012-01-01

    The importance of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) as effectors of metformin (Met) action on glucose uptake (GU) in skeletal muscle cells was investigated. GU in L6 myotubes was stimulated 2-fold following 16 h of Met treatment and acutely enhanced by insulin in an additive fashion. Insulin-stimulated GU was sensitive to PI3K inhibition, whereas that induced by Met was not. Met and its related biguanide, phenformin, stimulated AMPK activation/phosphorylation to a level comparable with that induced by the AMPK activator, 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR). However, the increase in GU elicited by AICAR was significantly lower than that induced by either biguanide. Expression of a constitutively active AMPK mimicked the effects of AICAR on GU, whereas a dominant interfering AMPK or shRNA silencing of AMPK prevented AICAR-stimulated GU and Met-induced AMPK signaling but only repressed biguanide-stimulated GU by ∼20%. Consistent with this, analysis of GU in muscle cells from α1−/−/α2−/− AMPK-deficient mice revealed a significant retention of Met-stimulated GU, being reduced by ∼35% compared with that of wild type cells. Atypical PKCs (aPKCs) have been implicated in Met-stimulated GU, and in line with this, Met and phenformin induced activation/phosphorylation of aPKC in L6 myotubes. However, although cellular depletion of aPKC (>90%) led to loss in biguanide-induced aPKC phosphorylation, it had no effect on Met-stimulated GU, whereas inhibitors targeting novel/conventional PKCs caused a significant reduction in biguanide-induced GU. Our findings indicate that although Met activates AMPK, a significant component of Met-stimulated GU in muscle cells is mediated via an AMPK-independent mechanism that involves novel/conventional PKCs. PMID:22511782

  10. Maternal high-salt diet altered PKC/MLC20 pathway and increased ANG II receptor-mediated vasoconstriction in adult male rat offspring.

    PubMed

    Li, Weisheng; Lv, Juanxiu; Wu, Jue; Zhou, Xiuwen; Jiang, Lin; Zhu, Xiaolin; Tu, Qing; Tang, Jiaqi; Liu, Yanping; He, Axin; Zhong, Yuan; Xu, Zhice

    2016-07-01

    High-salt diet (HSD) is associated with cardiovascular diseases. This study aims at ascertaining the influence of maternal HSD on offspring's angiotensin II (ANG II)-mediated vasoconstriction and the underlying mechanisms. In comparison to a normal-salt diet, HSD used in pregnancy in rats changed the ultrastructures of the coronary artery (CA) in 5-month-old male offspring, and increased ANG II-mediated CA contractility. Measurement of [Ca(2+) ]i in CA using fluorescent fura-2, a Ca(2+) indicator, showed that ANG II-mediated increases in [Ca(2+) ]i were the same between HSD and normal-salt diet groups, but the ratio of diameter change/[Ca(2+) ]i induced by ANG II were significantly higher in HSD groups. Angiotensin II receptor type 1, not angiotensin II receptor type 2, caused ANG II-mediated vasoconstriction. Protein kinase C (PKC) inhibitor GF109203X attenuated the ANG II-mediated vasoconstriction, PKC agonist phorbol12,13-dibutyrate produced a greater contraction. There was an increase in PKCβ mRNA and the corresponding protein abundance in the offspring, whereas other PKC subunits PKCα, PKCδ, and PKCε did not change. Moreover, 20 kDa myosin light chain phosphorylation levels were increased in HSD group. Maternal HSD affected the developmental programing for the offspring CA, with increased ANG II-mediated vasoconstrictions. The angiotensin II receptor type 1-PKC-20 kDa myosin light chain phosphorylation pathway was the possible mediated cellular mechanism. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. 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.

  12. Upregulation of TLR4 via PKC activation contributes to impaired wound healing in high-glucose-treated kidney proximal tubular cells

    PubMed Central

    Peng, Jianping; Zheng, Hang; Wang, Xia; Cheng, Zhixiang

    2017-01-01

    Acute kidney injury (AKI) leads to a worse prognosis in diabetic patients compared with prognoses in non-diabetic patients, but whether and how diabetes affects kidney repair after AKI remains unknown. Here, we used scratch-wound healing and transwell migration models to examine whether and how wound healing is affected by high glucose levels in cultured kidney proximal tubular cells (RPTC). The results show that scratch-wound healing and transwell migration were significantly slower in high-glucose-treated kidney tubular cells (30 mM glucose) than in low-glucose-treated cells (5.5 mM). Toll-like receptor 4 (TLR4), MyD88, phospho-protein kinase C (PKC), phospho-p38 MAPK and monocyte chemoattractant protein-1 (MCP-1) mRNA levels were upregulated after high glucose treatments. Staurosporine, a selective PKC inhibitor, inhibited TLR4, MyD88 and p-p38 upregulation in the high-glucose-treated cells, indicating the involvement of PKC in high-glucose-induced TLR4 upregulation. The pharmacological inhibition of TLR4 or shRNA-mediated TLR4 knockdown improved wound healing and transwell migration in high-glucose-treated RPTC. In contrast, the overexpression of TLR4 in low-glucose-treated RPTC suppressed wound healing, mimicking the effects of high glucose levels. These results suggest that the upregulation of TLR4 expression via PKC activation contributes to defective wound healing in high-glucose-treated kidney tubular cells. PMID:28542370

  13. PKA, novel PKC isoforms, and ERK is mediating PACAP auto-regulation via PAC1R in human neuroblastoma NB-1 cells.

    PubMed

    Georg, Birgitte; Falktoft, Birgitte; Fahrenkrug, Jan

    2016-12-01

    The neuropeptide PACAP is expressed throughout the central and peripheral nervous system where it modulates diverse physiological functions including neuropeptide gene expression. We here report that in human neuroblastoma NB-1 cells PACAP transiently induces its own expression. Maximal PACAP mRNA expression was found after stimulation with PACAP for 3h. PACAP auto-regulation was found to be mediated by activation of PACAP specific PAC1Rs as PACAP had >100-fold higher efficacy than VIP, and the PAC1R selective agonist Maxadilan potently induced PACAP gene expression. Experiments with pharmacological kinase inhibitors revealed that both PKA and novel but not conventional PKC isozymes were involved in the PACAP auto-regulation. Inhibition of MAPK/ERK kinase (MEK) also impeded the induction, and we found that PKA, novel PKC and ERK acted in parallel and were thus not part of the same pathways. The expression of the transcription factor EGR1 previously ascribed as target of PACAP signalling was found to be transiently induced by PACAP and pharmacological inhibition of either PKC or MEK1/2 abolished PACAP mediated EGR1 induction. In contrast, inhibition of PKA mediated increased PACAP mediated EGR1 induction. Experiments using siRNA against EGR1 to lower the expression did however not affect the PACAP auto-regulation indicating that this immediate early gene product is not part of PACAP auto-regulation in NB-1 cells. We here reveal that in NB-1 neuroblastoma cells, PACAP induces its own expression by activation of PAC1R, and that the signalling is different from the PAC1R signalling mediating induction of VIP in the same cells. PACAP auto-regulation depends on parallel activation of PKA, novel PKC isoforms, and ERK, while EGR1 does not seem to be part of the PACAP auto-regulation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. 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

  15. Protein Kinase C Inhibitors as Modulators of Vascular Function and their Application in Vascular Disease.

    PubMed

    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 Ca(2+) concentration ([Ca(2+)]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 Ca(2+)-dependent and Ca(2+)-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 [Ca(2+)]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 metalloproteinase (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

  16. 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

  17. 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.

  18. 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.

  19. Go-6976 Reverses Hyperglycemia-Induced Insulin Resistance Independently of cPKC Inhibition in Adipocytes

    PubMed Central

    Robinson, Katherine A.; Hegyi, Krisztina; Hannun, Yusuf A.; Buse, Maria G.; Sethi, Jaswinder K.

    2014-01-01

    Chronic hyperglycemia induces insulin resistance by mechanisms that are incompletely understood. One model of hyperglycemia-induced insulin resistance involves chronic preincubation of adipocytes in the presence of high glucose and low insulin concentrations. We have previously shown that the mTOR complex 1 (mTORC1) plays a partial role in the development of insulin resistance in this model. Here, we demonstrate that treatment with Go-6976, a widely used “specific” inhibitor of cPKCs, alleviates hyperglycemia-induced insulin resistance. However, the effects of mTOR inhibitor, rapamycin and Go-6976 were not additive and only rapamycin restored impaired insulin-stimulated AKT activation. Although, PKCα, (but not –β) was abundantly expressed in these adipocytes, our studies indicate cPKCs do not play a major role in causing insulin-resistance in this model. There was no evidence of changes in the expression or phosphorylation of PKCα, and PKCα knock-down did not prevent the reduction of insulin-stimulated glucose transport. This was also consistent with lack of IRS-1 phosphorylation on Ser-24 in hyperglycemia-induced insulin-resistant adipocytes. Treatment with Go-6976 did inhibit a component of the mTORC1 pathway, as evidenced by decreased phosphorylation of S6 ribosomal protein. Raptor knock-down enhanced the effect of insulin on glucose transport in insulin resistant adipocytes. Go-6976 had the same effect in control cells, but was ineffective in cells with Raptor knock-down. Taken together these findings suggest that Go-6976 exerts its effect in alleviating hyperglycemia-induced insulin-resistance independently of cPKC inhibition and may target components of the mTORC1 signaling pathway. PMID:25330241

  20. 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.

  1. 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

  2. Apelin-13 upregulates Egr-1 expression in rat vascular smooth muscle cells through the PI3K/Akt and PKC signaling pathways

    SciTech Connect

    Liu, Qi-Feng; Yu, Hong-Wei; Sun, Li-Li; You, Lu; Tao, Gui-Zhou; Qu, Bao-Ze

    2015-12-25

    Previous studies have shown that Apelin-13 upregulates early growth response factor-1 (Egr-1) via the extracellular signal-regulated protein kinase (ERK) signaling pathway. Apelin-13 induces proliferation and migration of vascular smooth muscle cells (VSMCs) as well as the upregulation of osteopontin (OPN) via the upregulation of Egr-1. This study was designed to further explore the activity of Apelin-13 in VSMCs by investigating members of the mitogen-activated protein kinase (MAPK) family, in particular Jun kinase (JNK) and p38 mitogen-activated protein kinase (P38). We also examined whether the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) and protein kinase C (PKC) signaling pathways were involved in the regulation of Egr-1 by Apelin-13. We treated rat aortic VSMCs with Apelin-13 and examined the expression of JNK, p-JNK, P38, and p-P38 to investigate whether Apelin-13-mediated increases in Egr-1 occurred through the JNK and P38 signaling pathways. We then pretreated VSMCs with the Gi protein inhibitor pertussis toxin (PTX) and the Gq inhibitor YM254890, added Apelin-13 and looked for changes in Egr-1 expression. Finally, we pretreated with the PI3K inhibitor LY294002 and the PKC inhibitor GF109203X, and treated with Apelin-13. Our results showed that JNK and P38 did not participate in Apelin-13-mediated increase in Egr-1. Instead, Apelin-13 upregulation of Egr-1 was mediated by a PTX-sensitive Gi protein. Apelin-13 did increase ERK phosphorylation through the PI3K/Akt and PKC signaling pathways, resulting in changes in Egr-1 expression. These data provide important targets for future studies to modulate vascular remodeling. - Highlights: • Apelin-13 mediates Egr-1 upregulation in vascular smooth muscle cells via ERK1/2. • The underlying mechanisms are unknown, but exclude Jnk or p38 pathway activation. • Apelin-13 binds to Gi, activating the PI3K/Akt and PKC signaling cascades. • Consequent ERK phosphorylation results in increased Egr-1

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. Cooperativity Between Oncogenic PKC Epsilon and Pten Loss in Prostate Cancer Progression

    DTIC Science & Technology

    2016-10-01

    pathway that correlates with motility and metastasis. Based on our preliminary data, PKCε overexpression and Pten loss individually and synergically confer...AWARD NUMBER: W81XWH-14-1-0535 TITLE: Cooperativity Between Oncogenic PKC Epsilon and Pten Loss in Prostate Cancer Progression PRINCIPAL...Sep 2015 - 29 Sep 2016 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Cooperativity Between Oncogenic PKC Epsilon and Pten Loss in Prostate Cancer

  11. 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.

  12. 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.

  13. PKC-η-MARCKS Signaling Promotes Intracellular Survival of Unopsonized Burkholderia thailandensis

    DOE PAGES

    Micheva-Viteva, Sofiya N.; Shou, Yulin; Ganguly, Kumkum; ...

    2017-06-07

    Pathogenic Burkholderia rely on host factors for efficient intracellular replication and are highly refractory to antibiotic treatment. To identify host genes that are required by Burkholderia spp. during infection, we performed a RNA interference (RNAi) screen of the human kinome and identified 35 host kinases that facilitated Burkholderia thailandensis intracellular survival in human monocytic THP-1 cells. We validated a selection of host kinases using imaging flow cytometry to assess efficiency of B. thailandensis survival in the host upon siRNA-mediated knockdown. We focused on the role of the novel protein kinase C isoform, PKC-η, in Burkholderia infection and characterized PKC-η/MARCKS signalingmore » as a key event that promotes the survival of unopsonized B. thailandensis CDC2721121 within host cells. While infection of lung epithelial cells with unopsonized Gram-negative bacteria stimulated phosphorylation of Ser175/160 in the MARCKS effector domain, siRNA-mediated knockdown of PKC-η expression reduced the levels of phosphorylated MARCKS by >3-fold in response to infection with Bt CDC2721121. We compared the effect of the conventional PKC-α and novel PKC-η isoforms on the growth of B. thailandensis CDC2721121 within monocytic THP-1 cells and found that ≥75% knock-down of PRKCH transcript levels reduced intracellular bacterial load 100% more efficiently when compared to growth in cells siRNA-depleted of the classical PKC-α, suggesting that the PKC-η isoform can specifically mediate Burkholderia intracellular survival. Based on imaging studies of intracellular B. thailandensis, we found that PKC-η function stimulates phagocytic pathways that promote B. thailandensis escape into the cytoplasm leading to activation of autophagosome flux. As a result, identification of host kinases that are targeted by Burkholderia during infection provides valuable molecular insights in understanding Burkholderia pathogenesis, and ultimately, in designing effective

  14. 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.

  15. 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

  16. A bidirectional antagonism between aPKC and Yurt regulates epithelial cell polarity

    PubMed Central

    Gamblin, Clémence L.; Hardy, Émilie J.-L.; Chartier, François J.-M.; Bisson, Nicolas

    2014-01-01

    During epithelial cell polarization, Yurt (Yrt) is initially confined to the lateral membrane and supports the stability of this membrane domain by repressing the Crumbs-containing apical machinery. At late stages of embryogenesis, the apical recruitment of Yrt restricts the size of the apical membrane. However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined. In this paper, we report that atypical protein kinase C (aPKC) phosphorylates Yrt to prevent its premature apical localization. A nonphosphorylatable version of Yrt dominantly dismantles the apical domain, showing that its aPKC-mediated exclusion is crucial for epithelial cell polarity. In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane. The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity. Thus, Yrt and aPKC are involved in a reciprocal antagonistic regulatory loop that contributes to segregation of distinct and mutually exclusive membrane domains in epithelial cells. PMID:24515345

  17. 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

  18. 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.

  19. Synthesis and biological activities of simplified analogs of the natural PKC ligands, bryostatin-1 and aplysiatoxin.

    PubMed

    Irie, Kazuhiro; Yanagita, Ryo C

    2014-04-01

    Protein kinase C (PKC) isozymes play central roles in signal transduction on the cell surface and could serve as promising therapeutic targets of intractable diseases like cancer, Alzheimer's disease, and acquired immunodeficiency syndrome (AIDS). Although natural PKC ligands like phorbol esters, ingenol esters, and teleocidins have the potential to become therapeutic leads, most of them are potent tumor promoters in mouse skin. By contrast, bryostatin-1 (bryo-1) isolated from marine bryozoan is a potent PKC activator with little tumor-promoting activity. Numerous investigations have suggested bryo-1 to be a promising therapeutic candidate for the above intractable diseases. However, there is a supply problem of bryo-1 both from natural sources and by organic synthesis. Recent approaches on the synthesis of bryo-1 have focused on its simplification, without decreasing the ability to activate PKC isozymes, to develop new medicinal leads. Another approach is to use the skeleton of natural PKC ligands to develop bryo-1 surrogates. We have recently identified 10-methyl-aplog-1 (26), a simplified analog of tumor-promoting aplysiatoxin (ATX), as a possible therapeutic lead for cancer. This review summarizes recent investigations on the simplification of natural PKC ligands, bryo-1 and ATX, to develop potential medicinal leads. Copyright © 2014 The Chemical Society of Japan and Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. 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.

  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. 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.

  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. Aldehyde dehydrogenase 2 activation and coevolution of its εPKC-mediated phosphorylation sites.

    PubMed

    Nene, Aishwarya; Chen, Che-Hong; Disatnik, Marie-Hélène; Cruz, Leslie; Mochly-Rosen, Daria

    2017-01-05

    Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a key enzyme for the metabolism of many toxic aldehydes such as acetaldehyde, derived from alcohol drinking, and 4HNE, an oxidative stress-derived lipid peroxidation aldehyde. Post-translational enhancement of ALDH2 activity can be achieved by serine/threonine phosphorylation by epsilon protein kinase C (εPKC). Elevated ALDH2 is beneficial in reducing injury following myocardial infarction, stroke and other oxidative stress and aldehyde toxicity-related diseases. We have previously identified three εPKC phosphorylation sites, threonine 185 (T185), serine 279 (S279) and threonine 412 (T412), on ALDH2. Here we further characterized the role and contribution of each phosphorylation site to the enhancement of enzymatic activity by εPKC. Each individual phosphorylation site was mutated to a negatively charged amino acid, glutamate, to mimic a phosphorylation, or to a non-phosphorylatable amino acid, alanine. ALDH2 enzyme activities and protection against 4HNE inactivation were measured in the presence or absence of εPKC phosphorylation in vitro. Coevolution of ALDH2 and its εPKC phosphorylation sites was delineated by multiple sequence alignments among a diverse range of species and within the ALDH multigene family. We identified S279 as a critical εPKC phosphorylation site in the activation of ALDH2. The critical catalytic site, cysteine 302 (C302) of ALDH2 is susceptible to adduct formation by reactive aldehyde, 4HNE, which readily renders the enzyme inactive. We show that phosphomimetic mutations of T185E, S279E and T412E confer protection of ALDH2 against 4HNE-induced inactivation, indicating that phosphorylation on these three sites by εPKC likely also protects the enzyme against reactive aldehydes. Finally, we demonstrate that the three ALDH2 phosphorylation sites co-evolved with εPKC over a wide range of species. Alignment of 18 human ALDH isozymes, indicates that T185 and S279 are unique ALDH2, εPKC

  6. Diazoxide ameliorates microcirculatory disturbances through PKC-dependent pathway in I/R-injured rat cremaster muscles.

    PubMed

    Wei, William; Wei, Fu-Chan; Hung, Li-Man

    2005-01-01

    Diazoxide is a selective mitochondria ATP-sensitive potassium (K(ATP)) channel opener, which has been reported to preserve the microvascular integrity of ischemia-reperfusion (I/R)-injured tissues. Our study aimed to assess diazoxide's effects on I/R-injured cremaster muscles and to further elucidate its underlying mechanisms. Male Sprague Dawley (SD) rats were randomized (n = 8 per group) into four groups: sham-operated control group, I/R group (4 h of pudic epigastic artery ischemia followed by 2 h of reperfusion), diazoxide + I/R group, and chelerythrine (PKC inhibitor)+diazoxide+I/R group. Microscopically, we observed that I/R markedly increased the number of rolling, adhering, and transmigrating leukocytes. I/R also markedly decreased the number of functional capillaries. Biochemically, we found that I/R significantly increased TNF-alpha, E-selectin,L-selectin and P-selectin expressions. However, I/R did not cause significant changes in ICAM-1 and PECAM-1 expressions. On the other hand, in I/R + diazoxide group, we found that diazoxide reduced the number of rolling, adhering, and transmigrating leukocytes. Furthermore, biochemical study revealed that diazoxide caused only a decrease in L-selectin expression but had no effect on TNF-alpha, E-selectin, P-selectin, ICAM-1, and PECAM-1 expressions. Finally, in chelerythrine + diazoxide + I/R group, we observed that diazoxide's protective effects were blocked by the addition of chelerythrine. Diazoxide's ability to protect against I/R injury was confirmed by the observation that it reduced the number of rolling, adhering, and transmigrating leukocytes, and increased the number of functional capillaries. Our results indicated that diazoxide operated via a PKC-dependent pathway to achieve protection against I/R injury.

  7. 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

  8. 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.

  9. Black Ink of Activated Carbon Derived From Palm Kernel Cake (PKC)

    NASA Astrophysics Data System (ADS)

    Selamat, M. H.; Ahmad, A. H.

    2009-06-01

    Recycling the waste from natural plant to produce useful end products will benefit many industries and help preserve the environment. The research reported in this paper is an investigation on the use of the natural waste of palm kernel cake (PKC) to produce carbon residue as a black carbon for pigment source by using pyrolysis process. The activated carbons (AC) is produced in powder form using ball milling process. Rheological spectra in ink is one of quality control process in determining its performance properties. Findings from this study will help expand the scientific knowledge-base for black ink production and formulation base on PKC. Various inks with different weight percentage compositions of AC will be made and tested against its respective rheological properties in order to determine ideal ink printing system. The items in the formulation used comprised of organic and bio-waste materials with added additive to improve the quality of the black ink. Modified Polyurethane was used as binder. The binder's properties highlighted an ideal vehicle to be applied for good black ink opacity performance. The rheological behaviour is a general foundation for ink characterization where the wt% of AC-PKC resulted in different pseudoplastic behaviors, including the Newtonian behavior. The result found that Newtonian field was located in between 2 wt% and 10 wt% of AC-PKC composition with binder. Mass spectroscopy results shown that the carbon content in PKC is high and very suitable for black performance. In the ageing test, the pigment of PKC perform fairly according to the standard pigment of Black carbon (CB) of ferum oxide pigment. The contact angle for substrate's wettability of the ink system shown a good angle proven to be a water resistive coating on paper subtrates; an advantage of the PKC ink pigment performance.

  10. Dap160/intersectin binds and activates aPKC to regulate cell polarity and cell cycle progression

    PubMed Central

    Chabu, Chiswili; Doe, Chris Q.

    2009-01-01

    The atypical protein kinase C (aPKC) is required for cell polarization of many cell types, and is upregulated in several human tumors. Despite its importance in cell polarity and growth control, relatively little is known about how aPKC activity is regulated. Here, we use a biochemical approach to identify Dynamin-associated protein 160 (Dap160; related to mammalian intersectin) as an aPKC-interacting protein in Drosophila. We show that Dap160 directly interacts with aPKC, stimulates aPKC activity in vitro and colocalizes with aPKC at the apical cortex of embryonic neuroblasts. In dap160 mutants, aPKC is delocalized from the neuroblast apical cortex and has reduced activity, based on its inability to displace known target proteins from the basal cortex. Both dap160 and aPKC mutants have fewer proliferating neuroblasts and a prolonged neuroblast cell cycle. We conclude that Dap160 positively regulates aPKC activity and localization to promote neuroblast cell polarity and cell cycle progression. PMID:18614576

  11. Physiological crosstalk between the AC/PKA and PLC/PKC pathways modulates melatonin-mediated, monochromatic-light-induced proliferation of T-lymphocytes in chickens.

    PubMed

    Guo, Qingyun; Wang, Zixu; Dong, Yulan; Cao, Jing; Chen, Yaoxing

    2017-06-28

    Previous study has demonstrated that melatonin plays a critical role in monochromatic-light-induced lymphocyte proliferation in response to T cell mitogen concanavalin A (ConA). However, its intracellular mechanism is still unclear. In this study, we investigate the intracellular signal pathways of melatonin receptor-mediated T-lymphocyte proliferation in the spleens of chicks exposed to different light wavelengths. Results showed that green light enhanced T-lymphocyte proliferation by 2.46-6.83% and increased splenic mRNA and protein expressions of melatonin receptor subtypes (Mel1a, Mel1b and Mel1c) by 16.05-40.43% compared with the white, red and blue light groups. However, pinealectomy resulted in a decrease in T-lymphocyte proliferation and melatonin receptor expression with no statistically significant differences between the different light groups. In vitro experiments showed that the Mel1b selective antagonist 4P-PDOT, the Mel1c selective antagonist prazosin and the mitogen-activated protein kinase kinase-1 (MEK-1) inhibitor PD98059 suppressed both melatonin-induced lymphocyte proliferation in response to ConA and melatonin- and ConA-stimulated extracellular signal-regulated kinase 1/2 (ERK1/2) activity but that the Mel1a/Mel1b non-selective antagonist luzindole did not. In addition, pretreatment with forskolin (FSK, the adenylyl cyclase activator), H89 (the PKA inhibitor), U73122 (the PLC inhibitor) or Go6983 (the broad spectrum PKC inhibitor) markedly attenuated melatonin- and ConA-stimulated T-lymphocyte proliferation and ERK1/2 activity. These results demonstrate that melatonin mediates green-light-induced T-lymphocyte proliferation via the Mel1b and Mel1c receptors by triggering crosstalk between the cAMP/PKA and PLC/PKC signal pathways followed by ERK1/2 activation.

  12. Niacin Activates the PI3K/Akt Cascade via PKC- and EGFR-Transactivation-Dependent Pathways through Hydroxyl-Carboxylic Acid Receptor 2

    PubMed Central

    Zhang, Wenjuan; Zhou, Qi; Yu, Yena; Shi, Ying; Offermanns, Stefan; Lu, Jianxin; Zhou, Naiming

    2014-01-01

    Niacin has been demonstrated to activate a PI3K/Akt signaling cascade to prevent brain damage after stroke and UV-induced skin damage; however, the underlying molecular mechanisms for HCA2-induced Akt activation remain to be elucidated. Using CHO-K1 cells stably expressing HCA2 and A431 cells, a human epidermoid cell line with high levels of endogenous expression of functional HCA2 receptors, we first demonstrated that niacin induced a robust Akt phosphorylation at both Thr308 and Ser473 in a time-dependent fashion, with a maximal activation at 5 min and a subsequent reduction to baseline by 30 min through HCA2, and that the activation was significantly blocked by pertussis toxin. The HCA2-mediated activation of Akt was also significantly inhibited by the PKC inhibitors GF109203x and Go6983 in both cell lines, by the PDGFR-selective inhibitor tyrphostin A9 in CHO-HCA2 cells and by the MMP inhibitor GM6001 and EGFR-specific inhibitor AG1478 in A431 cells. These results suggest that the PKC pathway and PDGFR/EGFR transactivation pathway play important roles in HCA2-mediated Akt activation. Further investigation indicated that PI3K and the Gβγ subunit were likely to play an essential role in HCA2-induced Akt activation. Moreover, Immunobloting analyses using an antibody that recognizes p70S6K1 phosphorylated at Thr389 showed that niacin evoked p70S6K1 activation via the PI3K/Akt pathway. The results of our study provide new insight into the signaling pathways involved in HCA2 activation. PMID:25375133

  13. 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.

  14. 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.

  15. Expression of syntaxin 1C, an alternative splice variant of HPC-1/syntaxin 1A, is enhanced by phorbol-ester stimulation in astroglioma: participation of the PKC signaling pathway.

    PubMed

    Nakayama, Takahiro; Mikoshiba, Katsuhiko; Yamamori, Tetsuo; Akagawa, Kimio

    2003-02-11

    Syntaxin 1C is an alternative splice variant of HPC-1/syntaxin 1A; the latter participates in neurotransmitter release and is assigned to the gene domain responsible for Williams' syndrome (WS). It is expressed in the soluble fraction extracted from human astroglioma cell lines T98G and U87MG. Quantitative immunoblot and indirect immunofluorescence analyses revealed that the expression of syntaxin 1C was upregulated by phorbol 12-myristate 13-acetate (PMA), but not by forskolin. A protein kinase C (PKC) inhibitor suppressed this enhancement. These results suggest that syntaxin 1C expression is regulated via the PKC signal pathway. This is the first report of a signal transduction system that directly affects the expression of syntaxin protein.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:26733150

  1. 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

  2. Fargesin exerts anti-inflammatory effects in THP-1 monocytes by suppressing PKC-dependent AP-1 and NF-ĸB signaling.

    PubMed

    Pham, Thu-Huyen; Kim, Man-Sub; Le, Minh-Quan; Song, Yong-Seok; Bak, Yesol; Ryu, Hyung-Won; Oh, Sei-Ryang; Yoon, Do-Young

    2017-01-15

    Fargesin is a lignan from Magnolia fargesii, an oriental medicine used in the treatment of nasal congestion and sinusitis. The anti-inflammatory properties of this compound have not been fully elucidated yet. This study focused on assessing the anti-inflammatory effects of fargesin on phorbal ester (PMA)-stimulated THP-1 human monocytes, and the molecular mechanisms underlying them. Cell viability was evaluated by MTS assay. Protein expression levels of inflammatory mediators were analyzed by Western blotting, ELISA, Immunofluorescence assay. mRNA levels were measured by Real-time PCR. Promoter activities were elucidated by Luciferase assay. It was found that pre-treatment with fargesin attenuated significantly the expression of two major inflammatory mediators, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Fargesin also inhibited the production of pro-inflammation cytokines (IL-1β, TNF-α) and chemokine (CCL-5). Besides, nuclear translocation of transcription factors nuclear factor-kappa B (NF-ĸB) and activator protein-1 (AP-1), which regulate multiple pro-inflammatory genes, was suppressed by fargesin in a PKC-dependent manner. Furthermore, among the mitogen-activated protein kinases (MAPKs), only c-Jun N-terminal kinase (JNK) was downregulated by fargesin in a PKC-dependent manner, and this reduction was involved in PMA-induced AP-1 and NF-ĸB nuclear translocation attenuation, demonstrated using a specific JNK inhibitor. Taken together, our results found that fargesin exhibits anti-inflammation effects on THP-1 cells via suppression of PKC pathway including downstream JNK, nuclear factors AP-1 and NF-ĸB. These results suggest that fargesin has anti-inflammatory properties with potential applications in drug development against inflammatory disorders. Copyright © 2016. Published by Elsevier GmbH.

  3. 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.

  4. 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

  5. The PDK1 master kinase is over-expressed in acute myeloid leukemia and promotes PKC-mediated survival of leukemic blasts.

    PubMed

    Zabkiewicz, Joanna; Pearn, Lorna; Hills, Robert K; Morgan, Rhys G; Tonks, Alex; Burnett, Alan K; Darley, Richard L

    2014-05-01

    PDK1 is a master kinase that activates at least six protein kinase groups including AKT, PKC and S6K and is a potential target in the treatment of a range of malignancies. Here we show overexpression of PDK1 in over 40% of myelomonocytic acute leukemia patients. Overexpression of PDK1 occurred uniformly throughout the leukemic population, including putative leukemia-initiating cells. Clinical outcome analysis revealed PDK1 overexpression was associated with poorer treatment outcome. Primary acute myeloid leukemia blasts over-expressing PDK1 showed improved in vitro survival and ectopic expression of PDK1 promoted the survival of myeloid cell lines. Analysis of PDK1 target kinases revealed that PDK1 overexpression was most closely associated with increased phosphorylation of PKC isoenzymes and inhibition of PKC strongly inhibited the survival advantage of PDK1 over-expressing cells. Membrane localization studies implicated PKCα as a major target for PDK1 in this disease. PDK1 over-expressing blasts showed differential sensitivity to PDK1 inhibition (in the low micromolar range) suggesting oncogene addiction, whilst normal bone marrow progenitors were refractory to PDK1 inhibition at effective inhibitor concentrations. PDK1 inhibition also targeted subpopulations of leukemic blasts with a putative leukemia-initiating cell phenotype. Together these data show that overexpression of PDK1 is common in acute myelomonocytic leukemia and is associated with poorer treatment outcome, probably arising from the cytoprotective function of PDK1. We also show that therapeutic targeting of PDK1 has the potential to be both an effective and selective treatment for these patients, and is also compatible with current treatment regimes.

  6. 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.

  7. Clematichinenoside Serves as a Neuroprotective Agent Against Ischemic Stroke: The Synergistic Action of ERK1/2 and cPKC Pathways

    PubMed Central

    Liu, Chao; Du, Qianming; Zhang, Xu; Tang, Zhichao; Ji, Hui; Li, Yunman

    2016-01-01

    There are numerous evidences suggesting that inhibition of apoptosis of neurons play a critical role in preventing the damage and even death of neurons after brain ischemia/reperfusion, which shows therapeutic potential for clinical treatment of brain injury induced by stroke. In this study, we aimed to investigate the neuroprotective effect of Clematichinenoside (AR) and its underlying mechanisms. MCAO mode was performed in rats and OGD/R model in primary cortical neurons to investigate the neuroprotective effect of AR. The rate of apoptotic cells was measured using TUNEL assay in cerebral cortex and flow cytometric assay in cortical neurons. Apoptosis-related proteins such as bcl-2, bcl-xl, and bax and the phosphorylation of ERK1/2, cPKC, p90RSK, and CREB in ischemic penumbra were assayed by western blot. Furthermore, we made a thorough inquiry about how these proteins play roles in the anti-apoptotic mechanism using targets-associated inhibitors step by step. The results revealed that AR could activate both ERK1/2 and cPKC which resulted in p90RSK phosphorylation and translocation into the nucleus. Moreover, CREB, a downstream target of p90RSK, was phosphorylated and then bound to cAMP-regulated enhancer (CRE) to activate apoptosis-related genes, and finally ameliorate ischemic stroke through preventing neuron death. In conclusion, these data strongly suggest that AR could be used as an effective neuroprotective agent to protect against ischemic stroke after cerebral I/R injury through regulating both ERK1/2 and cPKC mediated p90RSK/CREB apoptotic pathways. PMID:26793066

  8. Kibra and aPKC regulate starvation-induced autophagy in Drosophila

    SciTech Connect

    Jin, Ahrum; Neufeld, Thomas P.; Choe, Joonho

    2015-12-04

    Autophagy is a bulk degradation system that functions in response to cellular stresses such as metabolic stress, endoplasmic reticulum stress, oxidative stress, and developmental processes. During autophagy, cytoplasmic components are captured in double-membrane vesicles called autophagosomes. The autophagosome fuses with the lysosome, producing a vacuole known as an autolysosome. The cellular components are degraded by lysosomal proteases and recycled. Autophagy is important for maintaining cellular homeostasis, and the process is evolutionarily conserved. Kibra is an upstream regulator of the hippo signaling pathway, which controls organ size by affecting cell growth, proliferation, and apoptosis. Kibra is mainly localized in the apical membrane domain of epithelial cells and acts as a scaffold protein. We found that Kibra is required for autophagy to function properly. The absence of Kibra caused defects in the formation of autophagic vesicles and autophagic degradation. We also found that the well-known cell polarity protein aPKC interacts with Kibra, and its activity affects autophagy upstream of Kibra. Constitutively active aPKC decreased autophagic vesicle formation and autophagic degradation. We confirmed the interaction between aPKC and Kibra in S2 cells and Drosophila larva. Taken together, our data suggest that Kibra and aPKC are essential for regulating starvation-induced autophagy. - Highlights: • Loss of Kibra causes defects in autophagosome formation and autophagic degradation. • Constitutively-active aPKCs negatively regulate autophagy. • Kibra interacts with aPKC in vitro and in vivo. • Kibra regulates autophagy downstream of aPKC.

  9. Mechanism by which peripheral galanin increases acute inflammatory pain.

    PubMed

    Jimenez-Andrade, Juan Miguel; Zhou, Shengtai; Yamani, Ammar; Valencia de Ita, Sandra; Castañeda-Hernandez, Gilberto; Carlton, Susan M

    2005-09-21

    Galanin (GAL) is a neuropeptide involved in pain transmission. Intraplantar GAL at low doses enhances capsaicin (CAP)-induced pain behaviors in rat, suggesting an excitatory role for GAL under acute inflammatory conditions. The mechanisms underlying this pro-nociceptive action have not yet been elucidated. Thus, the present study investigated the role of protein kinase C (PKC) in the GAL enhancement of CAP-induced inflammatory pain. Ipsilateral, but not contralateral, calphostin C, a PKC inhibitor, blocked GAL-induced potentiation of CAP-evoked inflammatory pain in a dose-dependent fashion. Peripheral activation of PKC using the phorbol ester phorbol-12-myristate-13-acetate (PMA) mimicked the pro-nociceptive effect of GAL. These results suggest that GAL enhances acute inflammatory pain through activation of PKC intracellular pathways.

  10. Induction of hyper-adhesion attenuates autoimmune-induced keratinocyte cell-cell detachment and processing of adhesion molecules via mechanisms that involve PKC.

    PubMed

    Cirillo, Nicola; Lanza, Alessandro; Prime, Stephen S

    2010-02-15

    In confluent keratinocyte monolayers, desmosomal adhesion gradually becomes calcium-independent and this is associated with an increase in the strength of intercellular adhesion (hyper-adhesion). In this study, we investigated the functional and molecular significance of hyper-adhesion in a system challenged by autoimmune sera from patients with Pemphigus Vulgaris (PV), a disease primarily targeting desmosomal adhesion. The results show that keratinocytes with calcium-independent desmosomes are resistant to disruption of intercellular contacts (acantholysis) in experimental PV. Furthermore, both the desmosomal cadherins desmoglein (Dsg) 1 and Dsg3 and the adherens junction protein E-cadherin were decreased in confluent keratinocytes at Day 1, but not in hyper-adhesive cells (Day 6) after incubation with PV serum. Pharmacological induction of the hyper-adhesive state with the PKC inhibitor Go6976 reduced both the acantholysis rate and the processing of cell adhesion molecules induced by PV serum. When the establishment of the hyper-adhesive state was prevented by cell adhesion recognition (CAR) peptides that perturbed desmosomal interactions, Go6976 could still partially attenuate PV acantholysis. Taken together, these data demonstrate that keratinocyte hyper-adhesion decreases the morphological, functional and biochemical dys-cohesive effects of PV serum via mechanisms that involve, at least in part, the function of PKC. This suggests that reinforcing keratinocyte adhesion may be a promising way to inhibit the effects of this most debilitating disorder. Crown Copyright 2009. Published by Elsevier Inc. All rights reserved.

  11. 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.

  12. Diacylglycerol Metabolism and Signaling is a Driving Force Underlying FASN Inhibitor Sensitivity in Cancer Cells

    PubMed Central

    Benjamin, Daniel I.; Li, Daniel S.; Lowe, Wallace; Heuer, Timothy; Kemble, George; Nomura, Daniel K.

    2015-01-01

    Fatty acid synthase (FASN) generates the de novo source of lipids for cell proliferation and is a promising cancer therapy target. Development of FASN inhibitors, however, necessitates a better understanding of sensitive and resistant cancer types to optimize patient treatment. Indeed, testing the cytotoxic effects of FASN inhibition across human cancer cells revealed diverse sensitivities. We show here that metabolic incorporation of glucose into specific complex lipid species strongly predicts FASN inhibitor sensitivity. We also show that the levels of one of these lipid classes, protein kinase C (PKC) stimulators diacylglycerols, are lowered upon FASN inhibitor treatment in sensitive compared to resistant cells and that PKC activators and inhibitors rescue cell death in sensitive cells and sensitize resistant cells, respectively. Our findings not only reveal a biomarker for predicting FASN sensitivity in cancer cells, but also a put forth a heretofore unrecognized mechanism underlying the anti-cancer effects of FASN inhibitors. PMID:25871544

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. Drosophila aPKC regulates cell polarity and cell proliferation in neuroblasts and epithelia

    PubMed Central

    Rolls, Melissa M.; Albertson, Roger; Shih, Hsin-Pei; Lee, Cheng-Yu; Doe, Chris Q.

    2003-01-01

    Cell polarity is essential for generating cell diversity and for the proper function of most differentiated cell types. In many organisms, cell polarity is regulated by the atypical protein kinase C (aPKC), Bazooka (Baz/Par3), and Par6 proteins. Here, we show that Drosophila aPKC zygotic null mutants survive to mid-larval stages, where they exhibit defects in neuroblast and epithelial cell polarity. Mutant neuroblasts lack apical localization of Par6 and Lgl, and fail to exclude Miranda from the apical cortex; yet, they show normal apical crescents of Baz/Par3, Pins, Inscuteable, and Discs large and normal spindle orientation. Mutant imaginal disc epithelia have defects in apical/basal cell polarity and tissue morphology. In addition, we show that aPKC mutants show reduced cell proliferation in both neuroblasts and epithelia, the opposite of the lethal giant larvae (lgl) tumor suppressor phenotype, and that reduced aPKC levels strongly suppress most lgl cell polarity and overproliferation phenotypes. PMID:14657233

  19. 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

  20. 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.

  1. 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

  2. Protein kinase C in the wood frog, Rana sylvatica: reassessing the tissue-specific regulation of PKC isozymes during freezing

    PubMed Central

    Storey, Kenneth B.

    2014-01-01

    The wood frog, Rana sylvatica, survives whole-body freezing and thawing each winter. The extensive adaptations required at the biochemical level are facilitated by alterations to signaling pathways, including the insulin/Akt and AMPK pathways. Past studies investigating changing tissue-specific patterns of the second messenger IP3 in adapted frogs have suggested important roles for protein kinase C (PKC) in response to stress. In addition to their dependence on second messengers, phosphorylation of three PKC sites by upstream kinases (most notably PDK1) is needed for full PKC activation, according to widely-accepted models. The present study uses phospho-specific immunoblotting to investigate phosphorylation states of PKC—as they relate to distinct tissues, PKC isozymes, and phosphorylation sites—in control and frozen frogs. In contrast to past studies where second messengers of PKC increased during the freezing process, phosphorylation of PKC tended to generally decline in most tissues of frozen frogs. All PKC isozymes and specific phosphorylation sites detected by immunoblotting decreased in phosphorylation levels in hind leg skeletal muscle and hearts of frozen frogs. Most PKC isozymes and specific phosphorylation sites detected in livers and kidneys also declined; the only exceptions were the levels of isozymes/phosphorylation sites detected by the phospho-PKCα/βII (Thr638/641) antibody, which remained unchanged from control to frozen frogs. Changes in brains of frozen frogs were unique; no decreases were observed in the phosphorylation levels of any of the PKC isozymes and/or specific phosphorylation sites detected by immunoblotting. Rather, increases were observed for the levels of isozymes/phosphorylation sites detected by the phospho-PKCα/βII (Thr638/641), phospho-PKCδ (Thr505), and phospho-PKCθ (Thr538) antibodies; all other isozymes/phosphorylation sites detected in brain remained unchanged from control to frozen frogs. The results of this study

  3. Matrix metalloproteinase inhibitors.

    PubMed

    Wojtowicz-Praga, S M; Dickson, R B; Hawkins, M J

    1997-01-01

    extremely poor water solubility, which required intraperitoneal administration of the drug as a detergent emulsion. Marimastat belongs to a second generation of MMP inhibitors. In contrast to batimastat, marimastat is orally available. Both of these agents are currently in Phase I/II trials in US, Europe and Canada. Some other new agents, currently in clinical trials, have been shown to inhibit MMP production. Bryostatins, naturally occurring macrocyclic lactones, have both in vitro and in vivo activity in numerous murine and human tumors. In culture, bryostatin-1 has been shown to induce differentiation and halt the growth of several malignant cell lines. While the exact mechanism responsible for anti-tumor activity is unclear, an initial event in the action of bryostatin-1 is activation of protein kinase C (PKC), followed by its down regulation. Bryostatin-1 does not directly affect the activity of MMPs, but it can inhibit the production of MMP-1, 3, 9, 10 and 11 by inhibiting PKC. TIMP-1 levels could also be modulated by bryostatin-1, as it is encoded by a PKC responsive gene.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. Does PKC activation increase the homologous desensitization of μ opioid receptors?

    PubMed

    Arttamangkul, Seksiri; Birdsong, William; Williams, John T

    2015-01-01

    This study examined the role of agents known to activate PKC on morphine-induced desensitization of μ-opioid receptors (MOP receptors) in brain slices containing locus coeruleus neurons. Intracellular recordings were obtained from rat locus coeruleus neurons. Two measurements were used to characterize desensitization, the decline in hyperpolarization induced by application of a saturating concentration of agonist (acute desensitization) and the decrease in hyperpolarization induced by a subsaturating concentration of [Met](5) enkephalin (ME) following washout of the saturating concentration (sustained desensitization). Internalization of MOP receptors was studied in brain slices prepared from transgenic mice expressing Flag-MOP receptors. The subcellular distribution of activated PKC was examined using a novel fluorescent sensor of PKC in HEK293 cells. The phorbol esters (PMA and PDBu) and muscarine increased acute desensitization induced by a saturating concentration of morphine and ME. These effects were not sensitive to staurosporine. Staurosporine did not block the decline in hyperpolarization induced by muscarine. PDBu and muscarine did not affect sustained desensitization induced by ME nor did phorbol esters or muscarine change the trafficking of MOP receptors induced by morphine or ME. The distribution of activated PKC measured in HEK293 cells differed depending on which phorbol ester was applied. This study demonstrates a distinct difference in two measurements that are often used to evaluate desensitization. The measure of decline correlated well with the reduction in peak amplitudes caused by PKC activators implicating the modification of other factors rather than MOP receptors. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. © 2014 The British Pharmacological Society.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

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

    PubMed Central

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

    2013-01-01

    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. PMID:24157969

  15. 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.

  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. Proteasome inhibitors induce peroxisome proliferator-activated receptor transactivation through RXR accumulation and a protein kinase C-dependent pathway

    SciTech Connect

    Tsao, W.-C.; Wu, H.-M.; Chi, K.-H.; Chang, Y.-H.; Lin, W.-W. . E-mail: wwl@ha.mc.ntu.edu.tw

    2005-03-10

    Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), a member of nuclear hormone receptors, forms a heterodimeric DNA binding complex with retinoid X receptor (RXR) and serves as a transcriptional regulator of gene expression. In this study, using luciferase assay of a reporter gene containing PPAR response element (PPRE), we found PPRE transactivity was additively induced by PPAR{gamma} activator (15dPGJ{sub 2}) and RXR activator (9-cis retinoic acid, 9-cis RA). Proteasome inhibitors MG132 and MG262 also stimulate PPRE transactivity in a concentration-dependent manner, and this effect is synergistic to 15dPGJ{sub 2} and 9-cis RA. PKC activation by 12-myristate 13-acetate (PMA) and ingenol 3,20-dibenzoate (IDB) also led to an increased PPRE activation, and this action was additive to PPAR{gamma} activators and 9-cis RA, but not to proteasome inhibitors. Results indicate that the PPAR{gamma} enhancing effect of proteasome inhibitors was attributed to redox-sensitive PKC activation. Western blot analysis showed that the protein level of RXR{alpha}, but not PPAR{gamma}, RXR{beta}, or PKC isoforms, was accumulated in the presence of proteasome inhibitors. Taken together, we conclude that proteasome inhibitors can upregulate PPRE activity through RXR{alpha} accumulation and a PKC-dependent pathway. The former is due to inhibition of RXR{alpha} degradation through ubiquitin-dependent proteasome system, while the latter is mediated by reactive oxygen species (ROS) production.

  18. Phosphatidylinositol 3-kinase, phosphoinositide-specific phospholipase-Cgamma and protein kinase-C signal myelin phagocytosis mediated by complement receptor-3 alone and combined with scavenger receptor-AI/II in macrophages.

    PubMed

    Makranz, Chen; Cohen, Goni; Baron, Ayellet; Levidor, Lital; Kodama, Tatsuhiko; Reichert, Fanny; Rotshenker, Shlomo

    2004-03-01

    Complement-receptor-3 (CR3/MAC-1), scavenger-receptor-AI/II (SRAI/II) and Fcgamma-receptor (FcgammaR) can mediate phagocytosis of degenerated myelin in macrophages and microglia. However, CR3/MAC-1 and SRAI/II, but not FcgammaR, mediate phagocytosis after axonal injury. We tested for phosphatidylinositol 3-kinase (PI3K), phosphoinositide-specific phospholipase-Cgamma (PLCgamma) and protein kinase-C (PKC) signaling in myelin phagocytosis mediated by CR3/MAC-1 alone and by CR3/MAC-1 combined with SRAI/II. Phagocytosis was inhibited by PI3K inhibitors wortmannin and LY-294002, PLCgamma inhibitor U-73122, classical PKC (cPKC) inhibitor Go-6976, general PKC inhibitors Ro-318220 and calphostin-C, and BAPTA/AM which chelates intracellular Ca(2+) required for cPKC activation. PKC activator PMA augmented phagocytosis and further alleviated inhibitions induced by PI3K and PLCgamma inhibitors. Overall, altering PKC activity modulated phagocytosis 4- to 6-fold between inhibition and augmentation. PLCgamma activation did not require tyrosine phosphorylation. Thus, signaling of myelin phagocytosis mediated by CR3/MAC-1 alone and by CR3/MAC-1 combined with SRAI/II involves PI3K, PLCgamma and cPKC, the cascade PI3K-->PLCgamma-->cPKC, and wide-range modulation by PKC. This pathway may thus be targeted for in vivo modulation, which may explain differences in the efficiency of CR3/MAC-1-mediated myelin phagocytosis in different pathological conditions.

  19. 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

  20. High concentration of glucose inhibits glomerular endothelial eNOS through a PKC mechanism.

    PubMed

    Chu, Shaoyou; Bohlen, H Glenn

    2004-09-01

    Kidney glomeruli are important targets of diabetic nephropathy. We hypothesized a high concentration of glucose could suppress glomerular endothelial nitric oxide synthase (eNOS) by a protein kinase C (PKC) mechanism, as has been found in other tissues. Mouse kidney slices (150-200 microm) were bathed in Hanks' solution with 100 microM L-arginine and exposed to either 5 or 20-30 mM D-glucose. Immunofluorescence identified only eNOS in normal mouse glomeruli. Measurements of glomerular NO concentration with NO-sensitive fluorescent dye (4,5-diaminofluorescein diacetate) using confocal microscopy and NO-sensitive microelectrodes verified that resting glomeruli had active production of NO that was inhibited by N(G)-nitro-L-arginine methyl ester. High-concentration (20-30 mM) D-glucose inhibited 60-70% of the NO production within 15-30 min; L-glucose at the same concentration did not have any effect. Inhibition of PKC-beta with 100 nM ruboxistaurin prevented eNOS suppression in high-glucose media. Activation of PKC with 100 nM phorbol ester also suppressed the glomerular NO concentration. We concluded that eNOS in the renal glomerular capillary endothelial cells is suppressed by activity of PKC at high-glucose concentrations comparable to those in diabetic animals and humans. The consequence is a rapid decline in the generation of NO in the glomerular endothelial cells in the presence of a high concentration of glucose.

  1. Local control of TRPV4 channels by AKAP150-targeted PKC in arterial smooth muscle

    PubMed Central

    Mercado, Jose; Baylie, Rachael; Navedo, Manuel F.; Yuan, Can; Scott, John D.; Nelson, Mark T.

    2014-01-01

    Transient receptor potential vanilloid 4 (TRPV4) channels are Ca2+-permeable, nonselective cation channels expressed in multiple tissues, including smooth muscle. Although TRPV4 channels play a key role in regulating vascular tone, the mechanisms controlling Ca2+ influx through these channels in arterial myocytes are poorly understood. Here, we tested the hypothesis that in arterial myocytes the anchoring protein AKAP150 and protein kinase C (PKC) play a critical role in the regulation of TRPV4 channels during angiotensin II (AngII) signaling. Super-resolution imaging revealed that TRPV4 channels are gathered into puncta of variable sizes along the sarcolemma of arterial myocytes. Recordings of Ca2+ entry via single TRPV4 channels (“TRPV4 sparklets”) suggested that basal TRPV4 sparklet activity was low. However, Ca2+ entry during elementary TRPV4 sparklets was ∼100-fold greater than that during L-type CaV1.2 channel sparklets. Application of the TRPV4 channel agonist GSK1016790A or the vasoconstrictor AngII increased the activity of TRPV4 sparklets in specific regions of the cells. PKC and AKAP150 were required for AngII-induced increases in TRPV4 sparklet activity. AKAP150 and TRPV4 channel interactions were dynamic; activation of AngII signaling increased the proximity of AKAP150 and TRPV4 puncta in arterial myocytes. Furthermore, local stimulation of diacylglycerol and PKC signaling by laser activation of a light-sensitive Gq-coupled receptor (opto-α1AR) resulted in TRPV4-mediated Ca2+ influx. We propose that AKAP150, PKC, and TRPV4 channels form dynamic subcellular signaling domains that control Ca2+ influx into arterial myocytes. PMID:24778429

  2. Twins/PP2A regulates aPKC to control neuroblast cell polarity and self-renewal

    PubMed Central

    Chabu, Chiswili; Doe, Chris Q.

    2009-01-01

    Asymmetric cell division is a mechanism for generating cell diversity as well as maintaining stem cell homeostasis in both Drosophila and mammals. In Drosophila, larval neuroblasts are stem cell-like progenitors that divide asymmetrically to generate neurons of the adult brain. Mitotic neuroblasts localize atypical protein kinase C (aPKC) to their apical cortex. Cortical aPKC excludes cortical localization of Miranda and its cargo proteins Prospero and Brain tumor, resulting in their partitioning into the differentiating, smaller ganglion mother cell (GMC) where they are required for neuronal differentiation. In addition to aPKC, the kinases Aurora-A and Polo also regulate neuroblast self-renewal, but the phosphatases involved in neuroblast self-renewal have not been identified. Here we report that aPKC is in a protein complex in vivo with Twins, a Drosophila B-type protein phosphatase 2A (PP2A) subunit, and that Twins and the catalytic subunit of PP2A, called Microtubule star (Mts), are detected in larval neuroblasts. Both Twins and Mts are required to exclude aPKC from the basal neuroblast cortex: twins mutant brains, twins mutant single neuroblast mutant clones, or mts dominant negative single neuroblast clones all show ectopic basal cortical localization of aPKC. Consistent with ectopic basal aPKC is the appearance of supernumerary neuroblasts in twins mutant brains or twins mutant clones. We conclude that Twins/PP2A is required to maintain aPKC at the apical cortex of mitotic neuroblasts, keeping it out of the differentiating GMC, and thereby maintaining neuroblast homeostasis. PMID:19374896

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

    DTIC Science & Technology

    2009-02-01

    cells of hairy cell leukemia . Am. J. Pathol. 170: 745-754 (2007). 14. Kampfer S, Windegger M, Hochholdinger F, Schwaiger W, Pestell RG, Baier G...0119 TITLE: Role of the chemokine MCP-1 in sensitization of PKC-mediated apoptosis in prostate cancer cells ...of 5a. CONTRACT NUMBER PKC-mediated apoptosis in prostate cancer cells b. GRANT NUMBER W81XWH-07-1-0119 5c. PROGRAM ELEMENT

  4. 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.

  5. 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

  6. LeY oligosaccharide upregulates DAG/PKC signaling pathway in the human endometrial cells.

    PubMed

    Li, Yali; Ma, Keli; Sun, Ping; Liu, Shuai; Qin, Huamin; Zhu, Zhengmei; Wang, Xiaoqi; Yan, Qiu

    2009-11-01

    LeY oligosaccharide is stage specifically expressed by the embryo and uterine endometrium, and it plays important roles in embryo implantation. In addition to participating in the recognition and adhesion on fetal-maternal interface, LeY potentially regulates the expression of some implantation-related factors. However, it remains elusive whether it can mediate the involved signaling pathway. In this study, agarose-LeY beads were used to mimic the embryos, and the effects of LeY oligosaccharide on DAG/PKC signaling pathway was studied in human endometrial epithelial cells. Results showed that LeY could significantly trigger the activation of cPKCalpha and cPKCbeta2, and their translocation from the cytosol to the plasma membrane. The cellular DAG content was also upregulated, and the activation of PLCgamma1 was promoted. On the contrary, DAG/PKC signaling pathway was significantly inhibited when anti-LeY antibody was used after confirmation of LeY expression in human endometrial epithelial cells by immunohistochemistry and flow cytometry. These results suggest that LeY oligosaccharide acts as a signal molecule to modulate DAG/PKC signaling pathway.

  7. Keratins control intercellular adhesion involving PKC-α–mediated desmoplakin phosphorylation

    PubMed Central

    Kröger, Cornelia; Loschke, Fanny; Schwarz, Nicole; Windoffer, Reinhard; Leube, Rudolf E.

    2013-01-01

    Maintenance of epithelial cell adhesion is crucial for epidermal morphogenesis and homeostasis and relies predominantly on the interaction of keratins with desmosomes. Although the importance of desmosomes to epidermal coherence and keratin organization is well established, the significance of keratins in desmosome organization has not been fully resolved. Here, we report that keratinocytes lacking all keratins show elevated, PKC-α–mediated desmoplakin phosphorylation and subsequent destabilization of desmosomes. We find that PKC-α activity is regulated by Rack1–keratin interaction. Without keratins, desmosomes assemble but are endocytosed at accelerated rates, rendering epithelial sheets highly susceptible to mechanical stress. Re-expression of the keratin pair K5/14, inhibition of PKC-α activity, or blocking of endocytosis reconstituted both desmosome localization at the plasma membrane and epithelial adhesion. Our findings identify a hitherto unknown mechanism by which keratins control intercellular adhesion, with potential implications for tumor invasion and keratinopathies, settings in which diminished cell adhesion facilitates tissue fragility and neoplastic growth. PMID:23690176

  8. Numb controls E-cadherin endocytosis through p120 catenin with aPKC

    PubMed Central

    Sato, Kazuhide; Watanabe, Takashi; Wang, Shujie; Kakeno, Mai; Matsuzawa, Kenji; Matsui, Toshinori; Yokoi, Keiko; Murase, Kiyoko; Sugiyama, Ikuko; Ozawa, Masayuki; Kaibuchi, Kozo

    2011-01-01

    Cadherin trafficking controls tissue morphogenesis and cell polarity. The endocytic adaptor Numb participates in apicobasal polarity by acting on intercellular adhesions in epithelial cells. However, it remains largely unknown how Numb controls cadherin-based adhesion. Here, we found that Numb directly interacted with p120 catenin (p120), which is known to interact with E-cadherin and prevent its internalization. Numb accumulated at intercellular adhesion sites and the apical membrane in epithelial cells. Depletion of Numb impaired E-cadherin internalization, whereas depletion of p120 accelerated internalization. Expression of the Numb-binding fragment of p120 inhibited E-cadherin internalization in a dominant-negative fashion, indicating that Numb interacts with the E-cadherin/p120 complex and promotes E-cadherin endocytosis. Impairment of Numb induced mislocalization of E-cadherin from the lateral membrane to the apical membrane. Atypical protein kinase C (aPKC), a member of the PAR complex, phosphorylated Numb and inhibited its association with p120 and α-adaptin. Depletion or inhibition of aPKC accelerated E-cadherin internalization. Wild-type Numb restored E-cadherin internalization in the Numb-depleted cells, whereas a phosphomimetic mutant or a mutant with defective α-adaptin-binding ability did not restore the internalization. Thus, we propose that aPKC phosphorylates Numb to prevent its binding to p120 and α-adaptin, thereby attenuating E-cadherin endocytosis to maintain apicobasal polarity. PMID:21775625

  9. Design, synthesis, and evaluation of potent bryostatin analogs that modulate PKC translocation selectivity.

    PubMed

    Wender, Paul A; Baryza, Jeremy L; Brenner, Stacey E; DeChristopher, Brian A; Loy, Brian A; Schrier, Adam J; Verma, Vishal A

    2011-04-26

    Modern methods for the identification of therapeutic leads include chemical or virtual screening of compound libraries. Nature's library represents a vast and diverse source of leads, often exhibiting exquisite biological activities. However, the advancement of natural product leads into the clinic is often impeded by their scarcity, complexity, and nonoptimal properties or efficacy as well as the challenges associated with their synthesis or modification. Function-oriented synthesis represents a strategy to address these issues through the design of simpler and therefore synthetically more accessible analogs that incorporate the activity-determining features of the natural product leads. This study illustrates the application of this strategy to the design and synthesis of functional analogs of the bryostatin marine natural products. It is specifically directed at exploring the activity-determining role of bryostatin A-ring functionality on PKC affinity and selectivity. The resultant functional analogs, which were prepared by a flexible, modular synthetic strategy, exhibit excellent affinity to PKC and differential isoform selectivity. These and related studies provide the basic information needed for the design of simplified and thus synthetically more accessible functional analogs that target PKC isoforms, major targets of therapeutic interest.

  10. Design, synthesis, and evaluation of potent bryostatin analogs that modulate PKC translocation selectivity

    PubMed Central

    Wender, Paul A.; Baryza, Jeremy L.; Brenner, Stacey E.; DeChristopher, Brian A.; Loy, Brian A.; Schrier, Adam J.; Verma, Vishal A.

    2011-01-01

    Modern methods for the identification of therapeutic leads include chemical or virtual screening of compound libraries. Nature’s library represents a vast and diverse source of leads, often exhibiting exquisite biological activities. However, the advancement of natural product leads into the clinic is often impeded by their scarcity, complexity, and nonoptimal properties or efficacy as well as the challenges associated with their synthesis or modification. Function-oriented synthesis represents a strategy to address these issues through the design of simpler and therefore synthetically more accessible analogs that incorporate the activity-determining features of the natural product leads. This study illustrates the application of this strategy to the design and synthesis of functional analogs of the bryostatin marine natural products. It is specifically directed at exploring the activity-determining role of bryostatin A-ring functionality on PKC affinity and selectivity. The resultant functional analogs, which were prepared by a flexible, modular synthetic strategy, exhibit excellent affinity to PKC and differential isoform selectivity. These and related studies provide the basic information needed for the design of simplified and thus synthetically more accessible functional analogs that target PKC isoforms, major targets of therapeutic interest. PMID:21415363

  11. Removal of Potential Phosphorylation Sites does not Alter Creatine Transporter Response to PKC or Substrate Availability.

    PubMed

    Santacruz, Lucia; Darrabie, Marcus D; Mishra, Rajashree; Jacobs, Danny O

    2015-01-01

    Creatine, Phosphocreatine, and creatine kinases, constitute an energy shuttle that links ATP production in mitochondria with cellular consumption sites. Myocytes and neurons cannot synthesize creatine and depend on uptake across the cell membrane by a specialized transporter to maintain intracellular creatine levels. Although recent studies have improved our understanding of creatine transport in cardiomyocytes, the structural elements underlying the creatine transporter protein regulation and the relevant intracellular signaling processes are unknown. The effects of pharmacological activation of kinases or phosphatases on creatine transport in cardiomyocytes in culture were evaluated. Putative phosphorylation sites in the creatine transporter protein were identified by bioinformatics analyses, and ablated using site-directed mutagenesis. Mutant transporter function and their responses to pharmacological PKC activation or changes in creatine availability in the extracellular environment, were evaluated. PKC activation decreases creatine transport in cardiomyocytes in culture. Elimination of high probability potential phosphorylation sites did not abrogate responses to PKC activation or substrate availability. Modulation of creatine transport in cardiomyocytes is a complex process where phosphorylation at predicted sites in the creatine transporter protein does not significantly alter activity. Instead, non-classical structural elements in the creatine transporter and/or interactions with regulatory subunits may modulate its activity. © 2015 S. Karger AG, Basel.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. PI3K signalling in GnRH actions on dispersed goldfish pituitary cells: relationship with PKC-mediated LH and GH release and regulation of long-term effects on secretion and total cellular hormone availability.

    PubMed

    Pemberton, Joshua G; Orr, Michael E; Stafford, James L; Chang, John P

    2014-09-01

    Goldfish pituitary cells are exposed to two GnRHs, salmon (s)GnRH and chicken (c)GnRH-II. Phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC) both participate in acute sGnRH- and cGnRH-II-stimulated LH and GH release. Using goldfish pituitary cells, we examined the relationship between PI3K and PKC in acute LH and GH secretion, and PI3K involvement in chronic hormone release and total LH and GH availability. The PI3K inhibitor LY294002 did not affect PKC agonists-induced LH or GH release, and PKC agonists did not alter PI3K p85 phosphorylation, suggesting PKC activation is not upstream of PI3K in acute hormone release. In 2, 6, 12 and 24h treatments, LY294002 did not affect LH release but stimulated total LH availability at 6h. sGnRH stimulatory actions on LH release and total availability at 12 and 24h, and cGnRH-II effects on these parameters at 6h were inhibited by LY294002. LY294002 enhanced basal GH release at 2 and 6h, but reduced total GH at 12 and 24h. Increased GH release was seen following 6, 12 and 24h of sGnRH, and 2, 6 and 24h of cGnRH-II treatment but total GH availability was only elevated by 24h cGnRH-II treatment. Whereas LY294002 inhibited GH release responses to sGnRH at 12h and cGnRH-II at 6h, it attenuated cGnRH-II-elicited, but not sGnRH-induced, effects on total GH. These results indicate that PI3K differentially modulates long-term basal and GnRH-stimulated hormone release, and total hormone availability, in a time-, cell-type-, and GnRH isoform-selective manner. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. 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.

  1. TNF-α Up-Regulates Protein Level and Cell Surface Expression of the Leptin Receptor by Stimulating Its Export via a PKC-Dependent Mechanism

    PubMed Central

    Gan, Lixia; Guo, Kaiying; Cremona, Maria Laura; McGraw, Timothy E.; Leibel, Rudolph L.

    2012-01-01

    Increasing evidence suggests that inflammation/cytokines may modulate hypothalamic responses to leptin, which is a key regulator of energy homeostasis and inflammatory/stress responses. We investigated a possible role of TNF-α, a key early mediator of inflammation, in regulating the expression and trafficking of the long-isoform leptin receptor (LEPRb), the primary mediator of leptin signaling, in cultured cells. We found that TNF-α in a wide range of concentrations up-regulated LEPRb protein level and soluble LEPR (sLEPR) release via ectodomain shedding of LEPRb in multiple cell types, including neuronal cells. TNF-α also acutely increased LEPRb cell surface expression and leptin-induced STAT3 phosphorylation. In contrast, TNF-α had no significant effects on the protein level or cell surface expression of several other transmembrane proteins, including the transferrin receptor and cadherin. The stimulatory effects of TNF-α on LEPRb cell surface expression and sLEPR release were not dependent on de novo protein synthesis or functional lysosomes but were blocked by brefeldin A, suggesting that an intact Golgi or continuous endoplasmic reticulum to Golgi transport of newly synthesized proteins is required for these effects. However, TNF-α did not increase the half-life of cell surface LEPRb. Protein kinase C (PKC) inhibitor GF109203X abrogated the effects of TNF-α, whereas the pan-PKC activator phorbol 12-myristate 13-acetate mimicked the TNF-α effects. Taken together, our results suggest that TNF-α, via activation of PKC, regulates anterograde trafficking and/or degradation of LEPRb in the biosynthetic pathway, leading to concomitant increases in LEPRb protein level, cell surface expression, and sLEPR production. The finding that LEPRb cell surface expression and sLEPR production, key modulators of leptin sensitivity and bioavailability, are direct targets of TNF-α signaling could have a potentially important implication in the regulation of leptin

  2. 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.

  3. A calcium and free fatty acid-modulated protein kinase as putative effector of the fusicoccin 14-3-3 receptor.

    PubMed Central

    van der Hoeven, P C; Siderius, M; Korthout, H A; Drabkin, A V; de Boer, A H

    1996-01-01

    A protein kinase that is activated by calcium and cis-unsaturated fatty acids has been characterized from oat (Avena sativa L.) root plasma membranes. The kinase phosphorylates a synthetic peptide with a motif (-R-T-L-S-) that can be phosphorylated by both protein kinase C (PKC) and calcium-dependent protein kinase (CDPK)-type kinases. Calphostin C and chelerythrine, two PKC inhibitors, completely inhibited the kinase activity with values of inhibitor concentration for 50% inhibition of 0.7 and 30 microns, respectively. At low Ca2+ concentrations cis-unsaturated fatty acids (linolenic acid, linoleic acid, arachidonic acid, and oleic acid) stimulated the kinase activity almost 10-fold. The two inhibitors of the kinase, calphostin C and chelerythrin, strongly reduced the fusicoccin (FC)-induced H+ extrusion, and the activators of the kinase, the cis-unsaturated fatty acids, prevented [3H]FC binding to the FC 14-3-3 receptor. CDPK antibodies cross-reacted with a 43-kD band in the plasma membrane and in a purified FC receptor fraction. A polypeptide with the same apparent molecular mass was recognized by a synthetic peptide that has a sequence homologous to the annexin-like domain from barely 14-3-3. The possibility of the involvement of a kinase, with properties from both CDPK and PKC, and a phospholipase A2 in the FC Signal transduction pathway is discussed. PMID:8754686

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. Translocation of Transfected GLUT2 to the Apical Membrane in Rat Intestinal IEC-6 Cells

    PubMed Central

    Zheng, Ye; Sarr, Michael G.

    2011-01-01

    In this study, we transfected the full length cDNA of GLUT2 into IEC-6 cells (which lack GLUT2 expression) to investigate GLUT2 translocation in enterocytes. AIM To investigate cellular mechanisms of GLUT2 translocation and its signaling pathway. METHODS Rat glut2 cDNA was transfected into IEC-6 cells. Glucose uptake was measured by incubating cell monolayers with glucose (0.5 to 50 mM), containing 14C-d-glucose and 3H-L-glucose to measure stereospecific, carrier-mediated and passive uptake, resp. We imaged GLUT2 immunoreactivity by confocal fluorescence microscopy. We evaluated the GLUT2 inhibitor (1mM phloretin), SGLT1 inhibitor (0.5 mM phlorizin), disrupting microtubular integrity (2 µM nocodazole and 0.5 µM cytochalasin B), PKC inhibitors (50 nM calphostin C and 10 µM chelerythrine), and PKC activator (50 nM phorbol 12-myristate 13-acetate: PMA). RESULTS In GLUT2-IEC cells, the Km (54.5 mM) increased compared with non-transfected IEC-6 cells (7.8 mM); phloretin (GLUT2 inhibitor) inhibited glucose uptake to that of non-transfected IEC-6 cells (p<0.05). Nocodazole and cytochalasin B (microtubule disrupters) inhibited uptake by 43–58% only at glucose concentrations ≥ 25 and 50 mM and the 10-min incubations. Calphostin C (PKC inhibitor) reproduced the inhibition of nocodazole; PMA (a PKC activator) enhanced glucose uptake by 69%. Exposure to glucose increased the GFP signal at the apical membrane of GLUT-1EC Cells. CONCLUSION IEC-6 cells lacking GLUT2 translocate GLUT2 apically when transfected to express GLUT2. Translocation of GLUT2 occurs through glucose stimulation via a PKC-dependent signaling pathway and requires integrity of the microtubular skeletal structure. PMID:22116644

  10. Translocation of transfected GLUT2 to the apical membrane in rat intestinal IEC-6 cells.

    PubMed

    Zheng, Ye; Sarr, Michael G

    2012-05-01

    In this study, we transfected the full length cDNA of glucose transporter 2 (GLUT2) into IEC-6 cells (which lack GLUT2 expression) to investigate GLUT2 translocation in enterocytes. The purpose of this study was to investigate cellular mechanisms of GLUT2 translocation and its signaling pathway. Rat GLUT2 cDNA was transfected into IEC-6 cells. Glucose uptake was measured by incubating cell monolayers with glucose (0.5-50 mM), containing (14)C-D-glucose and (3)H-L-glucose, to measure stereospecific, carrier-mediated and passive uptake. We imaged GLUT2 immunoreactivity by confocal fluorescence microscopy. We evaluated the GLUT2 inhibitor (1 mM phloretin), SGLT1 inhibitor (0.5 mM phlorizin), disrupting microtubular integrity (2 μM nocodazole and 0.5 μM cytochalasin B), protein kinase C (PKC) inhibitors (50 nM calphostin C and 10 μM chelerythrine), and PKC activator (50 nM phorbol 12-myristate 13-acetate: PMA). In GLUT2-IEC cells, the K(m) (54.5 mM) increased compared with non-transfected IEC-6 cells (7.8 mM); phloretin (GLUT2 inhibitor) inhibited glucose uptake to that of non-transfected IEC-6 cells (P < 0.05). Nocodazole and cytochalasin B (microtubule disrupters) inhibited uptake by 43-58% only at glucose concentrations ≥25 and 50 mM and the 10-min incubations. Calphostin C (PKC inhibitor) reproduced the inhibition of nocodazole; PMA (a PKC activator) enhanced glucose uptake by 69%. Exposure to glucose increased the GFP signal at the apical membrane of GLUT-1EC cells. IEC-6 cells lacking GLUT2 translocate GLUT2 apically when transfected to express GLUT2. Translocation of GLUT2 occurs through glucose stimulation via a PKC-dependent signaling pathway and requires integrity of the microtubular skeletal structure.

  11. 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

  12. 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.

  13. 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.

  14. Canonical transient receptor potential 3 channels activate NF-κB to mediate allergic airway disease via PKC-α/IκB-α and calcineurin/IκB-β pathways

    PubMed Central

    Song, Tengyao; Zheng, Yun-Min; Vincent, Peter A.; Cai, Dongsheng; Rosenberg, Paul; Wang, Yong-Xiao

    2016-01-01

    The purpose of this study was to determine the role of canonical transient receptor potential 3 (TRPC3) channel in allergen-induced airway disease (AIAD) and its underlying signaling mechanisms. The procedures included (1) intravenous injection of lentiviral TRPC3 channel or nonsilencing short hairpin ribonucleic acid (shRNA) to make the channel knockdown (KD) or control mice, (2) allergen sensitization/challenge to induce AIAD, (3) patch-clamp recording and Ca2+ imaging to examine the channel activity, and (4) gene manipulations and other methods to determine the underlying signaling mechanisms. The findings are that (1) intravenous or intranasal delivery of TRPC3 channel lentiviral shRNAs or blocker 1-[4-[(2,3,3-trichloro-1-oxo-2-propen-1-yl)amino]phenyl]-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid prevents AIAD in mice, (2) TRPC3 channel KD and overexpression, respectively, blocks and augments protein kinase C-α/nuclear factor of κ light polypeptide gene enhancer in B-cell inhibitor-α (PKC-α/IκB-α)-mediated or calcineurin/IκB-β–dependent, NF-κB–dependent allergen-induced airway smooth muscle cell (ASMC) hyperproliferation and cyclin D1 (an important cell proliferation molecule) induction, and (3) the changes of the major molecules of the PKC-α/IκBα- and calcineurin/IκB-β–dependent NF-κB signaling pathways are also observed in asthmatic human ASMCs. The conclusions are that TRPC3 channels plays an essential role in AIAD via the PKC-α/IκB-α– and calcineurin/IκB-β–dependent NF-κB signaling pathways, and lentiviral shRNA or inhibitor of TRPC3 channels may become novel and effective treatments for AIAD.—Song, T., Zheng, Y.-M., Vincent, P. A., Cai, D., Rosenberg, P., Wang, Y.-X. Canonical transient receptor potential 3 channels activate NF-κB to mediate allergic airway disease via PKC-α/IκB-α and calcineurin/IκB-β pathways. PMID:26373801

  15. 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.

  16. Converting enzyme inhibitor temocaprilat prevents high glucose-mediated suppression of human aortic endothelial cell proliferation.

    PubMed

    Yasunari, Kenichi; Maeda, Kensaku; Watanabe, Takanori; Nakamura, Munehiro; Asada, Akira; Yoshikawa, Junichi

    2003-12-01

    We examined the involvement of the oxidative stress in high glucose-induced suppression of human aortic endothelial cell proliferation. Chronic glucose treatment for 72 h concentration-dependently (5.6-22.2 mol/l) inhibited human coronary endothelial cell proliferation. Temocaprilat, an angiotensin-converting enzyme inhibitor, at 10 nmol/l to 1 micromol/l inhibited high glucose (22.2 mmol/l)-mediated suppression of human aortic endothelial cell proliferation. Temocaprilat at 1 micromol/l inhibited high glucose-induced membrane-bound protein kinase C activity in human aortic endothelial cells. The protein kinase C inhibitors calphostin C 100 nmol/l or chelerythrine 1 micromol/l inhibited high glucose-mediated suppression of human aortic endothelial cell proliferation. Chronic high glucose treatment for 72 h increased intracellular oxidative stress, directly measured by flow cytometry using carboxydichlorofluorescein diacetate bis-acetoxymethyl ester, and this increase was significantly suppressed by temocaprilat 10 nmol/l to 1 micromol/l. Bradykinin B2 receptor antagonist icatibant 100 nmol/l significantly reduced the action of temocaprilat; whereas bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin 100 nmol/l had no effect. These findings suggest that high glucose inhibits human aortic endothelial cell proliferation and that the angiotensin-converting enzyme inhibitor temocaprilat inhibits high glucose-mediated suppression of human aortic endothelial cell proliferation, possibly through suppression of protein kinase C, bradykinin B2 receptors and oxidative stress.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. Mutations in the cytoplasmic domain of P0 reveal a role for PKC-mediated phosphorylation in adhesion and myelination

    PubMed Central

    Xu, Wenbo; Shy, Michael; Kamholz, John; Elferink, Lisa; Xu, Gang; Lilien, Jack; Balsamo, Janne

    2001-01-01

    Mutations in P0 (MPZ), the major myelin protein of the peripheral nervous system, cause the inherited demyelinating neuropathy Charcot-Marie-Tooth disease type 1B. P0 is a member of the immunoglobulin superfamily and functions as a homophilic adhesion molecule. We now show that point mutations in the cytoplasmic domain that modify a PKC target motif (RSTK) or an adjacent serine residue abolish P0 adhesion function and can cause peripheral neuropathy in humans. Consistent with these data, PKCα along with the PKC binding protein RACK1 are immunoprecipitated with wild-type P0, and inhibition of PKC activity abolishes P0-mediated adhesion. Point mutations in the RSTK target site that abolish adhesion do not alter the association of PKC with P0; however, deletion of a 14 amino acid region, which includes the RSTK motif, does abolish the association. Thus, the interaction of PKCα with the cytoplasmic domain of P0 is independent of specific target residues but is dependent on a nearby sequence. We conclude that PKC-mediated phosphorylation of specific residues within the cytoplasmic domain of P0 is necessary for P0-mediated adhesion, and alteration of this process can cause demyelinating neuropathy in humans. PMID:11673479

  2. PAR3 and aPKC regulate Golgi organization through CLASP2 phosphorylation to generate cell polarity

    PubMed Central

    Matsui, Toshinori; Watanabe, Takashi; Matsuzawa, Kenji; Kakeno, Mai; Okumura, Nobumasa; Sugiyama, Ikuko; Itoh, Norimichi; Kaibuchi, Kozo

    2015-01-01

    The organization of the Golgi apparatus is essential for cell polarization and its maintenance. The polarity regulator PAR complex (PAR3, PAR6, and aPKC) plays critical roles in several processes of cell polarization. However, how the PAR complex participates in regulating the organization of the Golgi remains largely unknown. Here we demonstrate the functional cross-talk of the PAR complex with CLASP2, which is a microtubule plus-end–tracking protein and is involved in organizing the Golgi ribbon. CLASP2 directly interacted with PAR3 and was phosphorylated by aPKC. In epithelial cells, knockdown of either PAR3 or aPKC induced the aberrant accumulation of CLASP2 at the trans-Golgi network (TGN) concomitantly with disruption of the Golgi ribbon organization. The expression of a CLASP2 mutant that inhibited the PAR3-CLASP2 interaction disrupted the organization of the Golgi ribbon. CLASP2 is known to localize to the TGN through its interaction with the TGN protein GCC185. This interaction was inhibited by the aPKC-mediated phosphorylation of CLASP2. Furthermore, the nonphosphorylatable mutant enhanced the colocalization of CLASP2 with GCC185, thereby perturbing the Golgi organization. On the basis of these observations, we propose that PAR3 and aPKC control the organization of the Golgi through CLASP2 phosphorylation. PMID:25518939

  3. 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

  4. 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.

  5. 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

  6. HMG-CoA reductase inhibitors prevent migration of human coronary smooth muscle cells through suppression of increase in oxidative stress.

    PubMed

    Yasunari, K; Maeda, K; Minami, M; Yoshikawa, J

    2001-06-01

    In vitro and in vivo evidence of a decrease in vascular smooth muscle cell (SMC) migration induced by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors has been reported. When added to SMC cultures for 6 hours, the HMG-CoA reductase inhibitors fluvastatin, simvastatin, and pravastatin at 1 micromol/L resulted in a 48%, 50%, and 16% suppression, respectively, of human coronary SMC migration; these reductions mirrored the suppression in oxidative stress induced by 1 micromol/L lysophosphatidylcholine (lyso-PC) of 50%, 53% and 19%, respectively. The hydroxylated metabolites of fluvastatin, M(2) and M(3), at 1 micromol/L also suppressed the enhancement of SMC migration by 58% and 45% and the increase in oxidative stress induced by lyso-PC of 58% and 49%, respectively. Lyso-PC activated phospholipase D and protein kinase C (PKC), and this activation was also suppressed by HMG-CoA reductase inhibitors. The inhibition of phospholipase D and PKC was reversed by 100 micromol/L mevalonate, its isoprenoid derivative, farnesol, and geranylgeraniol but not by 10 micromol/L squalene. Antisense oligodeoxynucleotides at 5 micromol/L to PKC-alpha, but not those to the PKC-beta isoform, suppressed the lyso-PC-mediated increases in SMC migration and oxidative stress. These findings suggest that HMG-CoA reductase inhibitors have direct antimigratory effects on the vascular wall beyond their effects on plasma lipids and that they might exert such antimigratory effects via suppression of the phospholipase D- and PKC (possibly PKC-alpha)-induced increase in oxidative stress, which might in turn prevent significant coronary artery disease.

  7. 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.

  8. AdipoR-increased intracellular ROS promotes cPLA2 and COX-2 expressions via activation of PKC and p300 in adiponectin-stimulated human alveolar type II cells.

    PubMed

    Chen, Hsiao-Mei; Yang, Chuen-Mao; Chang, Jia-Feng; Wu, Chi-Sheng; Sia, Kee-Chin; Lin, Wei-Ning

    2016-08-01

    Adiponectin, an adipokine, accumulated in lung system via T-cadherin after allergens/ozone challenge. However, the roles of adiponectin on lung pathologies were controversial. Here we reported that adiponectin stimulated expression of inflammatory proteins, cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), and production of reactive oxygen species (ROS) in human alveolar type II A549 cells. AdipoR1/2 involved in adiponectin-activated NADPH oxidase and mitochondria, which further promoted intracellular ROS accumulation. Protein kinase C (PKC) may involve an adiponectin-activated NADPH oxidase. Similarly, p300 phosphorylation and histone H4 acetylation occurred in adiponectin-challenged A549 cells. Moreover, adiponectin-upregulated cPLA2 and COX-2 expression was significantly abrogated by ROS scavenger (N-acetylcysteine) or the inhibitors of NADPH oxidase (apocynin), mitochondrial complex I (rotenone), PKC (Ro31-8220, Gö-6976, and rottlerin), and p300 (garcinol). Briefly, we reported that adiponectin stimulated cPLA2 and COX-2 expression via AdipoR1/2-dependent activation of PKC/NADPH oxidase/mitochondria resulting in ROS accumulation, p300 phosphorylation, and histone H4 acetylation. These results suggested that adiponectin promoted lung inflammation, resulting in exacerbation of pulmonary diseases via upregulating cPLA2 and COX-2 expression together with intracellular ROS production. Understanding the adiponectin signaling pathways on regulating cPLA2 and COX-2 may help develop therapeutic strategies on pulmonary diseases. Copyright © 2016 the American Physiological Society.

  9. 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.

  10. 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

  11. Various Molecular Species of Diacylglycerol Hydroperoxide Activate Human Neutrophils via PKC Activation

    PubMed Central

    Kambayashi, Yasuhiro; Takekoshi, Susumu; Tanino, Yutaka; Watanabe, Keiichi; Nakano, Minoru; Hitomi, Yoshiaki; Takigawa, Tomoko; Ogino, Keiki; Yamamoto, Yorihiro

    2007-01-01

    We have proposed that diacylglycerol hydroperoxide-induced unregulated signal transduction causes oxidative stress-related diseases. In this study, we investigated which molecular species of diacylglycerol hydroperoxide activated human peripheral neutrophils. All diacylglycerol hydroperoxides, diacylglycerol hydroxides, and diacyglycerols tested in the present study induced superoxide production by neutrophils. The ability to activate neutrophils among molecular species containing the same fatty acid composition was as follows; diacylglycerol hydroperoxide>diacylglycerol hydroxide≥diacylglycerol. The diacylglycerol hydroperoxide composed of linoleate was a stronger activator for neutrophils than that composed of arachidonate. 1-Palmitoyl-2-linoleoylglycerol hydroperoxide (PLG-OOH) was the strongest stimulator for neutrophils. We reconfirmed that PLG-OOH activated protein kinase C (PKC) in neutrophils. PLG-OOH induced the phosphorylation of p47phox, a substrate of PKC and a cytosolic component of NADPH oxidase, in neutrophils, as did N-formyl-methionyl-leucyl-phenylalanine or 4β-phorbol-12β-myristate-13α-acetate. Moreover, the time course of p47phox phosphorylation was comparable to that of superoxide production. These results suggest that PLG-OOH activated intracellular protein kinase C. PLG-OOH, produced via an uncontrolled process, can act as a biological second messenger to cause inflammatory disease from oxidative stress. PMID:18392102

  12. p62 modulates Akt activity via association with PKC{zeta} in neuronal survival and differentiation

    SciTech Connect

    Joung, Insil . E-mail: ijoung@hanseo.ac.kr; Kim, Hak Jae; Kwon, Yunhee Kim . E-mail: kimyh@khu.ac.kr

    2005-08-26

    p62 is a ubiquitously expressed phosphoprotein that interacts with a number of signaling molecules and a major component of neurofibrillary tangles in the brain of Alzheimer's disease patients. It has been implicated in important cellular functions such as cell proliferation and anti-apoptotic pathways. In this study, we have addressed the potential role of p62 during neuronal differentiation and survival using HiB5, a rat neuronal progenitor cell. We generated a recombinant adenovirus encoding T7-epitope tagged p62 to reliably transfer p62 cDNA into the neuronal cells. The results show that an overexpression of p62 led not only to neuronal differentiation, but also to decreased cell death induced by serum withdrawal in HiB5 cells. In this process p62-dependent Akt phosphorylation occurred via the release of Akt from PKC{zeta} by association of p62 and PKC{zeta}, which is known as a negative regulator of Akt activation. These findings indicate that p62 facilitates cell survival through novel signaling cascades that result in Akt activation. Furthermore, we found that p62 expression was induced during neuronal differentiation. Taken together, the data suggest p62 is a regulator of neuronal cell survival and differentiation.

  13. 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

  14. 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.

  15. 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.

  16. Brain GLP-1 Signaling Regulates Femoral Artery Blood Flow and Insulin Sensitivity Through Hypothalamic PKC

    PubMed Central

    Cabou, Cendrine; Vachoux, Christelle; Campistron, Gérard; Drucker, Daniel J.; Burcelin, Rémy

    2011-01-01

    OBJECTIVE Glucagon-like peptide 1 (GLP-1) is a gut-brain hormone that regulates food intake, energy metabolism, and cardiovascular functions. In the brain, through a currently unknown molecular mechanism, it simultaneously reduces femoral artery blood flow and muscle glucose uptake. By analogy to pancreatic β-cells where GLP-1 activates protein kinase C (PKC) to stimulate insulin secretion, we postulated that PKC enzymes would be molecular targets of brain GLP-1 signaling that regulate metabolic and vascular function. RESEARCH DESIGN AND METHODS We used both genetic and pharmacological approaches to investigate the role of PKC isoforms in brain GLP-1 signaling in the conscious, free-moving mouse simultaneous with metabolic and vascular measurements. RESULTS In normal wild-type (WT) mouse brain, the GLP-1 receptor (GLP-1R) agonist exendin-4 selectively promotes translocation of PKC-δ (but not -βII, -α, or -ε) to the plasma membrane. This translocation is blocked in Glp1r−/− mice and in WT mice infused in the brain with exendin-9, an antagonist of the GLP-1R. This mechanism coordinates both blood flow in the femoral artery and whole-body insulin sensitivity. Consequently, in hyperglycemic, high-fat diet–fed diabetic mice, hypothalamic PKC-δ activity was increased and its pharmacological inhibition improved both insulin-sensitive metabolic and vascular phenotypes. CONCLUSIONS Our studies show that brain GLP-1 signaling activates hypothalamic glucose-dependent PKC-δ to regulate femoral artery blood flow and insulin sensitivity. This mechanism is attenuated during the development of experimental hyperglycemia and may contribute to the pathophysiology of type 2 diabetes. PMID:21810595

  17. 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.

  18. 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.

  19. Small-molecule GSK-3 inhibitor rescued apoptosis and neurodegeneration in anesthetics-injured dorsal root ganglion neurons.

    PubMed

    Yu, Tianchao; Lin, Wanchun

    2016-12-01

    Application of general anesthetics may induce neurotoxicity in dorsal root ganglia (DRG) neurons. In this study, we examined the possible protective mechanism and associated signaling pathways of small-molecule glycogen synthase kinase-3 (GSK-3) inhibitor, SB216763, in bupivacaine-injured mouse DRG neurons in vitro. In vitro DRG explant of 6-week old mice was treated with 5mM bupivacaine to induce neurotoxicity. The explants were also pre-treated with SB216763 for 72h. Neural protection of SB216763 on bupivacaine-injured DRG neurons was investigated by TUNEL assay, neurite outgrowth assay and western blot assay, respectively. Possible downstream gene of GSK-3 signaling pathway, protein kinase C (PKC) was knocked down by siRNA in DRG explant. Its function in regulating GSK-3 inhibition induced DRG neural protection was also examined by TUNEL, neurite outgrowth and western blot assays. Pre-treatment of SB216763 significantly ameliorated bupivacaine induced apoptosis and neurite loss in DRG neurons. Western blot showed that, in addition to the decrease of phosphorylated-GSK-3 α/β protein, SB216763 increased PKC and decreased caspase-3 (Casp-3) in bupivacaine-injured DRG neurons. SiRNA-mediated PKC knockdown was able to reverse the neural protection of SB216763 in bupivacaine-injured DRG neurons. Western blot showed that PKC knockdown increased phosphorylated-GSK-3 α/β and Casp-3 protein in DRG neurons, confirming that PKC was directly involved in GSK-3-inhibition induced neural protection in DRG. GSK-3 inhibitor SB216763, through PKC, is effective in protecting anesthetics-induced neurotoxicity in DRG. Copyright © 2016. Published by Elsevier Masson SAS.

  20. 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.

  1. ACE inhibitors

    MedlinePlus

    ... ACE inhibitors There are many different names and brands of ACE inhibitors. Most work as well as ... urac.org). URAC's accreditation program is an independent audit to verify that A.D.A.M. follows ...

  2. 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. © 2014 Wiley Periodicals, Inc.

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

    PubMed Central

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

    2014-01-01

    Atypical isoforms of protein kinase C (aPKCs; particularly protein kinase M zeta: PKMζ) have been hypothesised to be necessary and sufficient for the maintenance of long-term potentiation (LTP) and long term memory by maintaining postsynaptic AMPA receptors via the GluR2 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 GluR2-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 GluR2-independent target of ZIP (possibly PKCλ) in the mPFC during early memory formation. PMID:24729442

  4. 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.

  5. Activated PKC{delta} and PKC{epsilon} inhibit epithelial chloride secretion response to cAMP via inducing internalization of the Na+-K+-2Cl- cotransporter NKCC1.

    PubMed

    Tang, Jun; Bouyer, Patrice; Mykoniatis, Andreas; Buschmann, Mary; Matlin, Karl S; Matthews, Jeffrey B

    2010-10-29

    The basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is a key determinant of transepithelial chloride secretion and dysregulation of chloride secretion is a common feature of many diseases including secretory diarrhea. We have previously shown that activation of protein kinase C (PKC) markedly reduces transepithelial chloride secretion in human colonic T84 cells, which correlates with both functional inhibition and loss of the NKCC1 surface expression. In the present study, we defined the specific roles of PKC isoforms in regulating epithelial NKCC1 and chloride secretion utilizing adenoviral vectors that express shRNAs targeting human PKC isoforms (α, δ, ε) (shPKCs) or LacZ (shLacZ, non-targeting control). After 72 h of adenoviral transduction, protein levels of the PKC isoforms in shPKCs-T84 cells were decreased by ∼90% compared with the shLacZ-control. Activation of PKCs by phorbol 12-myristate 13-acetate (PMA) caused a redistribution of NKCC1 immunostaining from the basolateral membrane to intracellular vesicles in both shLacZ- and shPKCα-T84 cells, whereas the effect of PMA was not observed in shPKCδ- and shPKCε- cells. These results were further confirmed by basolateral surface biotinylation. Furthermore, activation of PKCs by PMA inhibited cAMP-stimulated chloride secretion in the uninfected, shLacZ- and shPKCα-T84 monolayers, but the inhibitory effect was significantly attenuated in shPKCδ- and shPKCε-T84 monolayers. In conclusion, the activated novel isoforms PKCδ or PKCε, but not the conventional isoform PKCα, inhibits transepithelial chloride secretion through inducing internalization of the basolateral surface NKCC1. Our study reveals that the novel PKC isoform-regulated NKCC1 surface expression plays an important role in the regulation of chloride secretion.

  6. 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.

  7. 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

  8. 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.

  9. aPKC restricts the basolateral determinant PtdIns(3,4,5)P3 to the basal region.

    PubMed

    Takahama, Shoukichi; Hirose, Tomonori; Ohno, Shigeo

    2008-04-04

    Both PtdIns(3,4,5)P3 (PIP3) and atypical protein kinase C (aPKC) play central roles in the polarization of many cell types. In epithelial cells, both PIP3 and aPKC are required for the development of apico-basolateral membrane polarity. However, the relationship between PIP3 and aPKC during the establishment and maintenance of polarized membrane domains remains to be clarified. We show that depolarized MDCK cells retain a polarized basal distribution of PIP3, supporting a role for PIP3 in determining the basolateral membrane domain. Importantly, overexpression of a kinase-negative mutant of aPKClambda (aPKClambda kn) impaired the basal distribution of PIP3, indicating that aPKC kinase activity is required for the restriction of PIP3 to the basal region. In support of this, overexpression of aPKClambda kn during polarization, but not after polarization, caused whole membrane distribution of PIP3 as well as defects in epithelial polarization.

  10. 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

  11. 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

  12. 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

  13. Absence of PKC-Alpha Attenuates Lithium-Induced Nephrogenic Diabetes Insipidus

    PubMed Central

    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. PMID:25006961

  14. 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.

  15. 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.

  16. 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

  17. Bryostatin-1 vs. TPPB: dose-dependent APP processing and PKC-α, -δ, and -ε isoform activation in SH-SY5Y neuronal cells.

    PubMed

    Yi, P; Schrott, L; Castor, T P; Alexander, J S

    2012-09-01

    Activation of the α-secretase processing pathway of amyloid precursor protein (APP) is recognized as an important mechanism which diverts APP processing from production of beta-amyloid (Aβ) to non toxic sAPPα, decreasing Alzheimer's disease (AD) plaque formation and AD-associated cognitive deficits. Two potent classes of PKC modulators can activate the α-secretase pathway, the benzo/indolactams and bryostatin/bryologues. While both modulate PKC-dependent APP processing, no direct comparisons of their relative pharmacological potencies have been accomplished which could assist in the development of AD therapies. In this study, we measured the activation of α-secretase APP processing and PKC-α, -δ, and -ε induced by the benzolactam-APP modulator TPPB and bryostatin-1 in the neuroblastoma cell line SH-SY5Y which expresses APP and α- and β-secretase processing mechanisms. Bryostatin-1 produced a more rapid, potent, and sustained activation of α-secretase APP processing than TPPB and selectively activated PKC-δ and PKC-ε. Although TPPB also activated α-secretase, its potency was approximately 10- to 100-fold lower, possibly reflecting lower PKC-δ and -ε activation. Because bryostatin-1 is a highly potent PKC-δ and -ε activator which activates α-secretase APP processing, further characterization of bryostatin-1/bryologues may help refine their use as important tools for the clinical management of AD.

  18. Overexpression of protein kinase C-zeta (PKC-zeta) inhibits invasive and metastatic abilities of Dunning R-3327 MAT-LyLu rat prostate cancer cells.

    PubMed

    Powell, C T; Gschwend, J E; Fair, W R; Brittis, N J; Stec, D; Huryk, R

    1996-09-15

    Previously, we reported that protein kinase C (PKC)-zeta mRNA levels are reduced markedly in metastatic Dunning R-3327 rat prostate tumors relative to the nonmetastatic Dunning H tumor and normal rat prostate (C.T. Powell et al., Cell Growth & Differ., 5: 143-149, 1994). To examine the effect of PKC-zeta on metastatic and invasive abilities of an aggressive Dunning R-3327 cell line, we generated stably transfected clones of MAT-LyLu cells that overexpress active PKC-zeta. PKC-zeta-overexpressing MAT-LyLu cells exhibited tumorigenicity and growth rates in syngeneic rats similar to those of MAT-LyLu cells transfected with vector alone or untransfected MAT-LyLu. However, nine independent clones of PKC-zeta-expressing cells exhibited an average 2-fold lower tendency to metastasize to lungs relative to vector-transfected MAT-LyLu cell clones, with about 2-fold and 4.5-fold fewer metastases per rat in two separate protocols. In addition, the ability of four PKC-zeta overexpressing MAT-LyLu clones to invade through Matrigel in a Boyden chamber assay was reduced an average of 12-fold relative to three vector-transfected clones. These results indicate that increased PKC-zeta expression can substantially suppress invasion and metastasis by an aggressive rat prostate tumor.

  19. Assessment of roles for the Rho-specific guanine nucleotide dissociation inhibitor 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

    2017-04-01

    On 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. Whereas 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. Copyright © 2017 the American Physiological Society.

  20. 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

  1. Quantitative properties and receptor reserve of the DAG and PKC branch of Gq-coupled receptor signaling

    PubMed Central

    Falkenburger, Björn H.; Dickson, Eamonn J.

    2013-01-01

    Gq protein–coupled receptors (GqPCRs) of the plasma membrane activate the phospholipase C (PLC) signaling cascade. PLC cleaves the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers diacylgycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), leading to calcium release, protein kinase C (PKC) activation, and in some cases, PIP2 depletion. We determine the kinetics of each of these downstream endpoints and also ask which is responsible for the inhibition of KCNQ2/3 (KV7.2/7.3) potassium channels in single living tsA-201 cells. We measure DAG production and PKC activity by Förster resonance energy transfer–based sensors, and PIP2 by KCNQ2/3 channels. Fully activating endogenous purinergic receptors by uridine 5′triphosphate (UTP) leads to calcium release, DAG production, and PKC activation, but no net PIP2 depletion. Fully activating high-density transfected muscarinic receptors (M1Rs) by oxotremorine-M (Oxo-M) leads to similar calcium, DAG, and PKC signals, but PIP2 is depleted. KCNQ2/3 channels are inhibited by the Oxo-M treatment (85%) and not by UTP (<1%), indicating that depletion of PIP2 is required to inhibit KCNQ2/3 in response to receptor activation. Overexpression of A kinase–anchoring protein (AKAP)79 or calmodulin (CaM) does not increase KCNQ2/3 inhibition by UTP. From these results and measurements of IP3 and calcium presented in our companion paper (Dickson et al. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210886), we extend our kinetic model for signaling from M1Rs to DAG/PKC and IP3/calcium signaling. We conclude that calcium/CaM and PKC-mediated phosphorylation do not underlie dynamic KCNQ2/3 channel inhibition during GqPCR activation in tsA-201 cells. Finally, our experimental data provide indirect evidence for cleavage of PI(4)P by PLC in living cells, and our modeling revisits/explains the concept of receptor reserve with measurements from all steps of GqPCR signaling. PMID:23630338

  2. 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.

  3. 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

  4. 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.

  5. Combinations of isoform-targeted histone deacetylase inhibitors and bryostatin analogues display remarkable potency to activate latent HIV without global T-cell activation.

    PubMed

    Albert, Brice J; Niu, Austin; Ramani, Rashmi; Marshall, Garland R; Wender, Paul A; Williams, Robert M; Ratner, Lee; Barnes, Alexander B; Kyei, George B

    2017-08-07

    Current antiretroviral therapy (ART) for HIV/AIDS slows disease progression by reducing viral loads and increasing CD4 counts. Yet ART is not curative due to the persistence of CD4+ T-cell proviral reservoirs that chronically resupply active virus. Elimination of these reservoirs through the administration of synergistic combinations of latency reversing agents (LRAs), such as histone deacetylase (HDAC) inhibitors and protein kinase C (PKC) modulators, provides a promising strategy to reduce if not eradicate the viral reservoir. Here, we demonstrate that largazole and its analogues are isoform-targeted histone deacetylase inhibitors and potent LRAs. Significantly, these isoform-targeted HDAC inhibitors synergize with PKC modulators, namely bryostatin-1 analogues (bryologs). Implementation of this unprecedented LRA combination induces HIV-1 reactivation to unparalleled levels and avoids global T-cell activation within resting CD4+ T-cells.

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. PAR3-aPKC regulates Tiam1 by modulating suppressive internal interactions.

    PubMed

    Matsuzawa, Kenji; Akita, Hiroki; Watanabe, Takashi; Kakeno, Mai; Matsui, Toshinori; Wang, Shujie; Kaibuchi, Kozo

    2016-05-01

    Tiam1 is one of the most extensively analyzed activators of the small GTPase Rac. However, fundamental aspects of its regulation are poorly understood. Here we demonstrate that Tiam1 is functionally suppressed by internal interactions and that the PAR complex participates in its full activation. The N-terminal region of Tiam1 binds to the protein-binding and catalytic domains to inhibit its localization and activation. Atypical PKCs phosphorylate Tiam1 to relieve its intramolecular interactions, and the subsequent stabilization of its interaction with PAR3 allows it to exert localized activity. By analyzing Tiam1 regulation by PAR3-aPKC within the context of PDGF signaling, we also show that PAR3 directly binds PDGF receptor β. Thus we provide the first evidence for the negative regulation of Tiam1 by internal interactions, elucidate the nature of Tiam1 regulation by the PAR complex, and reveal a novel role for the PAR complex in PDGF signaling. © 2016 Matsuzawa et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  13. PAR3-aPKC regulates Tiam1 by modulating suppressive internal interactions

    PubMed Central

    Matsuzawa, Kenji; Akita, Hiroki; Watanabe, Takashi; Kakeno, Mai; Matsui, Toshinori; Wang, Shujie; Kaibuchi, Kozo

    2016-01-01

    Tiam1 is one of the most extensively analyzed activators of the small GTPase Rac. However, fundamental aspects of its regulation are poorly understood. Here we demonstrate that Tiam1 is functionally suppressed by internal interactions and that the PAR complex participates in its full activation. The N-terminal region of Tiam1 binds to the protein-binding and catalytic domains to inhibit its localization and activation. Atypical PKCs phosphorylate Tiam1 to relieve its intramolecular interactions, and the subsequent stabilization of its interaction with PAR3 allows it to exert localized activity. By analyzing Tiam1 regulation by PAR3-aPKC within the context of PDGF signaling, we also show that PAR3 directly binds PDGF receptor β. Thus we provide the first evidence for the negative regulation of Tiam1 by internal interactions, elucidate the nature of Tiam1 regulation by the PAR complex, and reveal a novel role for the PAR complex in PDGF signaling. PMID:26941335

  14. miR-486 suppresses the development of osteosarcoma by regulating PKC-δ pathway.

    PubMed

    He, Ming; Wang, Guangbin; Jiang, Linlin; Qiu, Chuang; Li, Bin; Wang, Jiashi; Fu, Yonghui

    2017-05-01

    Osteosarcoma is one of the most highly malignant types of cancer in adolescents and young adults with a high mortality rate. Despite advances in surgery, radiation therapy and chemotherapy, the prognosis for patients with osteosarcoma has not significantly improved over the past several decades. It is necessary to find new indicators of prognosis and therapeutic targets of osteosarcoma. Through the analysis of 40 osteosarcoma tissues, we found that the expression of miR‑486 was low and the expression of PKC‑δ was high in osteosarcoma. Median survival of patients with low expression of miR-486 (30 months) was shorter than the patients with higher expression of miR‑486 (40 months). We further found that miR-486 can inhibit the targeting of PKC‑δ signaling pathways, and this inhibition can inhibit the growth and invasion of osteosarcoma cells. After transfection of miR‑486 for 24 h, the proliferation of osteosarcoma cells was inhibited by ~20%, and the migration was inhibited by ~15%. In the present investigation, we demonstrated that miR‑486 is negatively associated with the expression of PKC-δ and could regulate the development of osteosarcoma. miR-486 may be a potential target for the treatment of osteosarcoma.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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