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No / The L-glutamate transporter GLT-1 is an abundant central nervous system (CNS) membrane protein of the excitatory amino acid transporter (EAAT) family that controls extracellular L-glutamate levels and is important in limiting excitotoxic neuronal death. Using reverse transcription-polymerase chain reaction, we have determined that four mRNAs encoding GLT-1 exist in mouse brain, with the potential to encode four GLT-1 isoforms that differ in their N and C termini. We expressed all four isoforms (termed MAST-KREK, MPK-KREK, MAST-DIETCI, and MPK-DIETCI according to amino acid sequence) in a range of cell lines and primary astrocytes and show that each isoform can reach the cell surface. In transfected human embryonic kidney (HEK) 293 or COS-7 cells, all four isoforms support high-affinity sodium-dependent L-glutamate uptake with identical pharmacological and kinetic properties. Inserting a viral epitope (tagged with V5, hemagglutinin, or FLAG) into the second extracellular domain of each isoform allowed coimmunoprecipitation and time-resolved Forster resonance energy transfer (tr-FRET) studies using transfected HEK-293 cells. Here we show for the first time that each of the four isoforms is able to combine to form homomeric and heteromeric assemblies, each of which is expressed at the cell surface of primary astrocytes. After activation of protein kinase C by phorbol ester, V5-tagged GLT-1 is rapidly removed from the cell surface of HEK-293 cells and degraded. This study provides direct biochemical evidence for oligomeric assembly of GLT-1 and reports the development of novel tools to provide insight into the trafficking of GLT-1.

7 Abstract Protein kinase C alpha (PKCα) is a serine/threonine protein kinase. PKCα is an important protein regulating cell polarity, protein secretion, apoptosis, cell proliferation and differentiation and tumorogenesis. Previous research has shown a role of PKCα also in a cancer cell migration and cancer cell invasion. The aim of this study was to investigate the role of protein kinase C alpha (PKCα) played in amoeboid mode of cancer cell invasion. We showed that higher expression of PKCα resulted in mesenchymal-amoeboid transition of K2 and MDA mesenchymal cancer cell lines, which was accompanied with decreased cancer cell invasive capability in 3D collage matrix. PKCα overexpression had no effect on the cell morphology of A375m2, however, the results showed a trend in increased invasive potential of A375m2 cells. Conversely, the expression of dominant-negative PKCα resulted in amoeboid-mesenchymal transition of A375m2 cells, and it was associated with decreased invasive potential of K2 and MDA cell lines. Furthermore, a linkage between PKCα and phosphatidylinositol 3-kinase (PI3K) was tested. The results revealed that increased activity of PKCα was accompanied with decreased level of active Akt in K2 cell line. To summarize, our results suggest a probable role of PKCα in regulation of amoeboid...

Le trouble bipolaire est une maladie invalidante caractérisée par une alternance d’épisodes maniaques et dépressifs. Malgré des efforts de recherche notables, la physiopathologie et les mécanismes d’action des traitements du trouble bipolaire demeurent peu connus. La protéine kinase C (PKC) est récemment apparue comme une cible moléculaire potentielle pour le traitement du trouble bipolaire. Dans ce travail de thèse, nous avons cherché à étudier le rôle de la PKC dans les phases maniaque et dépressive du trouble bipolaire. Nous avons montré que l’inhibition de la PKC a un effet antimaniaque non seulement chez le rat naïf, mais aussi dans un modèle de manie basé sur une privation de sommeil, que nous avons validé au cours de notre étude. De plus, les inhibiteurs de la PKC sont capables de rétablir les déficits de prolifération cellulaire hippocampique que présentent les rats privés de sommeil. Ces effets prolifératifs et antimaniaques seraient indépendants, puisque le blocage de la prolifération cellulaire n’abolit pas l’efficacité antimaniaque des inhibiteurs de la PKC dans le modèle de privation de sommeil. En parallèle, nous avons montré que l’activation de la PKC a un effet antidépresseur chez le rat naïf, alors que son inhibition provoque un phénotype pseudodépressif qui s’accompagne d’une diminution de la prolifération cellulaire hippocampique. L’ensemble de ces données révèle une implication de la PKC dans les deux phases du trouble bipolaire, et soutient l’hypothèse qu’une suractivation du système PKC serait à l’origine des perturbations de neuroplasticité associées à la manie. / Bipolar disorder is a devastating long-term disease characterized by alternate episodes of mania and depression. Despite extensive research, the molecular and cellular underpinnings of bipolar disorder remain to be fully elucidated. Protein kinase C (PKC) has emerged as a potential molecular target for the treatment of bipolar disorder. The present study investigated the role of PKC in manic- and depressive-like behaviors. Our results showed that PKC inhibition produced an antimanic-like effect not only in naive rats, but also in an animal model of mania based on sleep deprivation, that we have validated in our study. Interestingly, PKC inhibitors rescued the hippocampal cell proliferation deficits displayed by sleep-deprived animals. These proliferative and antimanic effects were independent, since blockade of cell proliferation did not abolish the antimanic efficacy of PKC inhibitors in the sleep deprivation model. At the same time, we showed that PKC activation had an antidepressant-like effect in naive rats, whereas its inhibition caused a depressive-like phenotype accompanied by a decrease in hippocampal cell proliferation. Taken together, our results demonstrate the involvement of the PKC system in regulating opposite facets of bipolar disorder, and support the hypothesis that an overactivation of the PKC signaling system may be crucial for the deficits of neuroplasticity associated with mania.

Neste estudo foi verificado os efeito do α-tocoferol (AT) nas vias de sinalização celular, dependentes de proteína quinase C (PKC) e de tirosinas quinases (TK), associadas ao \"burst\" oxidativo de neutrófilos humanos. Foram realizados também estudos comparativos com o inibidor da PKC, estaurosporina, com o inibidor de tirosinas quinases, genisteína e, com o análogo solúvel da vitamina E, Trolox. Foi feita a incorporação de AT in vitro às células, e então, estas foram estimuladas ou não com acetato de forbol miristato (PMA) ou com zymosan opsonizado (Zy). AT (40 µM) inibiu a produção de espécies reativas de oxigênio (ERO) pelos neutróflos estimulados com PMA ou Zy. Estaurosporina (10 nM), genisteína (100 µM) e Trolox (40 µM) também tiveram efeitos inibitórios. A atividade da PKC foi inibida pelo AT e pela estaurosporina, entretanto, a atividade da enzima não foi afetada pela genisteína e pelo Trolox. PMA e Zy promoveram um aumento da fosforilação em resíduos de tirosina de proteínas de neutrófilos. AT e estaurosporina provocaram um aumento adicional na fosforilação PMA-dependente, enquanto a genisteína causou uma diminiução e, Trolox não produziu nenhum efeito. Por outro lado, os quatro compostos foram inibitórios na fosforilação Zy-dependente. A atividade de tirosina fosfatases (PTPs) foi medida em neutrófilos estimulados e não-estimulados. PMA e Zy causaram uma diminuição na atividade de PTPs. A pré-incubação com AT e Trolox causou uma reversão destes efeitos inibitórios. O inibidor de serina/treonina fosfatases, caliculina A, também foi utilizado. Nós mostramos que este composto foi capaz de reverter os efeitos inibitórios do AT na produção de ERO e na atividade de PKC dos neutrófilos. Os resultados deste trabalho mostram que AT modulam ambas as via de sinalização, PKC e TK-dependentes, associadas com o \"burst\" oxidativo de neutrófilos humanos e, que esta modulação pode ser devido a ativação de fosfatases pelo AT. / The effects of α-tocopherol succinate (TS) on the signaling pathways, dependent of protein kinase C (PKC) and tirosine kinases (TK), associated with the oxidative burst of human neutrophils were analysed. Comparative studies with the PKC inhibitor, staurosporine, the TK inhibitor, genistein and the soluble analogous of vitamin E, Trolox were also performed. TS was incorporated into neutrophils and cells were then, stimulated or not with phorbol myristate acetate (PMA) or with opsonized zymosan (OZ). TS (40 µmol/l) inhibited the production of reactive oxygen species (ROS) by PMA or OZ-stimulated neutrophils. Staurosporine (10 nmol/l), genistein (100 µmol/l) and Trolox (40 µmol/l) were also inhibitory. PKC activity was inhibited by TS and staurosporine, however, the enzyme activity was not affected by genistein and Trolox. PMA and OZ promoted tyrosine phosphorylation in neutrophil proteins. TS and staurosporine caused a further increase of tyrosine phosphorylation of proteins in PMA-stimulated neutrophils, whereas, genistein diminished the levels of phosphorylation, and Trolox did not alter them. On the other hand, the four compounds decreased the tyrosine phosphorylated proteins in OZ-stimulated neutrophils. Protein tyrosine phosphatases (PTP) activity was measured in both resting and stimulated cells. PMA and OZ-stimulated neutrophils showed a decrease on PTP activity. Pre-incubation with TS or with Trolox caused partial recovery of the basal activity of stimulated neutrophils. The serine/threonine phosphatase inhibitor, calyculin A, was also utilized, and we showed that this compound was capable of reversing the inhibitory effects of TS on ROS production and PKC activity by neutrophils. These results show that TS modulates both PKC- and TK-dependent signaling pathways associated with the oxidative burst in human neutrophils, and this modulation could be due the activation of phosphatases by TS.

Kar Wing To. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 200-223). / Abstracts in English and Chinese. / Acknowledgements --- p.i / List of Abbreviations --- p.ii / Abstract --- p.v / 撮要 --- p.viii / List of Tables --- p.x / List of Figures --- p.xi / Contents --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- The General Characteristics of Glial Cells --- p.1 / Chapter 1.1.1 --- Astrocytes --- p.1 / Chapter 1.1.2 --- Oligodendrocytes --- p.5 / Chapter 1.1.3 --- Microglial --- p.6 / Chapter 1.2 --- Brain Injury and Astrocyte Proliferation --- p.6 / Chapter 1.3 --- Reactive Astrogliosis and Glial Scar Formation --- p.9 / Chapter 1.4 --- Astrocytes and Immune Response --- p.10 / Chapter 1.5 --- Cytokines --- p.10 / Chapter 1.5.1 --- Cytokines and the Central Nervous System (CNS) --- p.12 / Chapter 1.5.2 --- Cytokines and brain injury --- p.13 / Chapter 1.5.3 --- Cytokines-activated astrocytes in brain injury --- p.13 / Chapter 1.5.4 --- Tumour Necrosis Factor-a --- p.14 / Chapter 1.5.4.1 --- Types of TNF-α receptor and their sturctures --- p.16 / Chapter 1.5.4.2 --- Binding to TNF-α --- p.17 / Chapter 1.5.4.3 --- Different Roles of the TNF-a Receptor Subtypes --- p.17 / Chapter 1.5.4.4 --- Role of TNF-α and Brain Injury --- p.19 / Chapter 1.5.4.5 --- TNF-α Stimulates Proliferation of Astrocytes and C6 Glioma Cells --- p.23 / Chapter 1.5.5 --- Interleukin-1 (IL-1) --- p.26 / Chapter 1.5.5.1 --- Interleukin-1 and Brain Injury --- p.27 / Chapter 1.5.6 --- Interleukin-6 (IL-6) --- p.28 / Chapter 1.5.6.1 --- Interleukin-6 and brain injury --- p.29 / Chapter 1.5.7 --- γ-Interferon (γ-IFN) --- p.30 / Chapter 1.5.7.1 --- γ-Interferon and Brain Injury --- p.30 / Chapter 1.6 --- Ion Channels and Astrocytes --- p.31 / Chapter 1.6.1 --- Roles of Sodium Channels in Astrocytes --- p.33 / Chapter 1.6.2 --- Role of Potassium Channels in Astrocytes --- p.33 / Chapter 1.6.3 --- Importance of Calcium Ion Channels in Astrocytes --- p.34 / Chapter 1.6.3.1 --- Function of Cellular and Nuclear Calcium --- p.34 / Chapter 1.6.3.2 --- Nuclear Calcium in Cell Proliferation --- p.36 / Chapter 1.6.3.3 --- Nuclear Calcium in Gene Transcription --- p.36 / Chapter 1.6.3.4 --- Nuclear Calcium in Apoptosis --- p.38 / Chapter 1.6.3.5 --- Spatial and Temporal Changes of Calcium-Calcium Oscillation --- p.39 / Chapter 1.6.3.6 --- Calcium Signalling in Glial Cells --- p.39 / Chapter 1.6.3.7 --- Calcium Channels in Astrocytes --- p.41 / Chapter 1.6.3.8 --- Relationship Between [Ca2+]i and Brain Injury --- p.43 / Chapter 1.6.3.9 --- TNF-α and Astrocyte [Ca2+]i --- p.45 / Chapter 1.6.3.10 --- Calcium-Sensing Receptor (CaSR) --- p.46 / Chapter 1.7 --- Protein Kinase C (PKC) Pathways --- p.49 / Chapter 1.7.1 --- PKC and Brain Injury --- p.50 / Chapter 1.7.2 --- Role of Protein Kinase C Activity in TNF-α Gene Expression in Astrocytes --- p.51 / Chapter 1.7.3 --- PKC and Calcium in Astrocytes --- p.52 / Chapter 1.8 --- Intermediate Early Gene (IEGs) --- p.54 / Chapter 1.8.1 --- IEGs Expression and Brain Injury --- p.54 / Chapter 1.8.2 --- IEGs Expression and Calcium --- p.55 / Chapter 1.9 --- The Rat C6 Clioma Cells --- p.56 / Chapter 1.10 --- The Aim of This Project --- p.58 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.61 / Chapter 2.1.1 --- Sources of the Chemicals --- p.61 / Chapter 2.1.2 --- Materials Preparation --- p.65 / Chapter 2.1.2.1 --- Rat C6 Glioma Cell Line --- p.65 / Chapter 2.1.2.2 --- C6 Glioma Cell Culture --- p.65 / Chapter 2.1.2.2.1 --- Complete Dulbecco's Modified Eagle Medium (CDMEM) --- p.65 / Chapter 2.1.2.2.2 --- Serum-free Dulbecco's Modified Eagle Medium --- p.66 / Chapter 2.1.2.3 --- Phosphate Buffered Saline (PBS) --- p.66 / Chapter 2.1.2.4 --- Recombinant Cytokines --- p.67 / Chapter 2.1.2.5 --- Antibodies --- p.67 / Chapter 2.1.2.5.1 --- Anti-TNF-Receptor 1 (TNF-R1) Antibody --- p.67 / Chapter 2.1.2.5.2 --- Anti-TNF-Receptor 2 (TNF-R2) Antibody --- p.67 / Chapter 2.1.2.6 --- Chemicals for Signal Transduction Study --- p.68 / Chapter 2.1.2.6.1 --- Calcium Ionophore and Calcium Channel Blocker --- p.68 / Chapter 2.1.2.6.2 --- Calcium-Inducing Agents --- p.68 / Chapter 2.1.2.6.3 --- Modulators of Protein Kinase C (PKC) --- p.69 / Chapter 2.1.2.7 --- Reagents for Cell Proliferation --- p.69 / Chapter 2.1.2.8 --- Reagents for Calcium Level Measurement --- p.70 / Chapter 2.1.2.9 --- Reagents for RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.71 / Chapter 2.1.2.10 --- Sense and Antisense Used --- p.72 / Chapter 2.1.2.11 --- Reagents for Electrophoresis --- p.74 / Chapter 2.2 --- Methods --- p.74 / Chapter 2.2.1 --- Maintenance of the C6 Cell Line --- p.74 / Chapter 2.2.2 --- Cell Preparation for Assays --- p.75 / Chapter 2.2.3 --- Determination of Cell Proliferation --- p.76 / Chapter 2.2.3.1 --- Determination of Cell Proliferation by [3H]- Thymidine Incorporation --- p.76 / Chapter 2.2.3.2 --- Measurement of Cell Viability Using Neutral Red Assay --- p.77 / Chapter 2.2.3.3 --- Measurement of Cell Proliferation by MTT Assay --- p.77 / Chapter 2.2.3.4 --- Protein Assay --- p.78 / Chapter 2.2.3.5 --- Data Analysis --- p.79 / Chapter 2.2.3.5.1 --- The Measurement of Cell Proliferation by [3H]- Thymidine Incorporation --- p.79 / Chapter 2.2.3.5.2 --- The Measurement of Cell growth in Neutral Red and MTT Assays --- p.79 / Chapter 2.2.3.5.3 --- The Measurement of Cell Proliferationin Protein Assay --- p.79 / Chapter 2.2.4 --- Determination of Intracellular Calcium Changes --- p.80 / Chapter 2.2.4.1 --- Confocal Microscopy --- p.80 / Chapter 2.2.4.1.1 --- Procedures for Detecting Cell Activity by CLSM --- p.81 / Chapter 2.2.4.1.2 --- Precautions of CLSM --- p.82 / Chapter 2.2.5 --- Determination of Gene Expression by Reverse- Transcription Polymerase Chain Reaction (RT-PCR) --- p.83 / Chapter 2.2.5.1 --- RNA Preparation --- p.83 / Chapter 2.2.5.1.1 --- RNA Extraction --- p.83 / Chapter 2.2.5.1.2 --- Measurement of RNA Yield --- p.84 / Chapter 2.2.5.2 --- Reverse Transcription (RT) --- p.84 / Chapter 2.2.5.3 --- Polymerase Chain Reaction (PCR) --- p.85 / Chapter 2.2.5.4 --- Separation of PCR Products by Agarose Gel Electrophoresis --- p.85 / Chapter 2.2.5.5 --- Quantification of Band Density --- p.86 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- Effects of Different Drugs on C6 Cell Proliferation --- p.87 / Chapter 3.1.1 --- Effects of Cytokines on C6 Cell Proliferation --- p.87 / Chapter 3.1.1.1 --- Effect of TNF-α on C6 Proliferation --- p.88 / Chapter 3.1.1.2 --- Effects of Other Cytokines on C6 Cell Proliferation --- p.92 / Chapter 3.1.2 --- The Signalling Pathway of TNF-α induced C6 Cell Proliferation --- p.92 / Chapter 3.1.2.1 --- The Involvement of Calcium Ions in TNF-α-induced C6Cell Proliferation --- p.95 / Chapter 3.1.2.2 --- The Involvement of Protein Kinase C in TNF-α- induced C6 Cell Proliferation --- p.96 / Chapter 3.1.3 --- Effects of Anti-TNF Receptor Subtype Antibodies on C6 Cell Proliferation --- p.102 / Chapter 3.2 --- The Effect of in Tumour Necrosis Factor-α on Changesin Intracellular Calcium Concentration --- p.102 / Chapter 3.2.1 --- Release of Intracellular Calcium in TNF-α-Treated C6 Cells --- p.104 / Chapter 3.2.2 --- Effects of Calcium Ionophore and Calcium Channel Blocker on TNF-α-induced [Ca2+]i Release --- p.107 / Chapter 3.2.3 --- Effects of Other Cytokines on the Change in [Ca2+]i --- p.109 / Chapter 3.2.4 --- The Role of PKC in [Ca2+]i release in C6 Glioma Cells --- p.109 / Chapter 3.2.4.1 --- Effects of PKC Activators and Inhibitors on the Changes in [Ca2+]i --- p.114 / Chapter 3.3 --- Determination of Gene Expression by RT-PCR --- p.114 / Chapter 3.3.1 --- Studies on TNF Receptors Gene Expression --- p.117 / Chapter 3.3.1.1 --- Effect of TNF-α on TNF Receptors Expression --- p.117 / Chapter 3.3.1.2 --- Effects of Other Cytokines on the TNF Receptors Expression --- p.119 / Chapter 3.3.1.3 --- Effects of Anti-TNF Receptor Subtype Antibodies on the TNF-a-induced Receptors Expression --- p.121 / Chapter 3.3.1.4 --- Effect of Calcium Ions on TNF Receptors Expression --- p.121 / Chapter 3.3.1.4.1 --- Effect of Calcium Ionophore on TNF Receptors Expression --- p.126 / Chapter 3.3.1.4.2 --- Effect of TNF-α Combination with A23187 on the Expression of TNF Receptors --- p.128 / Chapter 3.3.1.4.3 --- Effects of Calcium Ionophore and Channel Blocker on TNF Receptors Expression --- p.130 / Chapter 3.3.1.4.4 --- Effects of Calcium-Inducing Agents on TNF Receptors Gene Expressions --- p.130 / Chapter 3.3.1.5 --- Effects of PKC Activator and Inhibitor on TNF-α- induced TNF Receptors Expressions --- p.135 / Chapter 3.3.1.6 --- Effect of PKC and Ro31-8220 on IL-l-induced TNF Receptors Expressions --- p.138 / Chapter 3.3.2 --- Expression of Calcium-sensing Receptor upon Different Drug Treatments --- p.138 / Chapter 3.3.2.1 --- Effect of TNF-α on the Calcium-sensing Receptor Expression --- p.141 / Chapter 3.3.2.2 --- Effects of Other Cytokines on CaSR Expression --- p.143 / Chapter 3.3.2.3 --- Effect of A23187 on CaSR Expression --- p.143 / Chapter 3.3.2.4 --- Effect of TNF-α and A23187 Combined Treatment on CaSR Expression --- p.146 / Chapter 3.3.2.5 --- Effects of Calcium-inducing Agents on CaSR Expression --- p.149 / Chapter 3.3.2.6 --- Effects of PKC Activator and PKC Inhibitor on CaSR Expression --- p.149 / Chapter 3.3.3 --- Expression of PKC Isoforms upon Treatment with Different Drugs --- p.153 / Chapter 3.3.3.1 --- Effect of TNF-α on the PKC Isoforms Expression --- p.155 / Chapter 3.3.3.2 --- Effect of A23187 on the PKC Isoforms Expression --- p.155 / Chapter 3.3.3.3 --- Effect of TNF-α and Calcium Ionophore Combined Treatment on PKC Isoforms Expression --- p.157 / Chapter 3.3.3.4 --- Effects of Calcium-inducing Agents on PKC Isoforms Expression --- p.159 / Chapter 3.3.4 --- Expression of the Transcription Factors c-fos and c-junin Drug Treatments --- p.161 / Chapter 3.3.4.1 --- Effect of TNF-a on c-fos and c-jun Expression --- p.163 / Chapter 3.3.4.2 --- Effect of A23187 on c-fos and c-jun Expression --- p.163 / Chapter 3.3.4.3 --- Effect of TNF-a in Combination with A23187 on c- fos and c-jun Expression --- p.165 / Chapter 3.3.4.4 --- Effects of Calcium-inducing Agents on c-fos and c- jun Expression --- p.167 / Chapter 3.3.5 --- Effects of Different Drugs on Endogenous TNF-α Expression --- p.167 / Chapter 3.3.5.1 --- Effect of TNF-α on Endogenous TNF-α Expression --- p.169 / Chapter 3.3.5.2 --- Effect of A23187 on Endogenous TNF-α Expression --- p.169 / Chapter 3.3.5.3 --- Effect of TNF-α in Combination with A23187 on the Expression of Endogenous TNF-α --- p.172 / Chapter 3.3.5.4 --- Effects of Calcium-inducing Agents on Endogenous TNF-α Expression --- p.172 / Chapter 3.3.6 --- Effect of Different Drugs on LL-1 Expression --- p.175 / Chapter 3.3.6.1 --- Effect of TNF-a on IL-lα Expression --- p.177 / Chapter 3.3.6.2 --- Effect of A23187 on the IL-lα Expression --- p.177 / Chapter 3.3.6.3 --- Effect of Calcium Ionophore and TNF-α Combined Treatment on IL-1α Expression --- p.179 / Chapter 3.3.6.4 --- Effects of Calcium-inducing Agents on IL-lα Expression --- p.179 / Chapter 3.3.6.5 --- Effect of PKC Activator on the IL-1α Expression --- p.181 / Chapter CHAPTER 4 --- DISCUSSIONS AND CONCLUSIONS / Chapter 4.1 --- "Effects of Cytokines, Ca2+ and PKC and Anti-TNF-α Antibodies on C6 Glioma Cells Proliferation" --- p.184 / Chapter 4.2 --- The Role of Calcium in TNF-α-induced Signal Transduction Pathways --- p.186 / Chapter 4.3 --- Gene Expressions in C6 Cells after TNF-a Stimulation --- p.187 / Chapter 4.3.1 --- "Expression of TNF-α, TNF-Receptors and IL-1" --- p.187 / Chapter 4.3.2 --- CaSR Expression --- p.190 / Chapter 4.3.3 --- PKC Isoforms Expressions --- p.192 / Chapter 4.3.4 --- Expression of c-fos and c-jun --- p.193 / Chapter 4.4 --- Conclusion --- p.194 / REFERENCES --- p.200

1. Abstract Protein kinase C α (PKCα) is a serine/threonine protein kinase regulating many different signaling pathways. The aim of this study was to investigate the potential role of PKCα in amoeboid morphology and invasion of cancer cells. It was observed, that expression of PKCα as well as its phosphorylation on Thr497 remained unchanged upon amoeboid-mesenchymal transition of A375m2 cells (induced by inhibition of ROCK kinase) both in 3D and in 2D environment. However, activation of PKCα by PKC activator treatment resulted in mesenchymal- amoeboid transition of K2 and MDA-MB-231 mesenchymal cell lines, although it did not change overall invasivity ability of cells to invade 3D collagen. Notably, PKCα activation significantly reduced matrix degrading abilities of A375m2 cells. Conversely, inhibition of PKCα by PKCα inhibitor treatment caused amoeboid-mesenchymal transition of amoeboid A375m2 cells and it was associated with decreased invasiveness of all three cell lines used. PKCα inhibitor did not have any effect on gelatin degradation area of A375m2 cells. Consistently, specific siRNA mediated downregulation of PKCα lead to transition from amoeboid to mesenchymal morphology of A375m2 cells and reduced invasiveness of cells into 3D collagen. Moreover, gelatin degrading abilities of A375m2 cells were...

La myopathie de Duchenne est une maladie génétique qui se caractérise principalement par une dégénérescence progressive des muscles squelettiques dont la cause est l’absence de dystrophine fonctionnelle dans les muscles. A ce jour, il n’existe toujours pas de traitement efficace contre ces maladies. Comme le plus grand gène connu chez l’Homme, la dystrophine code pour une protéine de 427kDa. La protéine connecte l’actine avec le DAPC (Dystrophin Associated Protein Complex) dans les muscles striés. Pour l’instant, il y a 3 hypothèses concernant le mécanisme du DMD. L’absence de la dystrophine peut supprimer le lien physique entre les protéines structurales de la membrane basale (laminines) et les protéines structurales du cytosquelette (filaments intermédiaires et actine), ou la distribution et la fonction des canaux ioniques, ou des voies de signalisation nécessaires à la survie du muscle. Caenorhabditis elegans ne possède qu’un homologue du gène de la dystrophine humaine, le gène dys-1. La protéine DYS-1 présente 37% d’homologie avec la dystrophine humaine. Le double mutant dys-1(cx18) ; hlh-1(cc561) présente une forte dégénérescence musculaire. Comme le sarcomère de C. elegans ressemble au sarcomère de mammifère, C. elegans est modèle pertinent d’étude la maladie. En vue de comprendre la raison du DMD chez les mammifères et chez les vers, le groupe L. SEGALAT a effectué des cribles pour identifier les molécules et les gènes qui peuvent supprimer la dégénérescence musculaire. On a trouvé un gène pkc-2 qui est capable de supprimer la dégénérescence musculaire chez C. elegans. La protéine PKC-2 est l’orthologue de la Protein Kinase C Alpha (PKC) humaine et appartient à la famille du serine/threonine protéine kinase. Afin d’étudier la fonction du gène pkc-2, on a analysé l’expression du gène avec les construits différents in vivo et a utilisé la technique de double-hybride dans la levure. De plus, le crible par EMS (éthane méthyle sulfonâtes) a identifié une molécule sérotonine (5-HT) qui est un neuromédiateur, et supprime partiellement la dégénérescence musculaire des doubles mutants dys-1; hlh-1. La sérotonine a aussi un effet fort sur le mutant pkc-2(ok328), puisqu’elle provoque un phénotype blister. Ça nous permet de rechercher le lien entre la signalisation sérotoninergique et pkc-2. Le crible génétique peut contribuer à la connaissance du rôle pkc-2. […]. Elle sert aussi de plate-forme de voie de signalisation intracellulaire. L’identification de Y59A8A.3 propose la possibilité que pkc-2 modifie la filamin A par l’intermédiaire de la filamin A interacting protéine 1. Le crible génétique par EMS pour rechercher des suppresseurs de l’effet blister de la sérotonine sur les mutants pkc-2(ok328) a donné 8 candidats sur 5000 F1s : cx253, cx254, cx259, cx263, cx267, cx268, cx270, cx276. Les mutations ont été localisées sur les chromosomes par SNP mapping avec une souche de C. elegans très polymorphe, mais le temps a manqué pour leur identification exacte. L’expérience valide notre approche à étudier le lien entre la signalisation sérotoninergique et pkc-2. En résumé, le but de la thèse était de rechercher la fonction du gène pkc-2 dans les mécanismes moléculaires conduisant à la nécrose musculaire en absence de dystrophine. Les résultats présentés dans la thèse apportent des réponses aux questions fondamentales sur pkc-2 et aussi demandent des expériences supplémentaires afin de élucider plus avant les mécanismes de la dégénérescence musculaire dystrophine-dépendante. / Duchenne Muscular Dystrophy (DMD) is an X-linked progressive muscle disease which is caused by mutations in the dystrophin gene. Until now, there is no effective therapy for DMD. As the largest gene in human beings, it produces a 427-kDa cytoskeleton protein: Dystrophin. Dystrophin links actin and dystrophin associated protein complex (DAPC) in muscles. Currently, there are 3 hypotheses to explain the mechanisms of DMD. They suggest that the absence of dystrophin could lead to periodic muscle cell membrane ruptures, or affect the distribution and function of ion channels, or perturb signal transduction pathways. In Caenorhabditis elegans, there is only one homologue of mammalian dystrophin gene named dys-1, and the nematode protein DYS-1 presents 37% similar to the human one. The double mutant dys-1; hlh-1 exhibits a severe progressive muscle degeneration. The protein composition of the sarcomere has been studied and it has revealed a high degree of similarity with mammalian sarcomere. These allow C. elegans be a relevant animal model to study DMD.To understand why the lack of dystrophin induces muscle degeneration in mammals and worms, and to find new drugs that might help in reducing muscle degeneration, L. Ségalat and his coworkers performed several screens for drugs and genes suppressing muscle degeneration. An interesting gene pkc-2 came out and was considered as a possible regulator in the process of muscle degeneration in C. elegans. The protein that is encoded by this gene in C. elegans is an orthologous of the human gene Protein Kinase C Alpha (PKC), which belongs to the family of serine/threonine specific protein kinases. To study the function of pkc-2, we generated different recombinant constructs, analyzed the expression pattern of pkc-2 with immunocytochemistry, and performed yeast two-hybrid to search for PKC-2 binding partners. In addition, a neurotransmitter serotonin (5-HT) was found by drug screening to be an active blocker of striated muscle degeneration. As C. elegans lacking PKC-2 displays a severe blister phenotype in exogenous 5-HT, studying the correlation between PKC-2 and 5-HT therefore seems to be an opportunity to explore the reasons of muscle degeneration. A genetic screen with EMS (ethane methyl sulfonate) to search serotonin resistant mutant in strain pkc-2 (ok328) would help us study further about the role of pkc-2.In this thesis, different clones myo3::pkc-2 and pkc-2::gfp were made to inject into wild-type animals. The results revealed that pkc-2 expressed intensely in neurons and pharynx, but was not found in body-wall muscles. Mutants dys-1;hlh-1 fed with pkc-2 RNAi did not reduce muscle degeneration statistically comparing to triple mutant pkc-2;dys-1;hlh-1. This indicated that PKC-2 may be dominantly acting in neurons. A yeast two-hybrid screen identified the gene Y59A8A.3, which is a homologue to mammalian filamin A interacting protein 1 isoform 3, as a binding partner of PKC-2. Filamin A is a cytoskeleton protein, anchoring various trans-membrane proteins to the actin cytoskeleton and may also function as an important signaling scaffold. The result suggested that PKC-2 may therefore modulate filamin A activity through the filamin interacting protein 1. Genetic screen by EMS presented 8 candidates named cx253, cx254, cx259, cx263, cx267, cx268, cx270, cx276, which were mapped on chromosomes by SNP mapping using a polymorphic C. elegans strain, but time was too short to identify these genes formally. The experiment also offered possibilities of searching links between PKC-2 and serotonin pathways.In summary, this work studied the gene pkc-2 in order to reveal the function of PKC-2 and its involvement in muscle degeneration. The present results answered some questions about pkc-2, and needed further researches to elucidate the in vivo role of PKC-2 protein and its interaction with other proteins in the mechanism of muscle dystrophy in C. elegans.

Cellular senescence is a permanent cell cycle arrest that is induced as a response to cellular stress. Replicative senescence is a well-described mechanism that limits the replicative capacity of cells and must be overcome by cancer cells. Oncogene-induced senescence (OIS) is a form of premature senescence and a potent tumor suppressor mechanism. OIS is induced in normal cells as a result of deregulated oncogene or tumor suppressor gene expression. An exciting area of research is the identification of novel targets that induce senescence in cancer cells as a therapeutic approach. In this study, a novel mechanism is described where the inhibition of Hsp90 in small cell lung cancer (SCLC) cells induced premature senescence rather than cell death. The senescence induced following Hsp90 inhibition was p21-dependent and the loss of p21 allowed SCLC cells to bypass the induction of senescence. Additionally, we identified a novel mechanism where the depletion of PKCι induced senescence in glioblastoma multiforme (GBM) cells. PKCι depletion-induced senescence did not activate the DNA-damage response pathway and was p21-dependent. Further perturbations of mitosis, using an aurora kinase inhibitor, increased the number of senescent cells when combined with PKCι depletion. This suggests that PKCι depletion-induced senescence involves defects in mitotic progression. Senescent glioblastoma cells at a basal level of senescence in culture, induced by p21 overexpression, and induced after PKCι depletion had aberrant centrosomes. Mitotic slippage is an early exit from mitosis without cell division that occurs when the spindle assembly checkpoint (SAC) is not satisfied. Senescent glioblastoma cells had multiple markers of mitotic slippage. Therefore, PKCι depletion-induced senescence involves mitotic slippage and results in aberrant centrosomes. A U87MG cell line with a doxycycline-inducible shRNA targeting PKCι was developed to deplete PKCι in established xenografts. PKCι was depleted in established glioblastoma xenografts in mice and resulted in decreased cell proliferation, delayed tumor growth and improved survival. This study has demonstrated that both Hsp90 and PKCι are novel targets to induce senescence in cancer cells as a potential therapeutic approach.

El objetivo general de este trabajo ha sido estudiar la posible implicación de diferentes cascadas de proteín kinasas en las modificaciones cardiacas que se producen tras la administración de naloxona a ratas dependientes de morfina. Los datos obtenidos indican que durante la abstinencia a morfina se produce un aumento del turnover de NA, de la actividad TH y de su fosforilación en serina 40 y 31, lo que sugiere la puesta en marcha de mecanismos post-transcripcionales. Por otra parte, la vía de la PKA estaría implicada en el incremento del turnover de NA, en el aumento de TH total y en la fosforilación y activación de TH en serina 40 durante dicho síndrome. Por último, la vía de la PKC sería una de las vías implicadas en la expresión de c-fos, así como la de las ERK, que estaría también implicada en la activación de TH en serina 31. / The main aim of this work was to study the posible involvement of different protein kinases in the cardiac adaptive changes induced during morphine withdrawal. Our results show an increase of NA turnover, TH activity and TH phosphorylation at serine 31 and 40, suggesting starting post-trascriptional mechanisms. On the other hand, PKA transduction system could be implicated in the enhanced NA turnover, in the total TH increase and in the phosphorylation and activation of TH at serine 40 during this syndrome. Finally, PKC pathway would be involved in c-Fos expression as well as ERK system which would also be responsible for TH phosphorylation at serine 31.