Impaired activation of protein kinase C-zeta by insulin and phosphatidylinositol-3,4,5-(PO4)3 in cultured preadipocyte-derived adipocytes and myotubes of obese subjects.

Insulin resistance in obesity is partly due to diminished glucose transport in myocytes and adipocytes, but underlying mechanisms are uncertain. Insulin-stimulated glucose transport requires activation of phosphatidylinositol (PI) 3-kinase (3K), operating downstream of insulin receptor substrate-1. PI3K stimulates glucose transport through increases in PI-3,4,5-(PO(4))(3) (PIP(3)), which activates atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt). However, previous studies suggest that activation of aPKC, but not PKB, is impaired in intact muscles and cultured myocytes of obese subjects. Presently, we examined insulin activation of glucose transport and signaling factors in cultured adipocytes derived from preadipocytes harvested during elective liposuction in lean and obese women. Relative to adipocytes of lean women, insulin-stimulated [(3)H]2-deoxyglucose uptake and activation of insulin receptor substrate-1/PI3K and aPKCs, but not PKB, were diminished in adipocytes of obese women. Additionally, the direct activation of aPKCs by PIP(3) in vitro was diminished in aPKCs isolated from adipocytes of obese women. Similar impairment in aPKC activation by PIP(3) was observed in cultured myocytes of obese glucose-intolerant subjects. These findings suggest the presence of defects in PI3K and aPKC activation that persist in cultured cells and limit insulin-stimulated glucose transport in adipocytes and myocytes of obese subjects.

Networking Notch : regulation of Notch traffic and signaling crosstalk

Utvecklingen av flercelliga organismer är en mångfacetterad process som kräver kommunikation celler emellan. Under utvecklingen av en organism måste cellerna göra vissa val, vilket bestämmer riktningen för deras fortsatta utveckling. Utgående från dessa val erhåller cellerna egenskaper som är karaktäristiska för olika celltyper. Notch-signalräckan är en viktig reglerare av valet mellan olika cellöden. Notch-signalräckan aktiveras när Notch-receptorer som uttrycks på ytan av en cell binder till Notch-ligander som uttrycks på ytan av en annan närliggande cell. Denna avhandling belyser mekanismerna som reglerar omsättningen av såväl Notch-receptorer som -ligander till och från cellmembranen, samt ökar förståelsen för hur dessa mekanismer påverkar Notch-medierade cellöden i stamceller. Internalisering av Notch receptorer anses nödvändigt för fullständig aktivering av Notch-signalvägen. De bakomliggande molekylära mekanismerna är dock fortfarande oklara. Vi har upptäckt att atypiskt protein kinas Cζ (aPKCζ) reglerar internaliseringen av Notch-receptorer. aPKCζ fosforylerar Notch, vilket leder till receptorns internalisering, men effekten är beroende av receptorns signaleringsstatus. Vi visar att aPKCζ reglerar Notch-signaleringen och styr både neuroners och muskelcellers differentiering. Ytterligare har vi analyserat samspelet mellan cellskelettet och Notch-signalvägen. Våra resultat visar att intermediärfilamenten, en del av cellskelettet, är viktiga reglerare av Notch-signaleringen både under neuronal och vaskulär utveckling. Intermediärfilamenten vimentin och GFAP reglerar uttrycket av Notch-ligander vid cellmembranen i hjärnans stödceller, astrocyterna, och påverkar därmed neuronala stamcellers cellödesbeslut. Vimentin är även viktigt reglerare av Notch-signalräckan vid angiogenesen. Celler som saknar vimentin uppvisar avvikande Notch-signalering emedan möss som saknar vimentin påvisar en fördröjd utveckling av vaskulaturen under embryonalstadiet. ------------------------------------------------- Monisoluisten organismien kehittyminen on monimutkainen prosessi, joka vaatii viestintää solujen välillä. Kehittymisen aikana solut joutuvat tiettyjen valintojen eteen, mitkä tulevat määrittämään niiden erilaistumisen suunnan. Solut omaksuvat solutyypille ominaisia ominaisuuksia näihin valintoihin perustuen Notch-signalointireitti säätelee solujen erilaistumista eri suuntiin. Notch-signalointireitti aktivoituu, kun Notch-reseptori yhden solun pinnalla sitoo Notch-ligandin toisen, viereisen solun solukalvolla. Tutkimukseni lisää tuntemusta Notch-reseptoreiden ja ligandien solun sisäisestä liikennöinnistä ja sitä säätelevistä mekanismeista, sekä tämän säätelyn vaikutuksista kantasulojen erilaistumiseen. Notch-signalointireitin aktivoituminen vaatii reseptoreiden ja ligandien sisäistämisen solukalvolta, mutta taustalla olevat ja sisäistymistä säätelevät mekanismit ovat vielä epäselviä. Tutkimukseni osoittaa, että atyyppinen proteiinikinaasi Cζ (aPKCζ) säätelee Notch-reseptoreiden endosytoosia. Endosytoosin lopputulos riippuu siitä onko reseptori aktivoitunut ligandin välityksellä vai ei. Tuloksemme osoittavat aPKCζ säätelevän Notch-signalointia ja kontrolloivan sekä hermosolujen, että lihassolujen erilaistumista. Analysoimme myös Notch-signaloinnin ja solun tukirangan vuorovaikutuksia. Välikokoiset filamentit, jotka ovat osa tukirankaa, säätelevät Notch-signalointia neuronaalisen erilaistumisen sekä verisuonten uudismuodostumisen aikana. Vimentiini ja GFAP ovat välikokoisia säikeitä, jotka säätelevät Notch-ligandien ekspressiota astrosyyttien, eli aivojen hermotukisolujen solukalvolla. Vimentiini säätelee myös Notch-signalointireittiä angiogeneesin aikana. Vimentiiniä vailla olevilla soluilla ilmenee heikentynyttä Notch-signalointia, joka voidaan liittää hiirillä ilmenevään vimenttiinin puutteesta johtuvaan viivästyneeseen verisuonien kehitykseen.

Étude des mécanismes cellulaires activés par l'Angiopoïétine-1 et le VEGF régulant la perméabilité et la migration endothéliales

L’angiogenèse est la formation de nouveaux vaisseaux sanguins à partir d’un réseau vasculaire existant. C’est un phénomène essentiel pour des processus physiologiques et pathologiques. L’activation des cellules endothéliales est contrôlée par plusieurs facteurs de croissance. Le VEGF et son récepteur le VEGFR-2 ont été prouvés comme étant spécifiques et critiques pour la formation des vaisseaux sanguins alors que Tie2, le récepteur auquel se lie l’Ang-1, est requis aussi bien dans le développement vasculaire que dans l’angiogenèse tumorale. Il est connu que l’activation de Tie2 est nécessaire à la stabilisation finale de la vascularisation en inhibant la perméabilité vasculaire induite par le VEGFR-2. Nous avons premièrement découvert que le facteur de croissance pro-angiogénique, l’Ang-1 contrecarre les effets de perméabilité cellulaire induits par le VEGF en inhibant la production de NO dans les cellules endothéliales. Cet effet inhibiteur de Tie2 intervient directement au niveau de l’activité de l’enzyme eNOS. Suite à l’activation de Tie2 par l’Ang-1, eNOS devient fortement phosphorylé sur la Thr497 après la phosphorylation et l’activation de la PKCζ. Nos résultats suggèrent que l’inhibition, par Tie2, de la perméabilité vasculaire durant l’angiogenèse serait due, en partie, à l’inhibition de la production de NO. Deuxièmement nous avons pu distinguer entre deux modes de migration cellulaire endothéliale induits par l’Ang-1 et le VEGF. À l’opposé du VEGF qui promeut une migration individuelle aléatoire, l’Ang-1 induit une migration collective directionnelle. Dans cette étude, nous avons identifié la β-caténine comme un nouveau partenaire moléculaire de la PKCζ. Cette association de la PKCζ à la β-caténine amène le complexe de polarité Par6-aPKC et le complexe des jonctions d’adhérences cellulaires à interagir ensemble à deux localisations différentes au niveau de la cellule endothéliale. Au niveau des contacts intercellulaires, le complexe PKCζ/β-caténine maintien la cohésion et l’adhésion cellulaire nécessaire pour le processus migratoire collectif. Ce complexe se retrouve aussi au niveau du front migratoire des cellules endothéliales afin d’assurer la directionalité et la persistance de la migration endothéliale en réponse à l’Ang-1. D’une manière intéressante, lors de l’inhibition de la PKCζ ou de la β-caténine on assiste à un changement du mode de migration en réponse à l’Ang-1 qui passe d’une migration directionnelle collective à une migration individuelle aléatoire. Ce dernier mode de migration est similaire à celui observé chez des cellules endothéliales exposées au VEGF. Ces résultats ont été corroborés in vivo par une polarité et une adhésion défectueuses au cours de la vasculogenèse chez le poisson zèbre déficient en PKCζ. En résumé, Ang-1/Tie2 module la signalisation et les réponses biologiques endothéliales déclenchées par le VEGF/VEGFR-2. L’identification des mécanismes moléculaires en aval de ces deux récepteurs, Tie2 et VEGFR-2, et la compréhension des différentes voies de signalisation activées par ces complexes moléculaires nous permettra de mettre la lumière sur des nouvelles cibles thérapeutiques pour le traitement des maladies angiogéniques.

AICAR and metformin, but not exercise, increase muscle glucose transport through AMPK-, ERK-, and PDK1-dependent activation of atypical PKC

Activators of 5'-AMP-activated protein kinase (AMPK) 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), metformin, and exercise activate atypical protein kinase C (aPKC) and ERK and stimulate glucose transport in muscle by uncertain mechanisms. Here, in cultured L6 myotubes: AICAR- and metformin-induced activation of AMPK was required for activation of aPKC and ERK; aPKC activation involved and required phosphoinositide-dependent kinase 1 (PDK1) phosphorylation of Thr410-PKC-zeta; aPKC Thr410 phosphorylation and activation also required MEK1-dependent ERK; and glucose transport effects of AICAR and metformin were inhibited by expression of dominant-negative AMPK, kinase-inactive PDK1, MEK1 inhibitors, kinase-inactive PKC-zeta, and RNA interference (RNAi)-mediated knockdown of PKC-zeta. In mice, muscle-specific aPKC (PKC-lambda) depletion by conditional gene targeting impaired AICAR-stimulated glucose disposal and stimulatory effects of both AICAR and metformin on 2-deoxyglucose/glucose uptake in muscle in vivo and AICAR stimulation of 2-[(3)H]deoxyglucose uptake in isolated extensor digitorum longus muscle; however, AMPK activation was unimpaired. In marked contrast to AICAR and metformin, treadmill exercise-induced stimulation of 2-deoxyglucose/glucose uptake was not inhibited in aPKC-knockout mice. Finally, in intact rodents, AICAR and metformin activated aPKC in muscle, but not in liver, despite activating AMPK in both tissues. The findings demonstrate that in muscle AICAR and metformin activate aPKC via sequential activation of AMPK, ERK, and PDK1 and the AMPK/ERK/PDK1/aPKC pathway is required for metformin- and AICAR-stimulated increases in glucose transport. On the other hand, although aPKC is activated by treadmill exercise, this activation is not required for exercise-induced increases in glucose transport, and therefore may be a redundant mechanism.

Activation of protein kinase C by tyrosine phosphorylation in response to H2O2

Protein kinase C (PKC) isoforms, α, βI, and γ of cPKC subgroup, δ and ɛ of nPKC subgroup, and ζ of aPKC subgroup, were tyrosine phosphorylated in COS-7 cells in response to H2O2. These isoforms isolated from the H2O2-treated cells showed enhanced enzyme activity to various extents. The enzymes, PKC α and δ, recovered from the cells were independent of lipid cofactors for their catalytic activity. Analysis of mutated molecules of PKC δ showed that tyrosine residues, which are conserved in the catalytic domain of the PKC family, are critical for PKC activation induced by H2O2. These results suggest that PKC isoforms can be activated through tyrosine phosphorylation in a manner unrelated to receptor-coupled hydrolysis of inositol phospholipids.

Retromer controls epithelial cell polarity by trafficking the apical determinant crumbs

The evolutionarily conserved apical determinant Crumbs (Crb) is essential for maintaining apicobasal polarity and integrity of many epithelial tissues [1]. Crb levels are crucial for cell polarity and homeostasis, yet strikingly little is known about its trafficking or the mechanism of its apical localization. Using a newly established, liposome-based system described here, we determined Crb to be an interaction partner and cargo of the retromer complex. Retromer is essential for the retrograde transport of numerous transmembrane proteins from endosomes to the trans-Golgi network (TGN) and is conserved between plants, fungi, and animals [2]. We show that loss of retromer function results in a substantial reduction of Crb in Drosophila larvae, wing discs, and the follicle epithelium. Moreover, loss of retromer phenocopies loss of crb by preventing apical localization of key polarity molecules, such as atypical protein kinase C (aPKC) and Par6 in the follicular epithelium, an effect that can be rescued by overexpression of Crb. Additionally, loss of retromer results in multilayering of the follicular epithelium, indicating that epithelial integrity is severely compromised. Our data reveal a mechanism for Crb trafficking by retromer that is vital for maintaining Crb levels and localization. We also show a novel function for retromer in maintaining epithelial cell polarity.

Development and characterisation of a neurogenic model of brain cancer

Primary glioblastoma (GB), the most common and aggressive adult brain tumour, is refractory to conventional therapies and characterised by poor prognosis. GB displays striking cellular heterogeneity, with a sub-population, called Glioblastoma Stem Cells (GSCs), intrinsically resistant to therapy, hence the high rate of recurrence. Alterations of the tumour suppressor gene PTEN are prevalent in primary GBM, resulting in the inhibition of the polarity protein Lgl1 due to aPKC hyperactivation. Dysregulation of this molecular axis is one of the mechanisms involved in GSC maintenance. After demonstrating that the PTEN/aPKC/Lgl axis is conserved in Drosophila, I deregulated it in different cells populations of the nervous system in order to individuate the cells at the root of neurogenic brain cancers. This analysis identified the type II neuroblasts (NBs) as the most sensitive to alterations of this molecular axis. Type II NBs are a sub-population of Drosophila stem cells displaying a lineage similar to that of the mammalian neural stem cells. Following aPKC activation in these stem cells, I obtained an adult brain cancer model in Drosophila that summarises many phenotypic traits of human brain tumours. Fly tumours are indeed characterised by accumulation of highly proliferative immature cells and keep growing in the adult leading the affected animals to premature death. With the aim to understand the role of cell polarity disruption in this tumorigenic process I carried out a molecular characterisation and transcriptome analysis of brain cancers from our fly model. In summary, the model I built and partially characterised in this thesis work may help deepen our knowledge on human brain cancers by investigating many different aspects of this complicate disease.