Depuis leur découverte, les glycogènes synthase kinases 3 (GSK3) ont été associées à de multiples fonctions cellulaires, incluant notamment la prolifération et la survie cellulaire. La délétion homozygote de GSK3? chez les souris est létale au stade embryonnaire E13,5 et il a été démontré que l'apoptose massive des hépatocytes due à une hypersensibilité au TNF? serait la principale cause de la létalité suggérant que l'activité de GSK3? est nécessaire pour la survie cellulaire. Plusieurs études suggèrent une activité des GSK3 plus élevée dans certains types de cancers, particulièrement au niveau des adénocarcinomes pancréatiques. Différentes études proposent que les GSK3 pourraient réguler la voie NF?B et influencer la prolifération et la survie des cellules pancréatiques tumorales. Mes travaux de maîtrise visaient à déterminer le rôle des GSK3 dans la survie et la prolifération des cellules pancréatiques tumorales humaines. Dans un premier temps, l'inhibition prolongée (24 à 72h) de l'activité des GSK3 a permis d'observer une diminution de l'activité métabolique des cellules pancréatiques tumorales humaines : MIA PaCa2 et PANC-1. Cette diminution semble être due en partie à une induction de l'apoptose et à une inhibition de la prolifération cellulaire. En effet, l'inhibition des GSK3 induit l'apoptose chez les cellules pancréatiques tumorales humaines (MIA PaCa2, PANC-1 et BxPC3) après 48 et 72h, mais non chez les cellules d'origine non-tumorale (HPDE6 et HEK293T). L'étude plus approfondie du mécanisme moléculaire de l'apoptose induit suite à l'inhibition des GSK3 par l'inhibiteur pharmacologique spécifique SB216763 démontre 1- l'activation des caspases 3 et 7, 2- l'augmentation de l'homologue BCL-2 pro-apoptotique BIM et 3- la diminution de l'homologue anti-apoptotique BCL-2. L'analyse des voies de signalisation associées à la survie cellulaire et à l'apoptose suite à l'inhibition des GSK3 a permis d'observer l'activation de la voie JNK-c-Jun qui contribuerait potentiellement à l'induction de l'apoptose. À cet égard, nous avons observé que la voie JNK est nécessaire 1- au clivage de PARP, 2- à l'activation de la caspase 7 et 3- à l'augmentation de BIM induit par l'inhibition des GSK3. De plus, la diminution de l'expression de c-Jun, une cible des JNK, à l'aide d'un siARN prévient la modulation de BIM par l'inhibition des GSK3, alors que l'induction de l'apoptose n'est pas bloquée. Donc, l'activation de la voie JNK-c-Jun semble également nécessaire à la régulation de l'homologue BCL-2 proapoptotique BIM. Nous avons ensuite observé que la voie MEKK1-JNK-c-Jun semble insuffisante pour induire l'apoptose des cellules PANC-1, suggérant que l'activation de la voie JNK pourrait requérir la régulation de d'autres voies afin d'induire l'apoptose. Dans un deuxième temps, nous avons observé que l'inhibition des GSK3 pendant 16h inhibe la prolifération des cellules pancréatiques tumorales humaines. En effet, nous avons observé que l'inhibition des GSK3 pendant 16h empêche l'atteinte du point de restriction en phase G1 du cycle cellulaire. Nous avons observé qu'en réponse au sérum les GSK3 sont inactivées de façon transitoire (0- 10h) avant d'être réactivées (10-16h). L'inhibition de l'activité des GSK3 lors de leur réactivation (10-16h) par le SB216763 prévient 1- l'hyperphosphorylation de pRb et de p130 et 2- l'augmentation d'E2F1 induites par le sérum et 3- diminue l'expression d'E2F4, suggérant un rôle des GSK3 dans la phase G1 tardive du cycle cellulaire. Également, nous avons observé la présence de complexes formés de GSK3-pRb et de GSK3?-E2F4 dans les cellules pancréatiques tumorales humaines. Ces observations suggèrent que l'activité des GSK3 pourrait contribuer à l'hyperphosphorylation de pRb et de p130 et pourrait également réguler directement l'activité des E2F. Bref, ces résultats suggèrent que l'activité des GSK3 est essentielle pour la survie et la prolifération des cellules pancréatiques tumorales humaines. Les auteurs suggèrent donc que l'inhibition des GSK3 pourrait s'avérer un traitement potentiel du cancer pancréatique, en ciblant les cellules cancéreuses. [Symboles non conformes]

v-Rel is the acutely oncogenic member of the NF-[kappa]B family of transcription factors and transforms cells through the altered regulation of pathways normally controlled by cellular NF-[kappa]B. Initial studies revealed that expression of v-Rel results in the strong and sustained activation of the ERK and JNK MAP kinases. This induction is critical for the v-Rel transformed phenotype, as suppression of MAPK activity with chemical inhibitors or siRNA severely limited colony formation of v-Rel transformed cell lines of hematopoietic origin. However, signaling must be maintained within a certain range in these cells, as strong additional activation of either pathway through expression of constitutively active MKK mutants also attenuated the transformed phenotype. Studies in primary spleen cells revealed that MAPK signaling is also required for the early stages of v-Rel-mediated transformation. However, constitutive MAPK activity further enhanced the transformation efficiency of v-Rel in primary cells. These studies, as well as analogous experiments in DT40 cells, indicate distinct requirements for MAPK activity at different stages of v-Rel-mediated transformation. The proto-oncoprotein, c-Rel, only weakly activates ERK and JNK signaling compared to v-Rel. Importantly, elevated MAPK activity enhanced transformation by c-Rel, indicating that the ability of v-Rel to induce MAPK signaling is a major contributor to its oncogenic potential. Taken together, this work demonstrates an important role for ERK and JNK activity in transformation by v-Rel. Additional studies examined mechanisms through which MAPK activity is regulated in v-Rel transformed cells. Feedback regulation of the ERK activator, MKK1, at T292 was shown to limit ERK activation in v-Rel transformed cells, preventing the detrimental effects of constitutive activity. This result is the first indication that this regulation may have a role in the maintenance of transformation. Further, several v-Rel induced cytokines were identified that activate ERK and JNK signaling in v-Rel transformed cells, revealing one means by which v-Rel-dependent transcriptional changes lead to MAPK activation. These studies demonstrate the integration of multiple mechanisms in achieving the optimal levels of MAPK activity that are essential for v-Rel-mediated transformation. / text

Aberrant JNK signaling can result in two main forms of disease in humans: 1) neurological, coronary, hepatobiliary, and respiratory diseases and 2) autoimmune, inflammatory, and cancer conditions. Enantiomers of the lignan zuonin A, (-)-zuonin A and (+)-zuonin A, have been shown to bind to JNK isoforms with similar affinity and disrupt protein-protein interactions at JNK's D-recruitment site, making them a good candidate for specific non-ATP competitive inhibitors. However, (-)-zuonin A inhibits 80% of JNK catalyzed reactions at saturating levels, while (+)-zuonin A only inhibits 15%. Molecular docking and molecular dynamics simulations were performed to gain a better understanding of how these inhibitors interact JNK. The results of this study provide an alternative binding mode for (-)-zuonin A, compared to one proposed in a previous study, that shows (-)-zuonin A interacting with JNK via an induced fit mechanism by forming a larger pocket for itself near the highly conserved [phi]A-X-[phi]B recognition site, a dynamic move not seen in (+)-zuonin A simulations, and may help explain their different inhibition patterns. / text

We hypothesized that with exercise training and the subsequent attenuation of hyperglycemia, β-cell adaptation to worsening insulin resistance would be maintained. Also, because classical stress-activated systems and oxidative stress are involved in hepatic insulin resistance we examined if exercise would be associated with improvements in hepatic markers of oxidative stress and inflammation. Exercise maintained fasted hyperinsulinemia and preserved normoglycemia in male Zucker diabetic fatty (ZDF) rats. β-cell function calculations indicate prolonged β-cell adaptation in exercised animals. Such improved β-cell function was associated with increased β-cell mass. Hypertrophy and replication contributed to expansion of β-cell mass; exercised animals had increased β-cell size and bromodeoxyuridine (BrdU) incorporation rates versus controls. Furthermore, we observed augmented β-cell-specific immunohistochemical staining of GLUT2 and Akt/PKB in exercised versus sedentary controls. We also observed large cytoplasmic ubiquitinated structures which form in response to oxidative stress in pancreatic tissue samples from hyperglycemic ZDF rats. In the exercised groups such aggregate numbers were reduced to numbers compared to those seen in younger non-diabetic basal ZDF animals and age-matched lean Zucker rats. With respect to the liver we investigated whether exercise alters kinases such as c-Jun NH2-terminal kinase (JNK) and IKKβ (as evidenced by IκBα levels) and related insulin receptor substrate-1 (IRS-1) serine phosphorylation which are associated with hepatic insulin resistance in obesity. On average, exercised animals ran 5250m/day which improved insulin sensitivity based on the homeostasis model assessment for insulin resistance (HOMA-IR) calculations, and maintained fed and fasted glucoregulation and glucose tolerance. Ten weeks of running decreased whole-body markers of inflammation and oxidative stress in the blood and in the liver. Exercise lowered circulating interleukin-6 (IL-6), haptoglobin, malondialdehyde (MDA) levels, and protein oxidation in the liver. Exercise reduced phosphorylated JNK (pJNK) indicating decreased JNK activity; in accordance serine phosphorylated IRS-1 was reduced in exercised rats. In conclusion, improvements in glucoregulation were associated with increased β-cell compensation at least in part due to a reduction in oxidative stress. Furthermore, we show exercise attenuates development of hyperglycemia in ZDF rats in association with decreases in plasma and hepatic markers of inflammation, oxidative stress, JNK activation, and serine phosphorylation of IRS-1.

Plusieurs études ont suggéré une implication des glycogène synthase kinases 3 (GSK3) dans la carcinogenèse, notamment du pancréas. Des études ont rapporté des résultats contradictoires quant à l’impact des GSK3 sur la survie cellulaire. Au niveau du pancréas, il a été observé que l’inhibition des GSK3 inhibe la croissance entre autres via la régulation de la voie JNK ou NFkB. Les inhibiteurs des GSK3 sont présentement à l’étude comme traitement de différentes pathologies, notamment pour le cancer pancréatique. Une meilleure compréhension des voies de signalisation régulées par les GSK3 sera donc nécessaire. Nous avons entrepris ces travaux afin de mieux comprendre les mécanismes impliqués dans la régulation de la survie des cellules pancréatiques tumorales par les GSK3. Nous avons démontré que l’inhibition des GSK3 induit l’apoptose et l’autophagie dans les cellules pancréatiques tumorales humaines. L’inhibition des GSK3 stimule l’autophagie autant dans les cellules pancréatiques tumorales que non tumorales, alors que l’apoptose est induite spécifiquement dans les cellules tumorales. Contrairement à l’apoptose, l’autophagie est induite indépendamment de la voie JNK-cJUN suite à l’inhibition des GSK3. Nos résultats démontrent que l’inhibition des GSK3 mène à l’inactivation de la voie mTORC1 qui pourrait contribuer à l’induction de l’autophagie. D’autre part, nos travaux ont démontré pour la première fois que les GSK3 régulent le facteur de transcription EB (TFEB) dans les cellules pancréatiques tumorales. En effet, l’inhibition des GSK3 entraîne la déphosphorylation de TFEB, notamment sur la Ser211, la dissociation des 14-3- 3 et sa translocation nucléaire. Nos résultats suggèrent que la régulation de TFEB par les GSK3 impliquerait des Ser/Thr phosphatases et pourrait être indépendante de l’activité mTORC1. L’inhibition de l’autophagie ou la déplétion de l’expression de TFEB sensibilise les cellules pancréatiques tumorales à l’apoptose induite suite à l’inhibition des GSK3 suggérant un rôle pro-survie de l’autophagie et de TFEB dans ces cellules. Enfin, l’inhibition des GSK3 semble mener à l’inhibition de la glycolyse qui contribuerait à l’induction de l’apoptose. En résumé, nos résultats démontrent que l’inhibition des GSK3 induit à la fois des signaux pro-apoptotiques et pro-survie suggérant que l’équilibre entre ces signaux dicterait l’impact des GSK3 sur la survie des cellules pancréatiques tumorales humaines.

Membrane depolarization is an effective neurotrophic stimulus, with its trophic effect on spiral ganglion neurons (SGNs) even surpassing that of neurotrophins. Thus, SGN cultures are a favorable system to investigate pro-survival signal transduction downstream of depolarization. Depolarization promotes SGN survival by recruiting three distinct kinase pathways: cyclic AMP-dependent protein kinase (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII) and CaMKIV. CaMKIV mediates the pro-survival effect of depolarization by activating CREB in nucleus. However, the mechanisms by which PKA and CaMKII promote survival are still not clear. By targeting constitutively active PKA or a PKA inhibitor (PKI) to the outer mitochondrial membrane (OMM), we showed that PKA activity at the OMM is sufficient to support SGN survival in the absence of other trophic factors and necessary for cAMP-dependent SGN survival. It has been suggested that PKA can promote survival by inactivating pro-apoptotic protein Bad. By cotransfection of SGNs with OMM-PKA and wild-type Bad, we showed that this was the case. We further showed that Ser112 and Ser136 in Bad, but not Ser155, a hypothetical PKA target, were necessary for functional inactivation of Bad by PKA. CaMKII mediates the third depolarization-dependent pro-survival pathway. A specific pro-survival target for CaMKII was identified through a separate investigation of the pro-apoptotic JNK-Jun signaling pathway, which we had identified as active in apoptotic SGNs in vivo. By measuring anti-phosphoJun immunofluorescence, we could quantify JNK-Jun activation in SGNs under different conditions. We showed that JNK inhibition or genetic deletion of JNK3 reduces SGN death after neurotrophic factor withdrawal. Neurotrophins have been shown to suppress JNK activation via their receptor protein tyrosine kinases (PTKs). By expressing constitutively active and dominant negative forms of candidate protein kinases, we identified a novel signaling pathway linking depolarization to JNK: Ca2+ entry - CaMKII - FAK/Pyk2 - PI-3-OH Kinase - Protein Kinase B - inhibition of MLKs (upstream activators of JNK). Thus, depolarization also recruits PTKs - the nonreceptor PTKs FAK and Pyk2 - to suppress JNK activation, implying a conserved PTK-PI3K-PKB pathway for suppression of pro-apoptotic JNK activation by neurotrophic stimuli.

The desmosome is one of the major cell adhesion junctions found in the epithelia, heart, and hair follicle. Described as a “rivet” that hold cells together, it provides these tissues with the integrity to withstand the tremendous forces they face in everyday life. Defects in this junction can lead to devastating diseases where patients are susceptible to skin infections and cardiovascular defects. Limited treatments exist for diseases of the desmosome, and strategies do not target all symptoms. Therefore, delineating the function and regulation of desmosomes is of paramount importance for the development of prevention and treatment strategies. The Xenopus laevis has been utilized for the study of embryonic development and tissue movements. This study takes advantage of the frog model to study a key desmosomal protein, desmoplakin (Dsp), in the epidermal development of the embryo. First, Xenopus embryonic epidermis has junctional desmosomes as early as the blastula stages. Desmosomes numbers per junction increase as the embryo develops. Dsp is present in many epidermally-derived structures in the embryo at varying levels. Xenopus embryos deficient in desmoplakin have phenotypic defects in epidermal structures and the heart, mimicking mammalian models. Embryos with reduced Dsp exhibit an increased susceptibility to epidermal damage under applied mechanical forces. Assays also reveal a potential role for desmosomes in radial intercalation, a process through which cells move from the inner to the outer epidermal layers. Embryos with reduced Dsp exhibit a slight reduction in intercalation and defects in intercalating cell types, including multiciliated cells and small secretory cells. Finally, c-Jun N-terminal kinase (JNK) may have a potential role in the regulation of desmosome assembly and adhesion. Embryos with deficient Dsp display a partial recovery of mechanical integrity when treated with a JNK inhibitor.

Patients with type 2 diabetes have reduced gene expression of Heat Shock Protein (HSP) 72 which correlates with reduced insulin sensitivity. Heat therapy, which activates HSP72, improves clinical parameters in these patients. Activation of several inflammatory signalling proteins such as c-jun amino terminal kinase (JNK) can induce insulin resistance but HSP72 can block the induction of these molecules in vitro. Whether up-regulation of HSP72 can protect against insulin resistance is not known. In experiments reported in this thesis we show that HSP72 protects against insulin resistance and blocks the activation of JNK in vivo. We first show that mice that underwent weekly heat shock therapy to increase intramuscular HSP72 protein expression were protected from high fat diet (HFD)-induced hyperinsulinemia, hyperglycemia and glucose intolerance, factors associated with reduced JNK phosphorylation. To determine whether the elevation in intramuscular HSP72 expressio n and protection from insulin resistance are causally linked, we studied muscle specific HSP72 overexpression mice (HSP72+/+). Compared with wild-type mice, HSP72+/+ mice were protected from hyperglycemia, hyperinsulinemia, glucose intolerance and insulin resistance when placed on a HFD, factors associated with a complete inhibition of HFD-induced JNK phosphorylation in skeletal muscle. Finally, we show that HSP72+/+ mice display greater mitochondrial enzyme activity in the liver, adipose tissue and skeletal muscle, corresponding to reduced plasma free fatty acid levels, white adipose tissue mass and alterations in circulating adipokines. These data identify HSP72 as being pivotal in protecting against obesity-induced insulin resistance possibly by blocking JNK and/or by up-regulation of mitochondrial oxidative capacity.

This study explored angiotensin IV’s (Ang IV) affects on the signaling pathways involved in the development of insulin resistance in 3T3-L1 adipocytes. Ang IV, working through the AT4 receptor, interferes with insulin signaling through the blockade of the phosphatidylinositol 3-kinases (PI3K)/Akt pathway and through activating mitogen activated protein kinases (MAPK): extracellular signal regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK) which are known to impair insulin receptor substrate-1 (IRS-1) signaling. The expression of AT4 receptors was confirmed by reverse transcription polymerase chain reaction. Ang IV’s effects were found by treating adipocytes with combinations of Ang IV, AT4 receptor inhibitor Norleual, and insulin. Cell lysates were resolved by SDS-PAGE electrophoresis and immunoblotted. Ang IV down-regulated the PI3K/Akt pathway. Insulin exerts its effects on adipocytes by activating this pathway, phosphorylating Akt (S473 and T308) residues. Pre-treatment with Ang IV blocked insulin’s effects, reversing Akt activation. Addition of Norleual blocked Ang IV’s inhibitory actions, leading to the phosphorylation of Akt residues. Studies show elevated MAPK levels produced by angiotensin peptides catalyze the phosphorylation of serine residues on IRS-1, impairing insulin signal transduction. Ang IV increased the activation of ERK 1/2 and JNK. This was reversed by pretreatment with Norleual. Ang IV’s effects on IRS-1 residues were found by immunoprecipitation of IRS-1 followed by SDS-PAGE immunoblotting. Ang IV increased the phosphorylation of IRS-1(S307 and S612). Pre-incubation with Norleual attenuated Ang IV’s effects. Ang IV’s stimulation of adipocytes quickly caused the phosphorylation of MAPK corresponding to serine residue phosphorylation on IRS-1, which may implicate Ang IV activation of MAPK in the development of insulin resistance. Ang IV is involved in the down-regulation of the insulin-signaling cascade by inhibiting insulin’s phosphorylation on Akt (S473 and T308). Ang IV increased phosphorylation of ERK 1/2 and JNK, corresponding with increases in serine phosphorylation of IRS-1. Pre-treatment with Norleual inhibited Ang IV’s negative effects on insulin signaling. This study elucidates a new mechanism that may lead to the development of insulin resistance in adipose tissue.

Ovarian cancer is the most lethal gynecological malignancy. Most of ovarian cancer patients relapse and subsequently die due to the development of resistance to chemotherapy. P73 belongs to the tumor suppressor p53 family. Like p53, the transcriptionally active TAp73 can bind specifically to p53 responsive elements and transactivates some of the p53 target genes, and finally leads to cell cycle arrest and apoptosis. TAp73 can be induced by DNA damage to enhance cellular sensitivity to anticancer agents in human cancer cells. However, the functions of TAp73 in ovarian cancer cells and the role in the regulation of cellular response to commonly used chemotherapeutic agents cisplatin are still poorly understood. The aims of this study were to examine the functions of TAp73 in ovarian cancer cells and its role in cellular response to cisplatin, as well as the relationship between TAp73 and p53 in ovarian cancer cells. Functional studies showed that over-expression of TAp73alpha (TAp73α) inhibited cell proliferation, colony formation ability and anchorage-independent growth of ovarian cancer cells, and this was irrespective of p53 expression status. In addition, TAp73α inhibited cell growth by arresting cell cycle at G2/M phase and up-regulating the expressions of G2/M regulators of p21, 14-3-3sigma and GADD45α. TAp73α enhanced the cellular sensitivity to cisplatin through the activation of JNK signaling pathway, at least partially, in ovarian cancer cells. TAp73α activated the JNK pathway through the up-regulation of its target gene GADD45α and subsequent activation of MKK4, the JNK up-stream kinase. Inhibition of JNK activity by a specific inhibitor (SP600125) or small interfering RNAs (siRNAs) significantly abrogated TAp73-mediated apoptosis induced by cisplatin. Moreover, the activations of MKK4, JNK and c-Jun were abolished when GADD45α was knocked down by siRNAs, and the JNK-dependent apoptosis was not observed. Collectively, these results supported that TAp73α was able to mediate apoptotic response to cisplatin through the GADD45α/MKK4/JNK signaling pathway, which was respective of p53 expression status. Further investigation on the relationship between TAp73α and p53 demonstrated that TAp73α increased p53 protein, but not mRNA expression by attenuating p53 protein degradation in wild-type p53 ovarian cancer cells. TAp73α could directly interact with p53 protein, which might interfere with the binding ability of MDM2 to p53, and consequently block the p53 protein degradation. In addition, TAp73α inactivated the Akt and ERK pathways and activated the p38 pathway in response to cisplatin in wild-type p53 OVCA433, but not in null-p53 SKOV3 cells, suggesting that the effect of TAp73α on these pathways might be p53-dependent. These results indicated that a functional cooperation of TAp73α and p53, to some extent, existed in ovarian cancer cells. In conclusion, this study demonstrated that TAp73α acted as a tumor suppressor in ovarian carcinogenesis. It promoted the cellular sensitivity to cisplatin via, at least partially, the activation of JNK signaling pathway. These TAp73α functions were irrespective of p53 expression. In addition, TAp73α was able to bind to p53 and increase p53 expression. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy