La voie de signalisation ERK/MAPK est connue pour son implication dans la progression du cycle cellulaire et dans le contrôle de la différenciation dans les cellules épithéliales intestinales. La phosphatase cytoplasmique DUSP6 a pour seuls substrats les kinases ERK1/2, kinases effectrices de la voie ERK/MAPK. Son rôle dans le contrôle des différents processus influencés par la signalisation ERK/MAPK n’a encore jamais été étudié dans l’épithélium intestinal et colique, malgré l’implication connue de cette voie de signalisation dans le contrôle de la prolifération et de la différenciation des cellules épithéliales intestinales. Pour étudier son rôle dans le maintien de l’homéostasie intestinale, un modèle de souris invalidée pour Dusp6 a été utilisé. De façon intéressante, une augmentation de la prolifération cryptale et un allongement des cryptes ont été observés dans le côlon de souris Dusp6-/- comparé aux souris Dusp6+/+. Cette augmentation de la prolifération cryptale permet notamment un développement plus rapide d’organoïdes de côlon démontrant ainsi une meilleure capacité de régénération épithéliale. De plus, une augmentation du nombre de cellules caliciformes a été observée dans le côlon des souris invalidées pour Dusp6. Une augmentation du nombre de cellules de Paneth et des cellules intermédiaires Paneth/caliciformes a été aussi observée dans l’iléon des souris Dusp6-/-. Cette augmentation de la prolifération, de la différenciation des cellules à mucus et de la capacité de régénération protège l’épithélium colique d’un stress induit par un traitement au dextran sulfate (DSS). Pour étudier l’implication de la phosphatase DUSP6 dans la tumorigenèse colorectale, un modèle de souris ApcMin/+;Dusp6-/- a été généré. De façon intéressante, ces souris ont développé plus de polypes dans l’intestin grêle et dans le côlon que les souris ApcMin/+;Dusp6+/+. De plus, l’expression de DUSP6 en ARNm a été analysée dans des tumeurs colorectales humaines et pairées avec une marge saine. De manière intéressante, le niveau des transcrits de DUSP6 est diminué dans les tumeurs colorectales et ce, de façon plus prononcée dans les stades avancés. Finalement, le rôle de DUSP6 dans différents processus associés à la carcinogenèse a été étudié dans des cellules cancéreuses colorectales sous-exprimant DUSP6 grâce à un ARN interférant. Une augmentation de l’activité ERK1/2, de la capacité de croissance en indépendance d’ancrage et de la capacité invasive a été observée chez les cellules HT29 sous-exprimant DUSP6, mais pas chez les HCT116. En conclusion, DUSP6 est impliqué dans le maintien de l’homéostasie grâce à son contrôle de la prolifération cryptale et de la différenciation cellulaire. De plus, en inhibant l’activité ERK1/2, cette phosphatase régularise négativement la tumorigenèse intestinale.

Multiple signaling pathways control cellular response to environmental cues such as nutrients, growth factors and stress. Interpretation of these cues requires coordinated regulation of intracellular signaling pathways. Our attempt to understand how cells coordinate different signaling pathways led to the discovery of two crosstalk mechanisms between different signaling cascades. We found that PI3K-AKT signaling reduces EGFR signaling to the parallel ERK-MAPK pathway by enhancing EGF induced EGFR degradation. At the molecular level AKT activates PIKfyve to facilitate EGFR trafficking from early endosomes to the lysosomes. Using a mass spectrometry based approach we also found growth factor signaling by EGF inhibits stress response. In particular, inhibiting RSK signaling downstream of EGF increased the activity of stress activated kinases p38, MSK2 and ERK5. We propose that when growth factors are present active RSK phosphorylates and inhibits a master regulator of stress response MEKK3, which leads to termination of MEKK3 signaling to downstream kinases. Our unbiased phosphoproteomic approach also lead to identification of many ERK and RSK substrates that will help us explain how growth factor signaling regulates a wide variety of biological processes.

Spinal muscle atrophy (SMA) is an autosomal recessive neurodegenerative disease which is characterized by the loss of α motor neurons from the anterior horn of the spinal cord, resulting in progressive muscle atrophy. The loss of functional Survival motor neuron (SMN) protein due to mutations or deletion in the SMN1 gene is the cause of SMA. A potential treatment strategy for SMA is to upregulate levels of the SMN protein originating from the copy gene SMN2 which can compensate in part for the absence of the functional SMN1 gene. I have shown a novel therapeutic strategy for SMA treatment through the activation of the p38 pathway by the bacterial antibiotic anisomycin which stabilizes and increases SMN mRNA levels in vitro. Activation of the p38 pathway by anisomycin leads to cytoplasmic accumulation of HuR protein which binds to the 3’UTR of SMN transcript resulting in increased SMN levels. This opens up a novel potential therapeutic strategy for SMA. I have also identified and demonstrated a significant induction of SMN protein levels in vitro and in vivo upon treatment with FDA approved drug celecoxib, which also activates the p38 pathway. Celecoxib mitigates disease severity along with increasing the lifespan of SMA mice. Sodium valproate, trichostatin A and aclarubicin, all agents which effectively enhance SMN2 expression, have been recently shown to activate STAT5 in SMA-like mouse embryonic fibroblasts and human SMN2-transfected NSC34 cells. Given that prolactin is also known to activate the STAT5 signalling pathway, can cross blood brain barrier and is FDA approved, we elected to assess its impact on SMN levels. In this manner, I have demonstrated a significant induction in SMN mRNA and protein levels in neuronal NT2 and MN-1 cells upon treatment with prolactin. I have also demonstrated that activation of the STAT5 pathway by prolactin is necessary for this transcriptional upregulation of the SMN gene. I have found that prolactin treatment induces SMN expression in brain and spinal cord samples and that it ameliorates the disease phenotype, improving motor neuron function and increasing survival in the SMA mouse model. Presently there is no cure for SMA. This study will help in the identification and characterization of potential therapeutic compounds for the treatment of SMA.

The binding of CD80/CD86 on the APC to CD28 on the T cell surface provides a second signal for T cell activation. While it was once believed that this interaction represented a one-way signal, resulting in T cell activation, recently, it has been investigated as a bidirectional signaling process. CD80/86 activation produces IL-6 in DCs, but its role in macrophage activation is unknown. Dysregulation of CD80/86 expression has been observed in autoimmune disorders and cancer, and may also influence the development of immune responses including production of cytokines in response to stimulation with TLR-4 ligand, LPS. Therefore, the focus of my project was twofold: 1) to investigate the role of CD80/86 as signaling receptors capable of transmitting extracellular signals, and 2) to determine the TLR-4 activated pathways that regulate CD80/86 expression in human monocyte-derived macrophages (MDMs). Since I demonstrated that activation of CD80/86 alone did not induce expression of the four cytokines investigated, I hypothesized that CD80/86 synergizes with other signaling pathways. I show for the first time that CD80/86 activation synergizes with TLR-4 signaling to produce IL-27 and IL-10 in human MDMs. Since cIAPs play a key role in TLR-4-mediated signaling, I investigated their role in TLR-4- and CD80/86-activated production of IL-10 and IL-27. Degradation of IAPs by SMAC mimetics inhibited LPS-induced IL-10 and IL-27 production in MDMs. However, it did not alter the TLR-4 and CD80/86 synergistic effect on IL-10 and IL-27 production suggesting that IAPs may not play a role in CD80/86 activation of macrophages. Since I have demonstrated this role for IAPs, I extended my studies by examining the involvement of IAPs and other upstream signaling molecules such as SHP-1, RIP1, TRAF2, in modulating the LPS-induced CD80/86 expression. I showed that cIAP2, SHP-1, RIP1, TRAF2 co-localize to form a complex that regulates the LPS-induced CD80 and CD86 expression through AKT-activated p38 MAPK in human macrophages. These findings may lead to the development of novel therapeutic interventions in the treatment of autoimmune diseases.

Mitogen-activated protein kinases (MAPKs) belong to the most conserved signaling pathways and are found in all eukaryotes, including humans where they play important roles in various diseases and cancer. Stimulation of this signal transduction pathway by microbe-associated molecular patterns (MAMP) results in a multitude of events to regulate innate immune responses in Arabidopsis thaliana stimulating large-scale changes in gene expression. Starting from a phosphoproteomic screen in Arabidopsis thaliana wild type and mpk3, mpk4 and mpk6 mutants following microbe-associated molecular pattern (MAMP) treatment, several novel chromatin-associated proteins were identified that are differentially phosphorylated by stress-induced protein kinases. Arabidopsis Ras GTPase-activating protein SH3-domain-binding protein (AtG3BP-1) is a downstream putative substrate of the MAMP-stimulated MAPK pathway that is phosphorylated by MPK3, 4 and 6 in in vitro kinase assays. AtG3BP1 belongs to a highly conserved family of RNA-binding proteins in eukaryotes that link kinase receptormediated signaling to RNA metabolism. Here, we report the characterization of the Arabidopsis homolog of human G3BP1 in plant innate immunity. AtG3BP1 negatively regulates plant immunity and defense immune responses. Atg3bp1 mutant lines show constitutive stomata closure, expression of a number of key defense marker genes, and accumulate salicylic acid but not jasmonic acid. Furthermore, Atg3bp1 plants exhibit enhanced resistance to the biotrophic pathogen Pseudomonas syringae pv. tomato. Pathogen resistance was mediated by stomatal and apoplastic immunity in Atg3bp1. More generally, our data reinforce that AtG3BP1 is a key mediator of plant defense responses and transient expression of AtG3BP1 delivered striking disease resistance in the absence of yield penalty, highlighting a potential application of this gene in crop protection.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23374号 / 医博第4743号 / 新制||医||1051(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 藤田 恭之, 教授 椛島 健治, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Stomata are cellular valves in plants that play an essential role in the regulation of gas exchange and are distributed in the epidermis of aerial organs. In Arabidopsis thaliana, stomatal production and development are coordinated by the mitogen-activated protein kinase (MAPK) signalling pathway, which modulates a variety of other processes, including cell proliferation, regulation of cytokinesis, programed cell death, and response to abiotic and biotic stress. The environment also plays a role in stomatal development, by influencing the frequency at which stomata develop in leaves. This thesis presents an analysis of stomatal development in Arabidopsis mutants in two MAPK pathways: MEKK1-MKK1/MKK2-MPK4, and MAP3K17/18-MKK3. Obtained results demonstrate the effect of stress conditions on stomatal development and specify the involvement of analysed MAPK in stomatal patterning. First, both analysed pathways modulate stomatal patterning in Arabidopsis cotyledons. Second, plant growth-promoting bacteria tested enhance stomatal density and affect guard cell morphology. Third, the sucrose or mannitol treatment increases defects in stomatal patterning. Finally, salt stress or high temperature can suppress stomatal defects in mutants of the MEKK1-MKK1/MKK2-MPK4 pathway.

The MAPK cascade is responsible for transmitting information in the cytoplasm of the cell and regulating important fate decisions like cell division and apoptosis. Due to scarce experimental data and limited knowledge about many complex biochemical processes, existing MAPK pathway models, which exhibit bistability, have a significant structural uncertainty. Often, small perturbations of network interactions or components can reduce the bistable region significantly or make it even disappear and small fluctuations of the input can make the system switch back, which reflects its low robustness. However, real biological systems have developed significant robustness through evolution and this robustness should be reflected by the models. The main goal of the present thesis is the development of a methodology for increasing the robustness of biochemical models, which exhibit bistability. Based on modifying existing network interactions or introducing new interactions to the system, several methods for both internal and external robustification are proposed. Internal robustness is addressed through a sensitivity analysis, which deals with a linearization of the model and can be used sequentially to introduce multiple modifications to the model. The methods for external robustness improvement are based on eigenvalue placement and slope modification (drawing on the linear model) and on the identification of feedback structures (nonlinear model). Further, a way to integrate static interaction changes to the nonlinear model, so that these perturbations have only a local impact on its behavior, is proposed. The application of the methods to existing MAPK models shows that, by introducing small modifications, the internal and external robustness of models can be increased significantly and thus provides knowledge about complex dynamics and interactions that play a key role for the inherent robustness of real biological systems. Furthermore, by employing a robustness analysis, stable steady-state branches can be recovered and bistability can be induced.

Palmitate (PA), one of the most prevalent saturated fatty acids, causes myocardial dysfunction. However, the mechanisms by which PA induces cell apoptosis and autophagy remain to be elucidated. We showed that autophagy was induced in an mTORC1-dependent way and played a protective role against PA-induced apoptosis, which was verified by pretreatment with 3-methyladenine (3MA) and rapamycin. However, p62 began to accumulate after 18 h treatment with PA, suggesting prolonged exposure to PA lead to an impairment of autophagic flux. PA enhanced ROS production as well as activated p38-mitogen-activated protein kinase (p38 MAPK) and c-jun NH2 terminal kinases (JNKs). The antioxidant N-Acety-L-Cysteine (NAC) was found to attenuate the JNK and p38 MAPK activation with a concomitant reduction of PA-induced autophagy and apoptosis. Furthermore, both JNK and p38 MAPK inhibitors were shown to directly abrogate caspase 7 cleavage as well as the conversion of LC3BI to LC3BII. Thus, we demonstrate that PA stimulates autophagy and apoptosis via ROS-dependent JNK and p38 MAPK pathways.

Valproic acid (VPA), a widely prescribed drug for seizures and bipolar disorder, induces apoptosis in microglia, but the underlying mechanism by which microglia apoptosis in response to VPA is not yet known. In this study, we found that the mitochondrial pathway played an important role in VPA-induced apoptosis in both BV-2 microglia and mouse primary microglial cells. In addition, VPA increased the level of phospho-p38 mitogen-activated protein kinase (MAPK), but had no effects on phospho-ERK and phospho-JNK MAPKs. Moreover, p38 inhibitor SB203580 strongly inhibited VPA-induced apoptosis and caspase-3 activation. Taken together, our results clearly demonstrated that VPA could induce apoptosis of microglia via p38 MAPK and mitochondrial apoptosis pathway.