Insulin and cellular stressors both activate p38 MAPK. Insulin protects cardiac tissue in a p38 MAPK-dependent manner. Paradoxically, inhibiting p38 MAPK is also protective. Hsp27 phosphorylation is regulated by p38 MAPK. Insulin was tested in H9c2 cardiomyocytes subjected to media exchange, 6 hours of oxygen-glucose deprivation, and reoxygenation. Insulin suppressed stress-induced phosphorylation of Hsp27 due to media-exchange or oxygen-glucose deprivation. Surprisingly, insulin increased Hsp27 phosphorylation during reoxygenation. Insulin also reduced total p38 MAPK levels. Insulin before oxygen-glucose deprivation prevented both localization of Hsp27 to the nucleus and localization of phospho-p38 MAPK to the cytoplasm. Insulin during oxygen-glucose deprivation caused the localization of phospho-p38 MAPK in the cytoplasm, but did not increase Hsp27 phosphorylation until reoxygenation. In conclusion, insulin may protect before oxygen-glucose deprivation by redirecting phospho-p38 MAPK to the nucleus away from damaging pathways in the cytoplasm and protects during oxygen-glucose deprivation by priming phospho-p38 MAPK to phosphorylate Hsp27. / Insulin was used on a model on H9c2 myotubes to determine the effect of oxygen-glucose deprivation and reoxygenation on the localization and phosphorylation of Hsp27 and p38 MAPK

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Normal cellular behaviour in multicellular organisms is achieved by tight control of signaling pathway networks. The mitogen-activated protein kinase (MAPK) signaling cascade is one of these signaling networks, that when deregulated can lead to cellular transformation. Activation of the RAS-RAF-MEK-MAPK (ERK) signal transduction pathway or the SAPK2/p38 pathway results in the activation of mitogen- and stress-activated protein kinases 1 and 2 (MSK1/2). Subsequently, MSKs go on to phosphorylate histone H3 at Ser10 and Ser28.Here, we demonstrate that the activities of ERK and MSK1, but not p38, are elevated in Hras-transformed cells (Ciras-3) relative to these activities in the parental 10T1⁄2 cells. Analyses of the subcellular distribution of MSK1 showed that the H3 kinase was similarly distributed in Ciras-3 and 10T1/2 cells, with most MSK1 being present in the nucleus. In contrast to many other chromatin modifying enzymes, MSK1 was loosely bound in the nucleus and was not a component of the nuclear matrix. Our results provide evidence that oncogene-mediated activation of the RAS-MAPK signal transduction pathway elevates the activity of MSK1, resulting in the increased steady-state levels of phosphorylated H3, which may contribute to the chromatin decondensation and aberrant gene expression observed in oncogene-transformed cells. Furthermore, upon activation of the ERK and p38 MAPK pathways, the MSK1/2- mediated nucleosomal response, including H3 phosphorylation at serine 28 or 10, is coupled with the induction of immediate-early gene transcription. The outcome of this response, varying with the stimuli and cellular contexts, ranges from neoplastic transformation to neuronal synaptic plasticity. Here, we used sequential co-immunoprecipitation assays and chromatin immunoprecipitation (ChIP) assays on mouse fibroblast 10T1/2, Ciras-3 and MSK1 knockdown 10T1/2 cells to show that H3 serine 28 and 10 phosphorylation leads to promoter remodeling. MSK1, in complexes with phospho-serine adaptor 14-3-3 proteins and BRG1 (the ATPase subunit of the SWI/SNF remodeler) is recruited to the promoter of target genes by transcription factors such as ELK-1 or NFκB. Following MSK1-mediated H3 phosphorylation, BRG1 associates with the promoter of target genes via 14-3-3 proteins, which act as scaffolds. The recruited SWI/SNF remodels nucleosomes at the promoter of immediate-early genes enabling the binding of transcription factors like JUN and the onset of transcription. Since RAS-MAPK activated MSKs mediate H3 phosphorylation that is required for expression of various immediate-early gene products involved in cellular transformation, inhibition of MSK activity may be a therapeutic target that could be exploited in cancers with upregulated RAS-MAPK signaling.

Le neutrophile humain est une composante essentielle du système immunitaire inné. Il joue un rôle-clé comme phagocyte professionnel pour la défense contre les agents externes. De plus, il a la capacité de libérer un large éventail de produits antimicrobiens et de produire également diverses protéines immunorégulatrices, dont une vaste gamme de cytokines (IL-8, MIP-1[alpha]/[beta], IP-10, I-TAC, TNF-[alpha], etc.). La génération de ces dernières permet le recrutement massif de neutrophiles et d'autres populations leucocytaires au site inflammatoire, contribuant ainsi au bon déroulement de la réponse inflammatoire. La génération de cytokines par le neutrophile humain est induite par différents agonistes, dont des molécules bactériennes (LPS, peptides N-formylés) ou les médiateurs inflammatoires (cytokines, chimiokines, facteurs de croissance). Ces molécules vont activer des récepteurs à la surface du neutrophile et déclancher ainsi plusieurs voies de signalisation et des facteurs transcriptionnels. Dans la présente étude, nous avons déterminé l'impact de la voie de signalisation MEK/ERK dans l'induction de l'expression des cytokines chez le neutrophile humain isolé du sang périphérique. Nous avons noté un découplage du module MEK/ERK suite à une stimulation avec certains agonistes pro-inflammatoires (LPS, TNF-[alpha]), mais par pour d'autres (fMLP, GM-CSF). L'utilisation de différentes classes d'agonistes et d'inhibiteurs pharmacologiques des voies de signalisation nous a permis de mettre en évidence les rôles différents de MEK et de ERK en ce qui concerne la sécrétion et la transcription de cytokines. Les kinases ERK et MEK sont toutes deux impliquées dans la sécrétion de cytokines, mais ERK est la seule des deux qui est associée à la transcription. Par contre, nous n'avons toujours pas identifié la kinase responsable de l'activation de ERK lorsque le module MEK/ERK est découplé. Enfin, à défaut d'identifier la kinase qui phosphoryle ERK, nous montrons que la MAP3K, TAK1, agit en amont de ERK et de MEK chez les neutrophiles. Nos résultats suggèrent que les thérapies basées sur l'inhibition de MEK devront être complémentées d'une inhibition de ERK, en particulier dans des maladies inflammatoires chroniques à forte prédominance neutrophilique.

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.

Normal cellular behaviour in multicellular organisms is achieved by tight control of signaling pathway networks. The mitogen-activated protein kinase (MAPK) signaling cascade is one of these signaling networks, that when deregulated can lead to cellular transformation. Activation of the RAS-RAF-MEK-MAPK (ERK) signal transduction pathway or the SAPK2/p38 pathway results in the activation of mitogen- and stress-activated protein kinases 1 and 2 (MSK1/2). Subsequently, MSKs go on to phosphorylate histone H3 at Ser10 and Ser28.Here, we demonstrate that the activities of ERK and MSK1, but not p38, are elevated in Hras-transformed cells (Ciras-3) relative to these activities in the parental 10T1⁄2 cells. Analyses of the subcellular distribution of MSK1 showed that the H3 kinase was similarly distributed in Ciras-3 and 10T1/2 cells, with most MSK1 being present in the nucleus. In contrast to many other chromatin modifying enzymes, MSK1 was loosely bound in the nucleus and was not a component of the nuclear matrix. Our results provide evidence that oncogene-mediated activation of the RAS-MAPK signal transduction pathway elevates the activity of MSK1, resulting in the increased steady-state levels of phosphorylated H3, which may contribute to the chromatin decondensation and aberrant gene expression observed in oncogene-transformed cells. Furthermore, upon activation of the ERK and p38 MAPK pathways, the MSK1/2- mediated nucleosomal response, including H3 phosphorylation at serine 28 or 10, is coupled with the induction of immediate-early gene transcription. The outcome of this response, varying with the stimuli and cellular contexts, ranges from neoplastic transformation to neuronal synaptic plasticity. Here, we used sequential co-immunoprecipitation assays and chromatin immunoprecipitation (ChIP) assays on mouse fibroblast 10T1/2, Ciras-3 and MSK1 knockdown 10T1/2 cells to show that H3 serine 28 and 10 phosphorylation leads to promoter remodeling. MSK1, in complexes with phospho-serine adaptor 14-3-3 proteins and BRG1 (the ATPase subunit of the SWI/SNF remodeler) is recruited to the promoter of target genes by transcription factors such as ELK-1 or NFκB. Following MSK1-mediated H3 phosphorylation, BRG1 associates with the promoter of target genes via 14-3-3 proteins, which act as scaffolds. The recruited SWI/SNF remodels nucleosomes at the promoter of immediate-early genes enabling the binding of transcription factors like JUN and the onset of transcription. Since RAS-MAPK activated MSKs mediate H3 phosphorylation that is required for expression of various immediate-early gene products involved in cellular transformation, inhibition of MSK activity may be a therapeutic target that could be exploited in cancers with upregulated RAS-MAPK signaling.

Prion diseases are neurodegenerative disorders that can affect humans and animals. The underlying event is a conformational change of the normal cellular prion protein (PrPC) into an aberrant isoform termed PrP-scrapie (PrPSc). PrPSc is thought to lead to neurodegeneration and activation of glial cells. Scrapie infection of neuroblastoma cells was shown to increase the expression of insulin receptor (IR). Additionally, a marked reduction of 125I-insulin binding sites was observed. Insulin stimulation showed alteration in both IR β-subunit tyrosine phosphorylation and extracellular signal regulated kinase-2 (ERK2) activity.  Furthermore, scrapie infection was shown to increase insulin-like growth factor-1(IGF-1) receptor (IGF-1R) expression, although the number of 125I-IGF-1-binding sites was reduced. Also binding affinity of 125I-IGF-1 to its receptor was reduced, and tyrosine phosphorylation of IGF-1R-β-subunit in response to IGF-1 was altered. The increased levels of neurotrophic receptors might represent a neuroprotective response to prion infection. However, scrapie infection instead leads to decreased function, decreased levels of functional receptors, or both, which could promote neurodegeneration in prion diseases, through attenuated neurotrophic support. In BV-2 microglial cells, LPS-induced iNOS (inducible nitric oxide synthase) expression and subsequent NO production were mainly mediated through c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathway. Antioxidant treatment indicates that oxidative suppressing mechanism(s) acts on JNK pathway possibly as a regulatory mechanism controlling the NO levels. The JNK pathway was also shown to play an important role in the survival of BV-2 cells. We show that BV-2 cells are protected from ongoing apoptosis by pro-survival activity mediated both by the JNK and p38 MAPK pathway during LPS-induced inflammation. This is very interesting findings since it is important for microglia to respond properly to a pathogen, without themselves being affected and undergo apoptosis.

The filamentous fungus Magnaporthe oryzae is a devastating pathogen of cultivated rice. M. oryzae elaborates a pressurized dome-shaped infection structure, called the appressorium, which physically ruptures the cuticle and gains entry into host tissue. Intracellular invasive hyphae invade neighbouring host cells through plasmodesmata. The Pmk1 MAPK cascade is well known for its roles in regulating the formation and function of the appressorium. Interestingly, ∆pmk1 mutants cannot infect host plant tissue through wounds, suggesting a role in invasive growth. Here, I define biological functions of the Pmk1 MAPK at various stages of the life cycle, by using a controllable version of Pmk1 that is specifically inhibited by a cell-permeable compound without disturbing other wild-type kinases. The Pmk1 MAPK signalling regulates morphogenesis of narrow invasive hyphae traversing the host cell wall, and modulates production of several putative secreted effectors, providing a direct link between the signalling cascade and effector-driven host immune suppression. These results indicate that the Pmk1 pathway is a central regulator of infection-related development necessary for many stages of plant infection including appressorium development, plant penetration, and importantly tissue colonisation. I also report the role of cell cycle progression in the development of plant infection structure. By using two novel conditional mutants that arrest in S and G2 phases, I defined that S-phase progression is crucial for appressorium-mediated plant penetration.

No description available.

abstract: The Erk/MAPK pathway plays a major role in cell growth, differentiation, and survival. Genetic mutations that cause dysregulation in this pathway can result in the development of Rasopathies, a group of several different syndromes including Noonan Syndrome, Costello Syndrome, and Neurofibromatosis Type-1. Since these mutations are germline and affect all cell types it is hard to differentiate the role that Erk/MAPK plays in each cell type. Previous research has shown that individual cell types utilize the Erk/MAPK pathway in different ways. For example, the morphological development of lower motor neuron axonal projections is Erk/MAPK-independent during embryogenesis, while nociceptive neuron projections require Erk/MAPK to innervate epidermal targets. Here, we tested whether Erk/MAPK played a role in the postnatal development of lower motor neurons during crucial periods of activity-dependent circuit modifications. We have generated Cre-dependent conditional Erk/MAPK mutant mice that exhibit either loss or gain of Erk/MAPK signaling specifically in ChAT:Cre expressing lower motor neurons. Importantly, we found that Erk/MAPK is necessary for the development of neuromuscular junction morphology by the second postnatal week. In contrast, we were unable to detect a significant difference in lower motor neuron development in Erk/MAPK gain-of-function mice. The data suggests that Erk/MAPK plays an important role in postnatal lower motor neuron development by regulating the morphological maturation of the neuromuscular junction. / Dissertation/Thesis / Masters Thesis Biology 2017