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1.  Differential effects of dopamine signalling on long-term memory formation and consolidation in rodent brain 
Proteome Science  2015;13:13.
Using auditory discrimination learning in gerbils, we have previously shown that activation of auditory-cortical D1/D5 dopamine receptors facilitates mTOR-mediated, protein synthesis-dependent mechanisms of memory consolidation and anterograde memory formation. To understand molecular mechanisms of this facilitatory effect, we tested the impact of local pharmacological activation of different D1/D5 dopamine receptor signalling modes in the auditory cortex. To this end, protein patterns in soluble and synaptic protein-enriched fractions from cortical, hippocampal and striatal brain regions of ligand- and vehicle-treated gerbils were analysed by 2D gel electrophoresis and mass spectrometry 24 h after intervention.
After auditory-cortical injection of SKF38393 – a D1/D5 dopamine receptor-selective agonist reported to activate the downstream effectors adenylyl cyclase and phospholipase C – prominent proteomic alterations compared to vehicle-treated controls appeared in the auditory cortex, striatum, and hippocampus, whereas only minor changes were detectable in the frontal cortex. In contrast, auditory-cortical injection of SKF83959 – a D1/D5 agonist reported to preferentially stimulate phospholipase C – induced pronounced changes in the frontal cortex. At the molecular level, we detected altered regulation of cytoskeletal and scaffolding proteins, changes in proteins with functions in energy metabolism, local protein synthesis, and synaptic signalling. Interestingly, abundance and/or subcellular localisation of the predominantly presynaptic protein α-synuclein displayed dopaminergic regulation. To assess the role of α-synuclein for dopaminergic mechanisms of memory modulation, we tested the impact of post-conditioning systemic pharmacological activation of different D1/D5 dopamine receptor signalling modes on auditory discrimination learning in α-synuclein-mutant mice. In C57BL/6JOlaHsd mice, bearing a spontaneous deletion of the α-synuclein-encoding gene, but not in the related substrains C57BL/6JCrl and C57BL/6JRccHsd, adenylyl cyclase-mediated signalling affected acquisition rates over future learning episodes, whereas phospholipase C-mediated signalling affected final memory performance.
Dopamine signalling modes via D1/D5 receptors in the auditory cortex differentially impact protein profiles related to rearrangement of cytomatrices, energy metabolism, and synaptic neurotransmission in cortical, hippocampal, and basal brain structures. Altered dopamine neurotransmission in α-synuclein-deficient mice revealed that distinct D1/D5 receptor signalling modes may control different aspects of memory consolidation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12953-015-0069-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4387680  PMID: 25852303
Dopamine agonist; Brain; Cortex; Hippocampus; Striatum; α-synuclein; Auditory discrimination learning; Memory consolidation; Proteome; 2D gel
2.  Structural modeling of the N-terminal signal–receiving domain of IκBα 
The transcription factor nuclear factor-κB (NF-κB) exerts essential roles in many biological processes including cell growth, apoptosis and innate and adaptive immunity. The NF-κB inhibitor (IκBα) retains NF-κB in the cytoplasm and thus inhibits nuclear localization of NF-κB and its association with DNA. Recent protein crystal structures of the C-terminal part of IκBα in complex with NF-κB provided insights into the protein-protein interactions but could not reveal structural details about the N-terminal signal receiving domain (SRD). The SRD of IκBα contains a degron, formed following phosphorylation by IκB kinases (IKK). In current protein X-ray structures, however, the SRD is not resolved and assumed to be disordered. Here, we combined secondary structure annotation and domain threading followed by long molecular dynamics (MD) simulations and showed that the SRD possesses well-defined secondary structure elements. We show that the SRD contains 3 additional stable α-helices supplementing the six ARDs present in crystallized IκBα. The IκBα/NF-κB protein-protein complex remained intact and stable during the entire simulations. Also in solution, free IκBα retains its structural integrity. Differences in structural topology and dynamics were observed by comparing the structures of NF-κB free and NF-κB bound IκBα-complex. This study paves the way for investigating the signaling properties of the SRD in the IκBα degron. A detailed atomic scale understanding of molecular mechanism of NF-κB activation, regulation and the protein-protein interactions may assist to design and develop novel chronic inflammation modulators.
PMCID: PMC4477481  PMID: 26157801
signal transduction; NF-κB; IκBα; secondary structure prediction; N-terminal extension; molecular dynamics simulation; protein-protein complex refinement; signal receiving domain
3.  Rapid Interaction of Helicobacter pylori with Microvilli of the Polar Human Gastric Epithelial Cell Line NCI-N87 
Infection with Helicobacter pylori results often in chronic gastritis, gastric ulcers or even gastric tumor development. Little is known about the initial interaction between gastric epithelial cells and H. pylori. The aim of the present study was to analyze the initial host contact to the bacteria. Monolayers of the human gastric epithelial cell line NCI-N87 grown on porous membranes were used and the apical side of the epithelium was exposed to the H. pylori wild-type strain P1 for 1 hr. Many epithelial cells were colonized by bacteria within the period of 60 min. Using scanning electron microscopy we detected that the bacteria were in close contact with the epithelia via microvilli. Further, transmission electron microscopy of the contact sites revealed no difference in the morphology of the microvilli in comparison to those not attached to the bacteria. The present study demonstrates the importance of microvilli on apical epithelial cells during the initial contact of the host by colonizing H. pylori. Anat Rec, 296:1800–1805, 2013. © 2013 The Authors. The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology published by Wiley Periodicals, Inc. on behalf of the American Association of Anatomists.
PMCID: PMC4225472  PMID: 24136815
gastric epithelial cells; scanning electron microscopy; tight junction; trans epithelial electrical resistance
4.  CYLD Enhances Severe Listeriosis by Impairing IL-6/STAT3-Dependent Fibrin Production 
PLoS Pathogens  2013;9(6):e1003455.
The facultative intracellular bacterium Listeria monocytogenes (Lm) may cause severe infection in humans and livestock. Control of acute listeriosis is primarily dependent on innate immune responses, which are strongly regulated by NF-κB, and tissue protective factors including fibrin. However, molecular pathways connecting NF-κB and fibrin production are poorly described. Here, we investigated whether the deubiquitinating enzyme CYLD, which is an inhibitor of NF-κB-dependent immune responses, regulated these protective host responses in murine listeriosis. Upon high dose systemic infection, all C57BL/6 Cyld−/− mice survived, whereas 100% of wildtype mice succumbed due to severe liver pathology with impaired pathogen control and hemorrhage within 6 days. Upon in vitro infection with Lm, CYLD reduced NF-κB-dependent production of reactive oxygen species, interleukin (IL)-6 secretion, and control of bacteria in macrophages. Furthermore, Western blot analyses showed that CYLD impaired STAT3-dependent fibrin production in cultivated hepatocytes. Immunoprecipitation experiments revealed that CYLD interacted with STAT3 in the cytoplasm and strongly reduced K63-ubiquitination of STAT3 in IL-6 stimulated hepatocytes. In addition, CYLD diminished IL-6-induced STAT3 activity by reducing nuclear accumulation of phosphorylated STAT3. In vivo, CYLD also reduced hepatic STAT3 K63-ubiquitination and activation, NF-κB activation, IL-6 and NOX2 mRNA production as well as fibrin production in murine listeriosis. In vivo neutralization of IL-6 by anti-IL-6 antibody, STAT3 by siRNA, and fibrin by warfarin treatment, respectively, demonstrated that IL-6-induced, STAT3-mediated fibrin production significantly contributed to protection in Cyld−/− mice. In addition, in vivo Cyld siRNA treatment increased STAT3 phosphorylation, fibrin production, pathogen control and survival of Lm-infected WT mice illustrating that therapeutic inhibition of CYLD augments the protective NF-κB/IL-6/STAT3 pathway and fibrin production.
Author Summary
Listeria monocytogenes causes high mortality in immunocompromised patients and fetuses. Murine studies have revealed that innate immune responses and fibrin, a major product of hepatocytes, are important to control Listeria. In the present study, we analysed whether the deubiquitinating enzyme CYLD impairs protective host responses in severe listeriosis and is a potential therapeutic target molecule. Using wildtype and Cyld−/− mice, we show that CYLD significantly reduced pathogen control and production of interferon (IFN)-γ, interleukin (IL)-6, and NOX2 mRNA in liver and spleen resulting in death of wildtype but not of Cyld−/− mice upon high-dose systemic infection. In vitro, CYLD impaired NF-κB-dependent pathogen control, reactive oxygen production, and IL-6 secretion in IFN-γ-stimulated, infected macrophages. We newly identified that CYLD directly removed K63-ubiquitin from STAT3, inhibited STAT3 activation and nuclear translocation resulting in reduced hepatocyte fibrin production. In Listeria-infected Cyld−/− mice, hepatic STAT3 K63-ubiquitination and activation, NF-κB activation, IL-6 production, and fibrin deposition were also increased. Neutralization experiments confirmed that the improved survival and pathogen control of Cyld−/− mice was dependent on IL-6-STAT3-mediated fibrin deposition. Finally, Cyld siRNA treatment of Listeria-infected wildtype mice significantly increased activated STAT3 and fibrin production, improved pathogen control and reduced mortality illustrating a therapeutic potential of CYLD inhibition.
PMCID: PMC3695090  PMID: 23825949
5.  Cyclin-Dependent Kinase 6 Phosphorylates NF-κB P65 at Serine 536 and Contributes to the Regulation of Inflammatory Gene Expression 
PLoS ONE  2012;7(12):e51847.
Nuclear factor kappa-B (NF-κB) activates multiple genes with overlapping roles in cell proliferation, inflammation and cancer. Using an unbiased approach we identified human CDK6 as a novel kinase phosphorylating NF-κB p65 at serine 536. Purified and reconstituted CDK6/cyclin complexes phosphorylated p65 in vitro and in transfected cells. The physiological role of CDK6 for basal as well as cytokine-induced p65 phosphorylation or NF-κB activation was revealed upon RNAi-mediated suppression of CDK6. Inhibition of CDK6 catalytic activity by PD332991 suppressed activation of NF-κB and TNF-induced gene expression. In complex with a constitutively active viral cyclin CDK6 stimulated NF-κB p65-mediated transcription in a target gene specific manner and this effect was partially dependent on its ability to phosphorylate p65 at serine 536. Tumor formation in thymi and spleens of v-cyclin transgenic mice correlated with increased levels of p65 Ser536 phosphorylation, increased expression of CDK6 and upregulaton of the NF-κB target cyclin D3. These results suggest that aberrant CDK6 expression or activation that is frequently observed in human tumors can contribute through NF-κB to chronic inflammation and neoplasia.
PMCID: PMC3530474  PMID: 23300567
6.  Dynamics of p53 and NF-κB regulation in response to DNA damage and identification of target proteins suitable for therapeutic intervention 
BMC Systems Biology  2012;6:125.
The genome is continuously attacked by a variety of agents that cause DNA damage. Recognition of DNA lesions activates the cellular DNA damage response (DDR), which comprises a network of signal transduction pathways to maintain genome integrity. In response to severe DNA damage, cells undergo apoptosis to avoid transformation into tumour cells, or alternatively, the cells enter permanent cell cycle arrest, called senescence. Most tumour cells have defects in pathways leading to DNA repair or apoptosis. In addition, apoptosis could be counteracted by nuclear factor kappa B (NF-κB), the main anti-apoptotic transcription factor in the DDR. Despite the high clinical relevance, the interplay of the DDR pathways is poorly understood. For therapeutic purposes DNA damage signalling processes are induced to induce apoptosis in tumour cells. However, the efficiency of radio- and chemotherapy is strongly hampered by cell survival pathways in tumour cells. In this study logical modelling was performed to facilitate understanding of the complexity of the signal transduction networks in the DDR and to provide cancer treatment options.
Our comprehensive discrete logical model provided new insights into the dynamics of the DDR in human epithelial tumours. We identified new mechanisms by which the cell regulates the dynamics of the activation of the tumour suppressor p53 and NF-κB. Simulating therapeutic intervention by agents causing DNA single-strand breaks (SSBs) or DNA double-strand breaks (DSBs) we identified candidate target proteins for sensitization of carcinomas to therapeutic intervention. Further, we enlightened the DDR in different genetic diseases, and by failure mode analysis we defined molecular defects putatively contributing to carcinogenesis.
By logic modelling we identified candidate target proteins that could be suitable for radio- and chemotherapy, and contributes to the design of more effective therapies.
PMCID: PMC3473366  PMID: 22979979
Topoisomerase inhibitors; Signal transduction; Cell cycle arrest; Apoptosis; Cancer; Logical model
7.  Chemoembolization with Drug-Eluting Beads Complicated by Intrahepatic Biloma 
Chemoembolization with drug-eluting beads is a type of locoregional therapy currently being used for the treatment of hepatocellular carcinoma and metastatic disease to the liver. This treatment has proven effectiveness in controlling tumor growth, extending survival time, and improving quality of life. Chemoembolization with drug-eluting beads have been shown to be safe, but like any other invasive procedure, can have associated complications. The authors present a case of intrahepatic biloma formation occurring as a result of treatment with drug-eluting beads.
PMCID: PMC3193327  PMID: 22654265
Biloma; drug-eluting beads; DEBs; chemoembolization
8.  Protective Toxoplasma gondii-Specific T-Cell Responses Require T-Cell-Specific Expression of Protein Kinase C-Theta▿  
Infection and Immunity  2010;78(8):3454-3464.
Protein kinase C-theta (PKC-θ) is important for the activation of autoreactive T cells but is thought to be of minor importance for T-cell responses in infectious diseases, suggesting that PKC-θ may be a target for the treatment of T-cell-mediated autoimmune diseases. To explore the function of PKC-θ in a chronic persisting infection in which T cells are crucial for pathogen control, we infected BALB/c PKC-θ−/− and PKC-θ+/+ wild-type mice with Toxoplasma gondii. The PKC-θ−/− mice succumbed to necrotizing Toxoplasma encephalitis due to an insufficient parasite control up to day 40, whereas the wild-type mice survived. The number of T. gondii-specific CD4 and CD8 T cells was significantly reduced in the PKC-θ−/− mice, resulting in the impaired production of protective cytokines (gamma interferon, tumor necrosis factor) and antiparasitic effector molecules (inducible nitric oxide, gamma interferon-induced GTPase) in the spleen and brain. In addition, Th2-cell numbers were reduced in infected the PKC-θ−/− mice, paralleled by the diminished GATA3 expression of PKC-θ−/− CD4 T cells and reduced T. gondii-specific IgG production in serum and cerebrospinal fluid. Western blot analysis of splenic CD4 and CD8 T cells revealed an impaired activation of the NF-κB, AP-1, and MAPK pathways in T. gondii-infected PKC-θ−/− mice. Adoptive transfer of wild-type CD4 plus CD8 T cells significantly protected PKC-θ−/− mice from death by increasing the numbers of gamma interferon-producing T. gondii-specific CD4 and CD8 T cells, illustrating a cell-autonomous, protective function of PKC-θ in T cells. These findings imply that PKC-θ inhibition drastically impairs T. gondii-specific T-cell responses with fatal consequences for intracerebral parasite control and survival.
PMCID: PMC2916261  PMID: 20498263
9.  Marginal and internal fit of zirconia based fixed dental prostheses fabricated with different concepts 
The purpose of this in vitro study was to compare the precision of fit of substructures milled from semi-sintered zirconia blocks, fabricated with two different fabrication concepts. Three-unit, posterior fixed dental prostheses (FDP) were fabricated for standardized dies (n = 10) with the laboratory Computer Aided Design (CAD)/Computer Aided Manufacturing (CAM) system Cercon® Brain (Brain) and the centralized CAD/CAM system Compartis Integrated Systems (Compartis). After cementation to the dies, the FDP were embedded and sectioned. Four cross-sections were made of each abutment tooth, and marginal and internal fit were evaluated under an optical microscope. A one-way analysis of variance (ANOVA) was used to compare data (α = 0.05). Mean gap dimensions at the marginal opening for Brain and Compartis were 56.0 (±34.5) μm and 51.7 (±45.2) μm, respectively. Mean internal gap dimensions of 62.8 (±37.5) μm to 164.6 (±33.4) μm were measured depending on the measurement location and the fabrication concept. Mean marginal openings and internal adaptations were not significantly different for both systems. Three out of four measurement locations showed significantly different cement gaps. Within the limitations of this study, the results suggest that the accuracy of both investigated systems is satisfactory for clinical use. The laboratory fabrication exhibited similar accuracy as the centralized manufacturing.
PMCID: PMC3645452  PMID: 23662077
zirconia; CAD; CAM; fit; FDP; all-ceramic
10.  Helicobacter pylori-induced activation of β-catenin involves low density lipoprotein receptor-related protein 6 and Dishevelled 
Molecular Cancer  2010;9:31.
The human microbial pathogen Helicobacter pylori resides in the stomach of about fifty percent of the world's population and represents a risk factor for chronic gastritis, peptic ulcers and, in rare cases, gastric cancer. Alterations of the Wnt/β-catenin signaling pathway have been described in almost every human cancer disease, due to the regulation of target genes being involved in cell cycle control, differentiation, cell migration or stem cell control. Our study aimed to elucidate the role of proximal Wnt signaling components low density lipoprotein receptor-related protein 6 (LRP6) and Dishevelled (Dvl) in the activation of β-catenin early after infection of gastric epithelial cells with H. pylori.
Infection of gastric epithelial NCI-N87 cells with H. pylori induces rapid phosphorylation of the Wnt/β-catenin pathway co-receptor LRP6 independent of the cytotoxin-associated gene A (CagA) or vacuolating cytotoxin A (VacA). However, bacteria lacking a functional type 4 secretion system (T4SS) failed to induce LRP6 phosphorylation. Further, we identified proteins of the Dvl family, namely Dvl2 and Dvl3, which are involved in LRP6 phosphorylation. H. pylori-induced nuclear accumulation of β-catenin and its transcriptional activation, and expression of Wnt target genes are strongly reduced in stable knockdown cell lines deficient for LRP6, Dvl2 or Dvl3.
We analysed the H. pylori-induced activation of Wnt-signaling factors and demonstrate for the first time that the canonical Wnt-signaling proteins LRP6 and Dvl2 and Dvl3 are involved in the regulation of β-catenin.
PMCID: PMC2825249  PMID: 20137080
11.  Logical network of genotoxic stress-induced NF-κB signal transduction predicts putative target structures for therapeutic intervention strategies 
Genotoxic stress is induced by a broad range of DNA-damaging agents and could lead to a variety of human diseases including cancer. DNA damage is also therapeutically induced for cancer treatment with the aim to eliminate tumor cells. However, the effectiveness of radio- and chemotherapy is strongly hampered by tumor cell resistance. A major reason for radio- and chemotherapeutic resistances is the simultaneous activation of cell survival pathways resulting in the activation of the transcription factor nuclear factor-kappa B (NF-κB). Here, we present a Boolean network model of the NF-κB signal transduction induced by genotoxic stress in epithelial cells. For the representation and analysis of the model, we used the formalism of logical interaction hypergraphs. Model reconstruction was based on a careful meta-analysis of published data. By calculating minimal intervention sets, we identified p53-induced protein with a death domain (PIDD), receptor-interacting protein 1 (RIP1), and protein inhibitor of activated STAT y (PIASy) as putative therapeutic targets to abrogate NF-κB activation resulting in apoptosis. Targeting these structures therapeutically may potentiate the effectiveness of radio-and chemotherapy. Thus, the presented model allows a better understanding of the signal transduction in tumor cells and provides candidates as new therapeutic target structures.
PMCID: PMC3169943  PMID: 21918620
apoptosis; Boolean network; cancer therapy; DNA-damage response; NF-κB
12.  Identification of novel Cyclooxygenase-2-dependent genes in Helicobacter pylori infection in vivo 
Molecular Cancer  2009;8:22.
Helicobacter pylori is a crucial determining factor in the pathogenesis of benign and neoplastic gastric diseases. Cyclooxygenase-2 (Cox-2) is the inducible key enzyme of arachidonic acid metabolism and is a central mediator in inflammation and cancer. Expression of the Cox-2 gene is up-regulated in the gastric mucosa during H. pylori infection but the pathobiological consequences of this enhanced Cox-2 expression are not yet characterized. The aim of this study was to identify novel genes down-stream of Cox-2 in an in vivo model, thereby identifying potential targets for the study of the role of Cox- 2 in H. pylori pathogenesis and the initiation of pre- cancerous changes.
Gene expression profiles in the gastric mucosa of mice treated with a specific Cox-2 inhibitor (NS398) or vehicle were analysed at different time points (6, 13 and 19 wk) after H. pylori infection. H. pylori infection affected the expression of 385 genes over the experimental period, including regulators of gastric physiology, proliferation, apoptosis and mucosal defence. Under conditions of Cox-2 inhibition, 160 target genes were regulated as a result of H. pylori infection. The Cox-2 dependent subset included those influencing gastric physiology (Gastrin, Galr1), epithelial barrier function (Tjp1, connexin45, Aqp5), inflammation (Icam1), apoptosis (Clu) and proliferation (Gdf3, Igf2). Treatment with NS398 alone caused differential expression of 140 genes, 97 of which were unique, indicating that these genes are regulated under conditions of basal Cox-2 expression.
This study has identified a panel of novel Cox-2 dependent genes influenced under both normal and the inflammatory conditions induced by H. pylori infection. These data provide important new links between Cox-2 and inflammatory processes, epithelial repair and integrity.
PMCID: PMC2667483  PMID: 19317916
13.  Host-pathogen systems biology: logical modelling of hepatocyte growth factor and Helicobacter pylori induced c-Met signal transduction 
The hepatocyte growth factor (HGF) stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of tissues, including epithelial cells, on binding to the receptor tyrosine kinase c-Met. Abnormal c-Met signalling contributes to tumour genesis, in particular to the development of invasive and metastatic phenotypes. The human microbial pathogen Helicobacter pylori can induce chronic gastritis, peptic ulceration and more rarely, gastric adenocarcinoma. The H. pylori effector protein cytotoxin associated gene A (CagA), which is translocated via a type IV secretion system (T4SS) into epithelial cells, intracellularly modulates the c-Met receptor and promotes cellular processes leading to cell scattering, which could contribute to the invasiveness of tumour cells. Using a logical modelling framework, the presented work aims at analysing the c-Met signal transduction network and how it is interfered by H. pylori infection, which might be of importance for tumour development.
A logical model of HGF and H. pylori induced c-Met signal transduction is presented in this work. The formalism of logical interaction hypergraphs (LIH) was used to construct the network model. The molecular interactions included in the model were all assembled manually based on a careful meta-analysis of published experimental results. Our model reveals the differences and commonalities of the response of the network upon HGF and H. pylori induced c-Met signalling. As another important result, using the formalism of minimal intervention sets, phospholipase Cγ1 (PLCγ1) was identified as knockout target for repressing the activation of the extracellular signal regulated kinase 1/2 (ERK1/2), a signalling molecule directly linked to cell scattering in H. pylori infected cells. The model predicted only an effect on ERK1/2 for the H. pylori stimulus, but not for HGF treatment. This result could be confirmed experimentally in MDCK cells using a specific pharmacological inhibitor against PLCγ1. The in silico predictions for the knockout of two other network components were also verified experimentally.
This work represents one of the first approaches in the direction of host-pathogen systems biology aiming at deciphering signalling changes brought about by pathogenic bacteria. The suitability of our network model is demonstrated by an in silico prediction of a relevant target against pathogen infection.
PMCID: PMC2254585  PMID: 18194572
14.  Helicobacter pylori CagA protein targets the c-Met receptor and enhances the motogenic response 
The Journal of Cell Biology  2003;161(2):249-255.
Infection with the human microbial pathogen Helicobacter pylori is assumed to lead to invasive gastric cancer. We find that H. pylori activates the hepatocyte growth factor/scatter factor receptor c-Met, which is involved in invasive growth of tumor cells. The H. pylori effector protein CagA intracellularly targets the c-Met receptor and promotes cellular processes leading to a forceful motogenic response. CagA could represent a bacterial adaptor protein that associates with phospholipase Cγ but not Grb2-associated binder 1 or growth factor receptor–bound protein 2. The H. pylori–induced motogenic response is suppressed and blocked by the inhibition of PLCγ and of MAPK, respectively. Thus, upon translocation, CagA modulates cellular functions by deregulating c-Met receptor signaling. The activation of the motogenic response in H. pylori–infected epithelial cells suggests that CagA could be involved in tumor progression.
PMCID: PMC2172921  PMID: 12719469
epithelial–mesenchymal transition; hepatocyte growth factor; motility; tumor invasion; motogenic response; PLCγ
15.  Coordinate Activation of Activator Protein 1 and Inflammatory Cytokines in Response to Neisseria gonorrhoeae Epithelial Cell Contact Involves Stress Response Kinases  
The Journal of Experimental Medicine  1998;188(7):1277-1286.
Neisseria gonorrhoeae (Ngo), the etiologic agent of gonorrhea, induce a number of proinflammatory cytokines by contact to epithelial cells. Cytokine genes and a variety of other immune response genes are activated as a result of the regulatory function of immediate early response transcription factors including activator protein 1 (AP-1). Since it is established that phosphorylation of c-Jun, the central component of AP-1, by the stress-activated c-Jun NH2-terminal kinase (JNK) increases the transcriptional activity of AP-1, we studied whether Ngo could induce stress response pathways involving JNK. We found that virulent Ngo strains induce phosphorylation and activation of JNK but not of p38 kinase. Analysis of a nonpathogenic Ngo strain revealed only weak JNK activation. In respect to the molecular components upstream of the JNK signaling cascade, we show that a dominant negative mutant of MAP kinase kinase 4 (MKK4) represses transcription of an AP-1–dependent reporter gene. Regarding upstream stress response factors involved in Ngo-induced MKK4/JNK/AP-1 activation, we identified p21-activated kinase (PAK) but not MAPK/ERK kinase kinase (MEKK1). Inhibition of small GTPases including Rac1 and Cdc42 by Toxin B prevented JNK and AP-1 activation. Our results indicate that Ngo induce the activation of proinflammatory cytokines via a cascade of cellular stress response kinases involving PAK, which directs the signal from the Rho family of small GTPases to JNK/AP-1 activation.
PMCID: PMC2212490  PMID: 9763607
Neisseria; inflammation; activator protein 1; c-Jun NH2-terminal kinase; p21-activated kinase
16.  Neisseria gonorrhoeae Epithelial Cell Interaction Leads to the Activation of the Transcription Factors Nuclear Factor κB and Activator Protein 1 and the Induction of Inflammatory Cytokines 
We have studied the effect of human bacterial pathogen Neisseria gonorrhoeae (Ngo) on the activation of nuclear factor (NF)-κB and the transcriptional activation of inflammatory cytokine genes upon infection of epithelial cells. During the course of infection, Ngo, the etiologic agent of gonorrhea, adheres to and penetrates mucosal epithelial cells. In vivo, localized gonococcal infections are often associated with a massive inflammatory response. We observed upregulation of several inflammatory cytokine messenger RNAs (mRNAs) and the release of the proteins in Ngo-infected epithelial cells. Moreover, infection with Ngo induced the formation of a NF-κB DNA–protein complex and, with a delay in time, the activation of activator protein 1, whereas basic leucine zipper transcription factors binding to the cAMP-responsive element or CAAT/enhancer-binding protein DNA-binding sites were not activated. In supershift assays using NF-κB–specific antibodies, we identified a NF-κB p50/p65 heterodimer. The NF-κB complex was formed within 10 min after infection and decreased 90 min after infection. Synthesis of tumor necrosis factor α and interluekin (IL)-1β occurred at later times and therefore did not account for NF-κB activation. An analysis of transiently transfected IL-6 promoter deletion constructs suggests that NF-κB plays a crucial role for the transcriptional activation of the IL-6 promoter upon Ngo infection. Inactivation of NF-κB conferred by the protease inhibitor N-tosyl-l-phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but not all, studied cyctokine genes. Activation of NF-κB and cytokine mRNA upregulation also occur in Ngo-infected epithelial cells that were treated with cytochalasin D, indicating an extracellular signaling induced before invasion.
PMCID: PMC2198971  PMID: 9221754
17.  Synaptic proteome changes in mouse brain regions upon auditory discrimination learning 
Proteomics  2012;12(15-16):2433-2444.
Changes in synaptic efficacy underlying learning and memory processes are assumed to be associated with alterations of the protein composition of synapses. Here, we performed a quantitative proteomic screen to monitor changes in the synaptic proteome of four brain areas (auditory cortex, frontal cortex, hippocampus striatum) during auditory learning. Mice were trained in a shuttle box GO/NO-GO paradigm to discriminate between rising and falling frequency modulated tones to avoid mild electric foot shock. Control-treated mice received corresponding numbers of either the tones or the foot shocks. Six hours and 24 h later, the composition of a fraction enriched in synaptic cytomatrix-associated proteins was compared to that obtained from naïve mice by quantitative mass spectrometry. In the synaptic protein fraction obtained from trained mice, the average percentage (±SEM) of downregulated proteins (59.9 ± 0.5%) exceeded that of upregulated proteins (23.5 ± 0.8%) in the brain regions studied. This effect was significantly smaller in foot shock (42.7 ± 0.6% down, 40.7 ± 1.0% up) and tone controls (43.9 ± 1.0% down, 39.7 ± 0.9% up). These data suggest that learning processes initially induce removal and/or degradation of proteins from presynaptic and postsynaptic cytoskeletal matrices before these structures can acquire a new, postlearning organisation. In silico analysis points to a general role of insulin-like signalling in this process.
PMCID: PMC3509369  PMID: 22696468
Animal proteomics; Auditory learning; Chemical synapse; Isotope-coded protein labelling; Learning and memory; Quantitative mass spectrometry
18.  Protein kinase C isozymes regulate matrix metalloproteinase-1 expression and cell invasion in Helicobacter pylori infection 
Gut  2012;62(3):358-367.
Protein kinase C (PKC) signalling is often dysregulated in gastric cancer and therefore represents a potential target in cancer therapy. The Gram-negative bacterium Helicobacter pylori, which colonises the human stomach, plays a major role in the development of gastritis, peptic ulcer and gastric adenocarcinoma.
To analyse the role of PKC isozymes as mediators of H pylori-induced pathogenesis.
PKC phosphorylation was evaluated by immunoblotting and immunohistochemistry. Gene reporter assays, RT-PCR and invasion assays were performed to assess the role of PKC in the regulation of activator protein-1 (AP-1), matrix metalloproteinase-1 (MMP-1) and the invasion of H pylori-infected epithelial cells.
H pylori induced phosphorylation of PKC isozymes α, δ, θ in AGS cells, which was accompanied by the phosphorylation of PKC substrates, including PKCμ and myristoylated alanine-rich C kinase substrate (MARCKS), in a CagA-independent manner. Phospholipase C, phosphatidylinositol 3-kinase and Ca2+ were crucial for PKC activation on infection; inhibition of PKC diminished AP-1 induction and, subsequently, MMP-1 expression. Invasion assays confirmed PKC involvement in H pylori-induced MMP-1 secretion. In addition, analysis of biopsies from human gastric mucosa showed increased phosphorylation of PKC in active H pylori gastritis and gastric adenocarcinoma.
The targeting of certain PKC isozymes might represent a suitable strategy to interfere with the MMP-1-dependent remodelling of infected tissue and to overcome the invasive behaviour of gastric cancer cells.
PMCID: PMC3585490  PMID: 22442164
AP-1; CagA; c-Fos; MARCKS; PLC; cell signalling; adenocarcinoma; helicobacter pylori; bacterial infection; matrix metalloproteinase; helicobacter pylori—pathogenesis; inflammation; nuclear factor kappa b; signal transduction; molecular oncology; gastro-oesophageal reflux disease; barretts metaplasia; barretts carcinoma; gastro-oesphageal junction; mucosal pathology; gastritis; gastric inflammation; inflammatory bowel disease; gastrointestinal cancer; gastric neoplasia; gastric pre-cancer

Results 1-18 (18)