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A subtly regulated and controlled course of cellular processes is essential for the healthy functioning not only of single cells, but also of organs being constituted thereof. In return, this entails the proper functioning of the whole organism. This implies a complex intra- and inter-cellular communication and signal processing that require equally multi-faceted methods to describe and investigate the underlying processes. Within the scope of this thesis, mathematical modeling of cellular signaling finds its application in the analysis of cellular processes and signaling cascades in different organisms. ...
Studies on receptor signaling and regulation in platelets and T cells from genetically modified mice
(2014)
Receptors with tyrosine-based signaling motifs control essential functions of hematopoietic cells, including lymphocytes and platelets. Downstream of the platelet receptor glycoprotein (GP) VI and the T cell receptor (TCR) the immunoreceptor tyrosine-based activation motif (ITAM) initiates a signaling cascade that involves kinases, adapter and effector proteins and finally leads to cellular activation. This thesis summarizes the results of three studies investigating different aspects of receptor signaling and regulation in platelets and T cells.
In the first part, the impact of constitutive Ca2+ influx on TCR signaling and T cell physiology was investigated using a transgenic mouse line with a mutation in the Ca2+ sensor stromal interaction molecule 1 (STIM1). The elevated cytoplasmic Ca2+ level resulted in an altered phosphorylation pattern of the key enzyme phospholipase (PL) Cγ1 in response to TCR stimulation, but without affecting its enzymatic activity. Withdrawal of extracellular Ca2+ or inhibition of the phosphatase calcineurin restored the normal phosphorylation pattern. In addition, there was a decrease in the release of Th2-type cytokines interleukin 4, 5 and 13 upon stimulation in vitro.
The second part of the thesis deals with the role of the adapter protein growth factor receptor-bound protein 2 (Grb2) in platelets using a megakaryocyte/platelet-specific knockout mouse line. Loss of Grb2 severely impaired signaling of GPVI and C-type lectin-like receptor 2 (CLEC-2), a related hemITAM receptor. This was attributed to defective stabilization of the linker for activation of T cells (LAT) signalosome and resulted in reduced adhesion, aggregation, Ca2+ mobilization and procoagulant activity downstream of (hem)ITAM-coupled receptors in vitro. In contrast, the signaling pathways of G protein-coupled receptors (GPCRs) and the integrin αIIbβ3, which do not utilize the LAT signalosome, were unaffected. In vivo, the defective (hem)ITAM signaling caused prolonged bleeding times, however, thrombus formation was only affected under conditions where GPCR signaling was impaired (upon acetylsalicylic acid treatment). These results establish Grb2 as an important adapter protein in the propagation of GPVI- and CLEC-2-induced signals.
Finally, the proteolytic regulation of the immunoreceptor tyrosine-based switch motif (ITSM)-bearing receptor CD84 in platelets was investigated. This study demonstrated that in mice CD84 is cleaved by two distinct and independent proteolytic mechanisms upon platelet activation: shedding of the extracellular part, which is exclusively mediated by a disintegrin and metalloproteinase (ADAM) 10 and cleavage of the intracellular C-terminus by the protease calpain. Finally, the analysis of soluble CD84 levels in the plasma of transgenic mice revealed that shedding of CD84 by ADAM10 occurs constitutively in vivo.
Neurotrophic factor signaling modulates differentiation, axon growth and maintenance, synaptic plasticity and regeneration of neurons after injury. Ciliary neurotrophic factor (CNTF), a Schwann cell derived neurotrophic factor, has an exclusive role in axon maintenance, sprouting and synaptic preservation. CNTF, but not GDNF, has been shown to alleviate motoneuron degeneration in pmn mutant mice carrying a missense mutation in Tbce gene, a model for Amyotrophic Lateral Sclerosis (ALS). This current study elucidates the distinct signaling mechanism by which CNTF rescues the axonal degeneration in pmn mutant mice. ...
In this century new experimental and computational techniques are adding an enormous amount of information, revealing many biological mysteries. The complexities of biological systems still broach new questions. Till now the main approach to understand a system has been to divide it in components that can be studied. The upcoming new paradigm is to combine the pieces of information in order to understand it at a global level. In the present thesis we have tried to study infectious diseases with such a global ‘Systems Biology’ approach. In the first part the apoptosis pathway is analyzed. Apoptosis (Programmed cell death) is used as a counter measure in different infections, for example viral infections. The interactions between death domain containing proteins are studied to address the following questions: i) How specificity is maintained - showing that it is induced through adaptors, ii) how proliferation/ survival signals are induced during activation of apoptosis – suggesting the pivotal role of RIP. The model also allowed us to detect new possible interacting surfaces. The pathway is then studied at a global level in a time step simulation to understand the evolution of the topology of activators and inhibitors of the pathway. Signal processing is further modeled in detail for the apoptosis pathway in M. musculus to predict the concentration time course of effector caspases. Further, experimental measurements of caspase-3 and viability of cells validate the model. The second part focuses on the phagosome, an organelle which plays an essential role in removal of pathogens as exemplified by M. tuberculosis. Again the problem is addressed in two main sections: i) To understanding the processes that are inhibited by M. tuberculosis; we focused on the phospholipid network applying a time step simulation in section one, which plays an important role in inhibition or activation of actin polymerization on the phagosome membrane. ii) Furthermore, actin polymers are suggested to play a role in the fusion of the phagosome with lysosome. To check this hypothesis an in silico model was developed; we find that the search time is reduced by 5 fold in the presence of actin polymers. Further the effect of length of actin polymers, dimensions of lysosome, phagosome and other model parameter is analyzed. After studying a pathway and then an organelle, the next step was to move to the system. This was exemplified by the host pathogen interactions between Bordetella pertussis and Bordetella bronchiseptica. The limited availability of quantitative information was the crucial factor behind the choice of the model type. A Boolean model was developed which was used for a dynamic simulation. The results predict important factors playing a role in Bordetella pathology especially the importance of Th1 related responses and not Th2 related responses in the clearance of the pathogen. Some of the quantitative predictions have been counterchecked by experimental results such as the time course of infection in different mutants and wild type mice. All these computational models have been developed in presence of limited kinetic data. The success of these models has been validated by comparison with experimental observations. Comparative models studied in chapters 6 and 9 can be used to explore new host pathogen interactions. For example in chapter 6, the analysis of inhibitors and inhibitory paths in three organism leads to the identification of regulatory hotspots in complex organisms and in chapter 9 the identification of three phases in B. bronchiseptica and inhibition of IFN-γ by TTSS lead us to explore similar phases and inhibition of IFN-γ in B. pertussis. Further an important significance of these models is to identify new components playing an essential role in host-pathogen interactions. In silico deletions can point out such components which can be further analyzed by experimental mutations.
Platelets are crucial to inhibit extensive blood loss at sites of vascular injury. However, under pathological conditions such as rupture of an atherosclerotic plaque, activated platelets form aggregates that may occlude the vessel. This can lead to heart attack and stroke. Various and complex signaling pathways in the cell are involved in the steps of platelet adhesion, activation and aggregation. Single aspects of these processes were studied in three different subprojects in this work. The Glycoprotein (GP) Ib-V-IX complex is responsible for the first contact of platelets with the vessel wall. Subsequently, GPVI can bind to collagen of the subendothelium, which initiates a signaling cascade leading to platelet activation, aggregation, characterized by integrin activation and granule secretion and platelet procoagulant activity. The latter is characterized by exposed phosphatidylserine (PS) on the platelet surface, which enhances thrombin generation and thereby the coagulation cascade. A controlled regulation of GP receptors on the platelet surface is vital for an intact response of the cell to platelet agonists. In the first subproject described here the regulation of GPV and GPVI on mouse platelets was investigated and it was found that both receptors are shed from the platelet surface in a metalloproteinase dependent manner. However, GPVI is shed upon mitochondrial injury, while GPV cleavage could be observed upon platelet stimulation. The metalloproteinase responsible for GPVI shedding remains unknown whereas the metallproteinase that sheds GPV was identified in this work as being ADAM17. This shows that the expression of both receptors underlies a controlled mechanism regulated through distinct metalloproteinases. In the second subproject the role of protein kinase C (PKC) in platelet activation and procoagulant response was investigated using PKC specific inhibitors. It was found that PKC blockage reduced platelet activation but enhanced platelet procoagulant activity. This is the first time that a dual role in platelet activation and procoagulant activity is defined for PKC. In the third project the role of the small GTPase Rac1 in platelet signaling was studied using conditional Rac1 knock out mice. It is reported here that Rac1 lies downstream of GPVI and is involved in integrin activation and cytsolic Ca2+ changes in vitro and platelet adhesion and thrombus formation in vivo. This is the first time that Rac1 is demonstrated to have a pivotal role in GPVI signaling and furthermore points to a novel, unknown pathway downstream of GPVI.
In mammals, KSR1 functions as an essential scaffold that coordinates the assembly of RAF/MEK/ERK complexes and regulates intracellular signal transduction upon extracellular stimulation. Aberrant activation of the equivalent MAPK signaling pathway has been implicated in multiple human cancers and some developmental disorders. The mechanism of KSR1 regulation is highly complex and involves several phosphorylation/dephosphorylation steps. In the present study, a number of novel in vivo phosphorylation sites were detected in mKSR1 by use of mass spectrometry analysis. Among others, Tyr728 was identified as a unique regulatory residue phosphorylated by LCK, a Src kinase family member. To understand how phosphorylation of Tyr728 may regulate the function of KSR1 in signal transduction and cellular processes, structural modeling and biochemical studies were integrated in this work.
Computational modeling of the mKSR1(KD) protein structure revealed strong hydrogen bonding between phospho-Tyr728 and the residues surrounding Arg649. Remarkably, this pattern was altered when Tyr728 was non-phosphorylated or substituted. As confirmed by biochemical analysis, Arg649 may serve as a major anchor point for phospho-Tyr728 in order to stabilize internal structures of KSR1. In line with the protein modeling results, mutational studies revealed that substitution of Tyr728 by phenylalanine leads to a less compact interaction between KSR1 and MEK, a facilitated KSR1/B-RAF binding and an increased phosphorylation of MEK in complex with KSR1. From these findings it can be concluded that phospho-Tyr728 is involved in tightening the KSR1/MEK interaction interface and in regulating the phosphorylation of KSR1-bound MEK by either RAF or KSR1 kinases.
Beside the Tyr728, Ser722 was identified as a novel regulatory phosphorylation site. Amino acid exchanges at the relevant position demonstrated that Ser722 regulates KSR1-bound MEK phosphorylation without affecting KSR1/MEK binding per se. Due to its localization, Ser722 might consequently control the catalytic activity of KSR1 by interfering with the access of substrate (possibly MEK) to the active site of KSR1 kinase. Together with Ser722, phosphorylated Tyr728 may further positively affect the kinase activity of KSR1 as a consequence of its vicinity to the activation and catalytic loop in the KSR1(KD). As revealed by structural modeling, phospho-Tyr728 builds a hydrogen bond with the highly conserved Lys685. Consequently, phospho-Tyr728 has a stabilizing effect on internal structures involved in the catalytic reaction and possibly enhances the phosphate transfer within the catalytic cleft in KSR1. Considering these facts, it seems very likely that the LCK-dependent phosphorylation of Tyr728 plays a crucial role in the regulation of KSR1 catalytic activity.
Results of fractionation and morphology analyses revealed that KSR1 recruits LCK to cytoskeleton for its phosphorylation at Tyr728 suggesting that this residue may regulate cytoskeleton dynamics and, consequently, cell motility. Beside that, phosphorylation of Tyr728 is involved in the regulation of cell proliferation, as shown by a significantly reduced population doubling time of KSR1-Y728F cells compared to cells expressing wild type KSR1.
Taken together, tyrosine phosphorylation in KSR1 uncovers a new link between Src family kinases and MAPK signaling. Tyr728, the novel regulatory phosphorylation site in murine KSR1, may coordinate the transition between the scaffolding and the catalytic function of KSR1 serving as a control point used to fine-tune cellular responses.
Zars and co-workers were able to localize an engram of aversive olfactory memory to the mushroom bodies of Drosophila (Zars et al., 2000). In this thesis, I followed up on this finding in two ways. Inspired by Zars et al. (2000), I first focused on the whether it would also be possible to localize memory extinction.While memory extinction is well established behaviorally, little is known about the underlying circuitry and molecular mechanisms. In extension to the findings by Zars et al (2000), I show that aversive olfactory memories remain localized to a subset of mushroom body Kenyon cells for up to 3 hours. Extinction localizes to the same set of Kenyon cells. This common localization suggests a model in which unreinforced presentations of a previously learned odorant intracellularly antagonizes the signaling cascades underlying memory formation. The second part also targets memory localization, but addresses appetitive memory. I show that memories for the same olfactory cue can be established through either sugar or electric shock reinforcement. Importantly, these memories localize to the same set of neurons within the mushroom body. Thus, the question becomes apparent how the same signal can be associated with different events. It is shown that two different monoamines are specificaly necessary for formation of either of these memories, dopamine in case of electric shock and octopamine in case of sugar memory, respectively. Taking the representation of the olfactory cue within the mushroom bodies into account, the data suggest that the two memory traces are located in the same Kenyon cells, but in separate subcellular domains, one modulated by dopamine, the other by octopamine. Taken together, this study takes two further steps in the search for the engram. (1) The result that in Drosophila olfactory learning several memories are organized within the same set of Kenyon cells is in contrast to the pessimism expressed by Lashley that is might not be possible to localize an engram. (2) Beyond localization, a possibible mechanism how several engrams about the same stimulus can be localized within the same neurons might be suggested by the models of subcellular organisation, as postulated in case of appetitive and aversive memory on the one hand and acquisition and extinction of aversive memory on the other hand.
Bone Morphogenetic Proteins (BMPs) are key regulators for a lot of diverse cellular processes. During embryonic development these proteins act as morphogens and play a crucial role particularly in organogenesis. BMPs have a direct impact on distinct cellular fates by means of concentration-gradients in the developing embryos. Using the diverse signaling input information within the embryo due to the gradient, the cells transduce the varying extracellular information into distinct gene expression profiles and cell fate decisions. Furthermore, BMP proteins bear important functions in adult organisms like tissue homeostasis or regeneration. In contrast to TGF-ß signaling, currently only little is known about how cells decode and quantify incoming BMP signals. There is poor knowledge about the quantitative relationships between signal input, transducing molecules, their states and location, and finally their ability to incorporate graded systemic inputs and produce qualitative responses. A key requirement for efficient pathway modulation is the complete comprehension of this signaling network on a quantitative level as the BMP signaling pathway, just like many other signaling pathways, is a major target for medicative interference. I therefore at first studied the subcellular distribution of Smad1, which is the main signal transducing protein of the BMP signaling pathway, in a quantitative manner and in response to various types and levels of stimuli in murine c2c12 cells. Results indicate that the subcellular localization of Smad1 is not dependent on the initial BMP input. Surprisingly, only the phospho-Smad1 level is proportionally associated to ligand concentration. Furthermore, the activated transducer proteins were entirely located in the nucleus. Besides the subcellular localization of Smad1, I have analyzed the gene expression profile induced by BMP signaling. Therefore, I examined two endogenous immediate early BMP targets as well as the expression of the stably transgenic Gaussia Luciferase. Interestingly, the results of these independent experimental setups and read-outs suggest oscillating target gene expression. The amplitudes of the oscillations showed a precise concentration-dependence for continuous and transient stimulation. Additionally, even short-time stimulation of 15’ activates oscillating gene-expression pulses that are detectable for at least 30h post-stimulation. Only treatment with a BMP type I receptor kinase inhibitor leads to the complete abolishment of the target gene expression. This indicated that target gene expression oscillations depend directly on BMP type I receptor kinase activity.
TWEAK is a typical member oft he TNF ligand family. Therefore it is initially expressed as a type II transmembrane protein, but a soluble variant can be released by proteolytic processing. In this work it is shown that oligomerized TWEAK is more competent than soluble, trimeric TWEAK regarding the activation of classical NFκB signaling pathway. However, both TWEAK variants are able to induce depletion of TRAF2 and processing of p100, which are hallmarks for the activation of the noncanonical NFκB pathway. Like other solube TNF ligands with no or poor activity on their corresponding receptor, TWEAK gains high activity upon oligomerization resembling the activity of the transmembrane ligand. TRAF2 has a key role in TWEAK-induced NFκB signaling. Depletion or degradation of TRAF2 is crucial for activation of the noncanonial or both, the classical and the noncanonical NFκB pathway. Blocking the TWEAK receptor Fn14 inhibits the activation of NFκB signaling, irrespective of the TWEAK form used for stimulation. This indicates that the different activities of the two TWEAK variants in activation of classical and noncanonical NFκB signaling are not caused by the use of different receptors. Therefore this study on TWEAK is the first reported case where one TNF ligand in different variants induces qualitatively different activities of the corresponding TNF receptor.
Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are exploited by human-specific pathogens to anchor themselves to or invade host cells. Interestingly, human granulocytes express a specific isoform, CEACAM3, that can direct efficient, opsonin-independent phagocytosis of CEACAM-binding Neisseria, Moraxella and Haemophilus species. As opsonin-independent phagocytosis of CEACAM-binding Neisseria depends on Src-family protein tyrosine kinase (PTK) phosphorylation of the CEACAM3 cytoplasmic domain, we hypothesized that an SH2-containing protein might be involved in CEACAM3-initiated, phagocytosis-promoting signals. Accordingly, we screened glutathione-S-transferase (GST) fusion proteins containing SH2 domains derived from a panel of signaling and adapter molecules for their ability to associate with CEACAM3. In vitro pull-down assays demonstrated that the SH2 domain of the adapter molecule Nck (GST-Nck SH2), but not other SH2 domains such as the Grb2 SH2 domain, interact with CEACAM3 in a phosphotyrosine-dependent manner. Either deletion of the cytoplasmic tail of CEACAM3, or point-mutation of a critical arginine residue in the SH2 domain of Nck (GST-NckSH2R308K) that disrupts phosphotyrosine binding, both abolished CEACAM3-Nck-SH2 interaction. Upon infection of human cells with CEACAM-binding Neisseria, full-length Nck comprising an SH2 and three SH3 domains co-localized with tyrosine phosphorylated CEACAM3 and associated bacteria as analyzed by immunofluorescence staining and confocal microscopy. In addition, Nck could be detected in CEACAM3 immunoprecipitates confirming the interaction in vivo. Importantly, overexpression of a GFP-fusion protein of the isolated Nck SH2 domain (GFP-Nck-SH2), but not GFP or GFP-Nck SH2 R308K reduced CEACAM3-mediated phagocytosis of CEACAM-binding Neisseria suggesting that the adaptor molecule Nck plays an important role in CEACAM3-initiated signaling leading to internalization and elimination of human-specific pathogens.
The transmission of proliferative and developmental signals from activated cell-surface receptors to initiation of cellular responses in the nucleus is synergically controlled by the coordinated action of a diverse set of intracellular signalling proteins. The Ras/Raf/MEK/MAPK signalling pathway has been shown to control the expression of genes which are crucial for the physiological regulation of cell proliferation, differentiation and apoptosis. Within this signalling cascade, the Raf protein family of serine/threonine kinases serves as a central intermediate which connects to many of other signal transduction pathways. To elucidate the signalling functions of the different Raf kinases in motoneurons during development, the expression, distribution and subcellular localization of Rafs in the spinal cord and the facial nucleus in brainstem of mice at various embryonic and postnatal stages were investigated. Moreover, we have investigated the intracellular redistribution of Raf molecules in isolated motoneurons from 13 or 14 day old mouse embryos, after addition or withdrawal of neurotrophic factors to induce Raf kinases activation in vitro. Furthermore, in order to investigate the potential anti-apoptotic function of Raf kinases on motoneurons, we isolated motoneurons from B-raf-/- and c-raf-1-/- mouse embryos and analysed the survival and differentiation effects of neurotrophic factors in motoneurons lacking B-Raf and c-Raf-1. We provide evidence here that all three Raf kinases are expressed in mouse spinal motoneurons. Their expression increases during the period of naturally occurring cell death of motoneurons. In sections of embryonic and postnatal spinal cord, motoneurons express exclusively B-Raf and c-Raf-1, but not A-Raf, and subcellularly Raf kinases are obviously colocalized with mitochondria. In isolated motoneurons, most of the B-Raf or c-Raf-1 immunoreactivity is located in the perinuclear space but also in the nucleus, especially after activation by addition of CNTF and BDNF in vitro. We found that c-Raf-1 translocation from the cytosol into the nucleus of motoneurons after its activation by neurotrophic factors is a distinct event. As a central finding of our study, we observed that the viability of isolated motoneurons from B-raf but not c-raf-1 knockout mice is lost even in the presence of CNTF and other neurotrophic factors. This indicates that B-Raf but not c-Raf-1, which is still present in B-raf deficient motoneurons, plays a crucial role in mediating the survival effect of neurotrophic factors during development. In order to prove that B-Raf is an essential player in this scenario, we have re-expressed B-Raf in mutant sensory and motor neurons by transfection. The motoneurons and the sensory neurons from B-raf knockout mouse which were transfected with exogenous B-raf gene revealed the same viability in the presence of neurotrophic factors as primary neurons from wild-type mice. Our results suggest that Raf kinases have important signalling functions in motoneurons in mouse CNS. In vitro, activation causes redistribution of Raf protein kinases, particularly for c-Raf-1, from motoneuronal cytoplasm into the nucleus. This redistribution of c-Raf-1, however, is not necessary for the survival effect of neurotrophic factors, given that B-raf-/- motor and sensory neurons can not survive despite the presence of c-Raf-1. We hypothesize that c-Raf-1 nuclear translocation may play a direct role in transcriptional regulation as a consequence of neurotrophic factor induced phosphorylation and activation of c-Raf-1 in motoneurons. Moreover, the identification of target genes for nuclear translocated c-Raf-1 and of specific cellular functions initiated by this mechanism awaits its characterization.
Extracellular signals are translated and amplified via cascades of serially switched protein kinases, MAP kinases (MAPKs). One of the MAP pathways, the classical RAS/RAF/MEK/ERK pathway, transduces signals from receptor tyrosine kinases and plays a central role in regulation of cell proliferation. RAF kinases (A-, B- and C-RAF) function atop of this cascade and convert signals emanating from conformational change of RAS GTPases into their kinase activity, which in turn phosphorylates their immediate substrate, MEK. Disregulated kinase activity of RAF can result in tumor formation, as documented for many types of cancer, predominantly melanomas and thyroid carcinomas (B-RAF). A-RAF is the least characterized RAF, possibly due to its low intrinsic kinase activity and comparatively mild phenotype of A-RAF knockout mice. Nevertheless, the unique phenotype of araf -/- mice, showed predominantly neurological abnormalities such as cerebellum disorders, suggesting that A-RAF participates in a specific process not complemented by activities of B- and CRAF. Here we describe the role of A-RAF in membrane trafficking and identify its function in a specific step of endocytosis. This work led to the discovery of a C-terminally truncated version of A-RAF, AR149 that strongly interfered with cell growth and polarization in yeast and with endocytosis and actin polymerization in mammalian cells. As this work was in progress two splicing isoforms of ARAF, termed DA-RAF1 and DA-RAF2 were described that act as natural inhibitors of RAS-ERK signaling during myogenic differentiation (Yokoyama et al., 2007). DA-RAF2 contains the first 153 aa of A-RAF and thus is nearly identical with AR149. AR149 localized specifically to the recycling endosomal compartments as confirmed by colocalization and coimmunoprecipitation with ARF6. Expression of AR149 interferes with recycling of endocytosed transferrin (Tfn) and with actin polymerization. The endocytic compartment, where internalized Tfn is trapped, was identified as ARF6- and RAB11- positive endocytic vesicles. We conclude that the inhibition of Tfn trafficking in the absence of A-RAF or under overexpression of AR149 occurs between tubular- and TGNassociated recycling endosomal compartments. siRNA-mediated depletion of endogenous A-RAF or inhibition of MEK by U0126 mimic the AR149 overexpression phenotype, supporting a role of ARAF regulated ERK signalling at endosomes that is controlled by AR149 and targets ARF6. Our data additionally suggest EFA6 as a partner of A-RAF during activation of ARF6. The novel findings on the A-RAF localization and the interaction with ARF6 have led to a new model of ARAF function were A-RAF via activation of ARF6 controls the recycling of endocytic vesicles.Endocytosis and rapid recycling of synaptic vesicles is critically important for the physiological function of neurons. The finding, that A-RAF regulates endocytic recycling open a new perspective for investigation of the role of A-RAF in the nervous system.
A successful therapy for colorectal cancer (CRC), one of the most common malignancies worldwide, requires the greatest possible research effort. Of critical importance is an understanding of the relevant intracellular networks of signaling cascades, their activation, and the resulting cellular changes that are a prerequisite for a more successful CRC therapy. Vascular endothelial growth factor (VEGF) and the appropriate VEGF receptors represent molecular targets that have already been successfully implemented in the clinic (i.e. using monoclonal antibodies, tyrosine kinase inhibitors). However, for platelet derived growth factor (PDGF) and the relevant PDGF receptors, there are currently no clinically approved molecular therapeutics available. However, there are preliminary data to show that PDGF and its associated signaling pathways play an important role in CRC progression. In particular, the PI3K/Akt/mTOR pathway is emerging as an important intracellular partner of PDGF with which to control proliferation, migration, and angiogenesis in tumor cells.
Therefore it was the objective of this work to investigate the multifactorial influence of PDGF on proliferation and metabolism, depending on CRC mutation status. The intention was to identify new therapeutic targets for future cancer therapy through analyses of PDGF-induced intracellular changes.
For this purpose two human colorectal cancer cell lines were analyzed at gene and/or protein level for components of the PI3K/Akt/mTOR and MAPK signaling pathway, c-Myc, p53, and HIF1α (hypoxia-inducible-factor 1α). Changes in proliferation and metabolism, either during stimulation with PDGF and/or PI3K/Akt/mTOR inhibition, were also investigated. Experiments conducted at protein level during PDGF stimulation and/or PI3K/Akt/mTOR inhibition revealed changes in signaling pathways and crosstalk. The influence of the tumor suppressors (retinoblastoma, Rb), oncogenes (c-Myc, p53mut), and HIF1α during stimulation with PDGF, and their interactions in the tumor cell with respect to proliferation and glycolysis warrant further examination in terms of clinical treatment options. Investigations at the gene level of ex vivo samples (UICC I-IV) complete the study with regards to the clinical relevance of PDGF.
PDGF stimulation increases tumor cell proliferation in HT29 cells via the PI3K/Akt/mTOR pathway rather than the MAPK pathway. However, if the PI3K/Akt/mTOR pathway is pharmacologically blocked, PDGF stimulation is mediated by inhibitory crosstalk through the MAPK pathway. Further analyses revealed that specific Akt inhibition impedes tumor cell growth, while PI3K inhibition had little effect on proliferation. Inhibitory crosstalk was found to be responsible for these different effects. Careful intervention strategies are therefore required if future therapies intend to make use of these specific signaling pathways. One aim of future research should be to gain a better understanding of the crosstalk between these signaling pathways. In this fashion, “over-inhibition” of the signal pathways, which would result in additional clinical side effects for patients, could be prevented.
In late stage UICC, more mutation events occur, with tumorigenicity promoted by an increased mutation rate. Given that PDGF is increasingly expressed in the late UICC stages, our data would indicate that PDGF's effects are amplified with increasing malignancy. The activating effect of PDGF on the PI3K/Akt/mTOR pathway and subsequent changes in the activity of p53mut, Rb, c-Myc, and HIF1α, lead to an unfavorable prognosis for colon cancer patients. PDGF acts on colon cancer cells in an Akt-activating, glycolysis-dependent manner. PDGF increases glycolysis and the ability of CRC cells to adjust their energy metabolism. These activities should be taken as possible starting points with which to design therapeutic interventions for CRC therapy.
PDGF, as another representative of the growth factor family, seems to play a similar role to VEGF in CRC. The data from this study underline the importance of the PDGF - PI3K/Akt/mTOR pathway-axis and its potential as a possible target in colorectal cancer. Thus PDGF represents an attractive therapeutic target, besides the VEGF/EGFR-based therapies already used in CRC.
A disturbance in the symbiotic mutualism between the intestinal microbiome and the human host’s organism (syn. dysbiosis) accompanies the development of a variety of inflammatory and metabolic diseases that comprise the Metabolic Syndrome, chronic inflammatory gut diseases like Crohn’s disease, Non-alcoholic fatty liver disease (NAFLD) and cardiovascular diseases, among others. The changed uptake and effectiveness of short chain fatty acids (SCFAs) as well as an increase of the intestinal permeability are common, interdependent disease elements in this regard. Short chain fatty acids are end-products of intestinal bacterial fermentation and affect the mucosal barrier integrity via numerous molecular mechanisms.
There is evidence to suggest, that SCFAs have a modulating influence on Signal transducer and activator of transcription 3 (STAT3) in intestinal epithelial cells. STAT3 is a central gene-transcription factor in signaling pathways of proliferation and inflammation. It can be activated by growth factors and other intercellular signaling molecules like the cytokine Oncostatin M (OSM). The mode of STAT3’s activation exhibits, finally, a decisive influence on the immunological balance at the intestinal mucosa. Therefore, the posttranslational modification of STAT3 under the influence of SCFAs is likely to be a very important factor within the development and -progression of dysbiosis-associated diseases.
In this study, a clear positive in vitro-effect of the short chain fatty acid butyrate on the posttranslational serine727-phosphorylation of STAT3 and its total protein amount in the human adenocarcinoma cell line CACO2 is verified. Moreover, an increased gene expression of the OSM-receptor subunit OSMRβ can be observed after butyrate incubation. Histone deacetylase inhibition is shown to have a predominant role in these effects. Furthermore, a subsequent p38 MAPK-activation by Butyrate is found to be a key molecular mechanism regarding the STAT3-phosphorylation at serine727-residues. To consider the portion of butyrate receptor signaling in this context in future assays, a CACO-2 cell 3D-culture model is introduced in which an improvement of the GPR109A-receptor expression in CACO-2 cells is accomplished.
Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine that is engaged in regulating versatile cellular processes that are pivotal for development and homeostasis of most tissues in multicellular organisms. TGF-ß signal transduction is initially propagated by binding of TGF-ß to transmembrane serine/threonine kinase receptors, designated TßRI and TßRII. Upon activation, the receptors phosphorylate Smad proteins which serve as downstream mediators that enter the nucleus and finally trigger transcriptional responses of specific genes. During the past years, it became evident that signaling cascades do not proceed in a linear fashion but rather represent a complex network of numerous pathways that mutually influence each other. Along these lines, members of the TGF-ß superfamily are attributed to synergize with neurotrophins. Together, they mediate neurotrophic effects in different populations of the nervous system, suggesting that an interdependence exists between TGF-ßs on the one hand and neurotrophins on the other. In the present work, the crosstalk of NGF and TGF-ß/Smad signaling pathways is characterized in rat pheochromocytoma cells (PC12) which are frequently used as a model system for neuronal differentiation. PC12 cells were found to be unresponsive to TGF-ß due to limiting levels of TßRII. However, stimulation with NGF results in initiation of Smad-mediated transcription independent of TGF-ß. Binding of NGF to functional TrkA receptors triggers activation of Smad3. This NGF-dependent Smad activation occurs by a mechanism which is different from being induced by TGF-ß receptors in that it provokes a different phosphorylation pattern of R-Smads. Together with an inferior role of TßRI, Smad3 is proposed to serve as a substrate for cellular kinases other than TßRI. Based on the presented involvement of components of both, the MAPK/Erk and the TAK1/MKK6 cascade, signal mediators of these pathways rank as candidates to mediate direct activation of Smad3. Smad3 is subsequently translocated to the nucleus and activates transcription in a Smad4-dependent manner. Negative regulation is provided by Smad7 which was found to act as a potent inhibitor of Smad signaling not only in TGF-ß- but also in NGF-mediated cascades. The potential of NGF to activate the Smad pathway independent of TGF-ß might be of special importance in regulating expression of genes that are essential for the development and function of neuronal cells or of other NGF-sensitive cells, in particular those which are TGF-ß-resistant.
The defense against invading pathogens is, amongst other things, mediated via the action of antibodies. Class-switched antibodies and antibodies of high affinity are produced by plasma cells descending from germinal center B (GCB) cells. GCB cells develop in the germinal center (GC), a specialized microstructure found in the B-cell follicle of secondary lymphoid organs. GCB-cell maturation and proliferation are supported by follicular T- helper (Tfh) cells. On the other hand, follicular regulatory T (Tfr) cells control this process in quantity and quality preventing, for instance, the formation of autoantibodies directed against endogenous structures. The development of GCB, Tfh and Tfr cells essentially depends on the migration into the GC, which is mediated via the expression of the chemokine receptor CXCR5.
One transcription factor highly expressed in follicular T cells, comprising Tfh and Tfr cells, is NFATc1. Tfr cells additionally express the transcriptional repressor Blimp-1, which is not expressed in Tfh cells. We found that NFATc1 is transactivating Cxcr5 via response elements in the promoter and enhancer in vitro. Blimp-1 binds to the same elements, transactivating Cxcr5 expression in cooperation with NFATc1, whilst mediating Cxcr5- repression on its own. In Tfr cells Blimp-1 suppresses CXCR5 expression in the absence of NFATc1. Blimp-1 itself is necessary to restrict Tfr-cell frequencies and to mediate Tfr- cell function as in mice with Blimp-1-ablated Tregs high frequencies of Tfr cells do not reduce GCB- or Tfh cell frequencies. NFATc1 and Blimp-1 double deficient Tfr cells show additional loss of function, which becomes visible in clearly expanded antibody titers.
To evaluate the function of NFATc1 in Tfr cells, we not only deleted it, but also overexpressed a constitutive active form of NFATc1/aA (caNFATc1/aA) in regulatory T cells (Tregs). The latter is leading to an upregulation of CXCR5 per cell, without changing Tfh or Tfr-cell frequencies. However, the high density of surface CXCR5 enhances the migration of Tfr cells deep into the GC, which results in a tighter control of the antigen- specific humoral immune response. Additionally, caNFATc1/aA increases the expression of genes coding for Tfr effector molecules like Il1rn, Il10, Tigit and Ctla4. Interestingly, this part of the transcriptional change is dependent on the presence of Blimp-1. Furthermore, Blimp-1 regulates the expression of multiple chemokine receptor genes on the background of caNFATc1/aA.
In contrast, when caNFATc1/aA is overexpressed in all T cells, the frequencies of Tfh- and GCB cells are dominantly reduced. This effect seems to stem from the conventional T- cell (Tcon) side, most probably originating from increased secretion of interleukin-2 (IL- 2) via the caNFATc1/aA overexpressing Tcons. IL-2 is known to hinder the germinal center reaction (GCR) and it might in its abundance not be neutralizable by Tfr cells.
Taken together, NFATc1 and Blimp-1 cooperate to control the migration of Tfr cells into the GC. Tfr cells in the GC depend on NFATc1 and Blimp-1 to perform their proper function. Overexpression of caNFATc1 in Tregs strengthens Tfr function in a Blimp-1-dependent manner, whilst overexpression of caNFATc1 in all T cells dominantly diminishes the GCR.
In physiological conditions platelets have a major role in maintaining haemostasis. Platelets prevent bleeding from wounds by distinguishing normal endothelial cells in vasculature from areas with lesions to which they adhere. Interaction of platelet agonists and their receptors is controlled by intracellular signaling molecules that regulate the activation state of platelets. Very important intracellular signaling molecules are cyclic nucleotides (cGMP and cAMP), both involved in inhibition of platelet activation. Formation of cGMP and cAMP in platelets is stimulated by endothelial-derived NO and prostacyclin (PGI2), which then mediate inhibition of platelets by activating protein kinase G (PKG) and protein kinase A (PKA). Recently, it has been suggested that reactive oxygen species (ROS) represent new modulators of cell signaling within different cell types. The work summarized here describes the involvement of platelet ROS production in platelet activation, the relation of NO/cGMP/PKG I pathway to ROS and to mitogen-activated protein kinases (MAP kinase) signaling, and the involvement of cyclic nucleotides in megakaryocyte and platelet development. Platelets activated with different agonists produce intracellular but not extracellular ROS by activation of NAD(P)H oxidase. In addition, ROS produced in platelets significantly affects αIIbβ3 integrin activation but not alpha/dense granule secretion and platelet shape change. Thrombin induced integrin αIIbβ3 activation is significantly decreased after pretreatment of platelets with NAD(P)H oxidase inhibitors and superoxide scavengers. These inhibitors also reduce platelet aggregation and thrombus formation on collagen under high shear and achieve their effects independently of the NO/cGMP pathway. ADP secreted from platelet dense granules with subsequent activation of P2Y12 receptors as well as thromboxane A2 release are found to be important upstream mediators of p38 MAP kinase activation by thrombin. However, p38 MAP kinase activation does not significantly contribute to calcium mobilization, P-selectin expression, αIIbβ3 integrin activation and aggregation of human platelets in response to thrombin. Finally, PKG activation does not stimulate, but rather inhibit, p38 and ERK MAP kinases in human platelets. Further study revealed that cyclic nucleotides not only inhibit platelet activation, but are also involved, albeit differentially, in megakaryocyte and platelet development. cAMP is engaged in haematopoietic stem cell differentiation to megakaryocytes, and cGMP has no impact on this process. While PKA is already present in stem cells, expression of proteins involved in cGMP signaling (soluble guanylyl cyclase, sGC; PKG) increases with maturation of megakaryocytes. In the final step of megakaryocyte maturation that includes release of platelets, cGMP and cAMP have mild but opposing effects: cGMP increases platelet production while cAMP decreases it indicating a finely regulated process that could depend on stimulus coming from adjacent endothelial cells of sinusoids in bone marrow. The results of this thesis contribute to a better understanding of platelet regulation and of the possible molecular mechanisms involved in megakaryocyte maturation in bone marrow vascular microenvironment.
The Immune system exerts its response against invading pathogens via a cumulative, sequential cooperation of immune cells coordinated by their secreted products. Immune cells, such as macrophages and dendritic cells (DCs), express toll-like receptors (TLRs) to sense the presence of pathogens through pathogen-associated molecular patterns (PAMPs). The interaction of PAMPs with TLRs elicits a cytosolic signaling cascade that enhances the expression of genes to stimulate inflammation. Interleukin 1 receptor-associated kinase 2 (IRAK2) is a component of the TLR signaling pathway. IRAK2 transduces the TLR signal via a direct interaction with TNF receptor-associated factor 6 (TRAF6) and subsequent enhancement of its ubiquitination.
During my PhD thesis, I determined that a 55-amino acid long stretch at the C-terminal end of IRAK2 is important for TLR signaling. Overexpression of C-terminal truncated IRAK2 (IRAK2Δ55) in the murine macrophage cell line RAW 264.7 led to impaired CD40 expression after TLR4 stimulation by Lipopolysaccharide (LPS). I observed attenuated competency of IRAK2Δ55 in restoring a full TLR signaling response i.e. IL-6 secretion, NO production and CD40 expression in IRAK2-deficient RAW cells generated via CRISPR-Cas9 approach. Additionally, diminished TLR4 induced activation of nuclear factor κB (NF-κB) and extracellular signal related kinase (ERK) was observed with IRAK2Δ55 reconstituted RAW cells as compared to cell reconstituted with wildtype IRAK2.
IRAK2Δ55 reconstituted RAW cells also exhibited reduced TLR4-induced cell death and phosphorylation of receptor interacting protein kinase 3 (RIP3). Co-immunoprecipitation experiments in HEK 293T cells showed that IRAK2Δ55 was still able to bind to TRAF6 alike IRAK2 but failed to induce ubiquitination of TRAF6. In conclusion, the results suggest that the IRAK2 TRAF6 interaction is not sufficient to sustain full TLR signaling. An C-terminus-dependent unknown molecular mechanism is also involved.
Through my PhD work, I also analyzed a B cell lineage-specific HECTD1 knock-out mice. HECTD1 is an E3 ubiquitin ligase for various substrate proteins, such as heat shock protein 90 (HSP90), adenomatous polyposis coli and phosphatidylinositol phosphate kinase type 1 γ. Hsp90 regulates a variety of signaling molecules in NF-κB activation pathways which are essential for an optimal B cell response.
HECTD1-deficient pro-B cells developed normally into mature B cells. However, TLR4 stimulated HECTD1-deficient B cells displayed reduced immunoglobulin (Ig) production in in vitro cultures. In addition, mice with HECTD1-deficient B cells showed a diminished Ig response after nitrophenylacetyl-keyhole limpet hemocyanin immunization. Thus, HECTD1 is necessary for efficient Ig secretion.
Flagellar motility and chemotaxis are essential virulence traits required for the ability of Helicobacter pylori to colonize the gastric mucosa. The flagellar regulatory network and the complex chemotaxis system of H. pylori are fundamentally different from other bacteria, despite many similarities. In H. pylori expression of the flagella is controlled by a complex regulatory cascade involving the two-component system FlgR-HP244, the sigma factors 54 and 28 and the anti-sigma 28 factor FlgM. Thus far, the input signal for histidine kinase HP244, which activates the transcriptional regulator FlgR, which triggers sigma factor 54-dependent transcription of the flagellar class 2 genes, is not known. Based on a yeast two-hybrid screen a highly significant protein-protein interaction between the H. pylori protein HP137 and both the histidine kinase HP244 and the flagellar hook protein HP908 (FlgE´) has been reported recently (Rain et al., 2001). So far, no function could be assigned to HP137. Interestingly, the interaction between HP137 and histidine kinase HP244 was observed in the characteristic block N sequence motif of the C-terminal ATP-binding kinase domain. In this work a potential role of HP137 in a feedback regulatory mechanism controlling the activity of histidine kinase HP244 in the flagellar regulation of H. pylori was investigated. Although the substitution of the gene encoding HP137 by a kanamycin cassette resulted in non-motile bacteria, the failure to restore motility by the reintroduction of hp137 in cis into the mutant strain, and the observation that HP137 has no significant effect on the activity of histidine kinase HP244 in vitro indicated that HP137 is not directly involved in flagellar regulation. Therefore, it was demonstrated that HP137 does not participate in the regulation of flagellar gene expression, neither in H. pylori nor in the closely related bacterium C. jejuni. Chemotactic signal transduction in H. pylori differs from the enterobacterial paradigm in several respects. In addition to a CheY response regulator protein (CheY1) H. pylori contains a CheY-like receiver domain (CheY2) which is C-terminally fused to the histidine kinase CheA. Furthermore, the genome of H. pylori encodes three CheV proteins consisting of an N-terminal CheW-like domain and a C-terminal receiver domain, while there are no orthologues of the chemotaxis genes cheB, cheR, and cheZ. To obtain insight into the mechanism controlling the chemotactic response of H. pylori the phosphotransfer reactions between the purified two-component signalling modules were investigated in vitro. Using in vitro phosphorylation assays it was shown that both H. pylori histidine kinases CheAY2 and CheA´ lacking the CheY-like domain (CheY2) act as ATP-dependent autokinases. Similar to other CheA proteins CheA´ shows a kinetic of phosphorylation represented by an exponential time course, while the kinetics of phosphorylation of CheAY2 is characterized by a short exponential time course followed by the hydrolysis of CheAY2~P. Therefore, it was demonstrated that the presence of the CheY2-like receiver domain influences the stability of the phosphorylated P1 domain of the CheA part of the bifunctional protein. Furthermore, it was proven that both CheY1 and CheY2 are phosphorylated by CheAY2 and CheA´~P and that the three CheV proteins mediate the dephosphorylation of CheA´~P, although with a clearly reduced efficiency as compared to CheY1 and CheY2. Moreover, CheA´ is capable of donating its phospho group to the CheY1 protein from C. jejuni and to CheY protein from E. coli. Retrophosphorylation experiments indicated that CheY1~P is able to transfer the phosphate group back to the HK CheAY2 and the receiver domain present in the bifunctional CheAY2 protein acts as a phosphate sink fine tuning the activity of the freely diffusible CheY1 protein, which is thought to interact with the flagellar motor. Hence, in this work evidence of a complex phosphorelay in the chemotaxis system was obtained which has similarities to other systems with multiple CheY proteins. The role of the CheV proteins remain unclear at the moment, but they might be engaged in a further fine regulation of the phosphate flow in this complex chemotaxis system and the independent function of the two domains CheA´ and CheY2 is not sufficient for normal chemotactic signalling in vivo.
Activation of mitogen-activated protein (MAP) kinases is a common reaction of plant cells in defense-related signal transduction pathways. Since the downstream events after the activation of MAP kinases are largely unknown in plants, the role of MAP kinases in the co-ordinate regulation of defense reactions and primary carbon metabolism by stress related stimuli has been analyzed in tomato. Thus, the relationship between mitogen activated protein kinases (LpMPK2 and LpMPK3) and extracellular invertases Lin6, as the key enzyme of an apoplasmic phloem unloading pathway, has been analyzed. The results showed that the mRNAs of LpMPK3 and Lin6 are sequentially induced by the same set of stress related stimuli (E-Fol, PGA,wounding, and KCl). The induction of the Lin6 promotor, as revealed by an increase in β-glucuronidase activity after 2 hours, was dependent both on the expression and activation of LpMPK3. These data suggest that the induction of extracellular invertase Lin6 by stress related stimuli requires LpMPK3. Glucose, metabolic molecule, was shown to result in the simultaneous induction of AtMPK4 and AtMPK6 activities that could be separated by anion-exchange chromatography, and characterized by differential cross-reaction with MAP kinase antibodies. Taken together, these data suggest that the activation of MAP inases play central roles in the regulation of sugar signaling. Stomatal movement is controlled by environmental signals including light intensity,humidity and atmospheric CO2 level. In Arabidopsis, a complete MAP kinase signaling cascade regulates stomatal development and patterning. However, the movement of stomata mediated by CO2 induced signaling pathways is not fully studied in higher plants. Here, we show that elevated levels of CO2 induce rapid and transient activation of SIPK and NtMPK4. The activation of both MAP kinases may regulate the anion channel activation for stomatal movement by the elevated level CO2. Up to now, the non-antioxidant function of tocopherol is not clear in higher plant,whereas the ability of tocopherol to modulate the stress tolerance mediated by function of antioxidant has been described in numerous studies. Thus, the function of α-tocopherol in stimuli-induced signal transduction pathways mediated by MAP kinase has been analyzed in tobacco. It has been shown that the activation of MAP kinase was induced by treatment of fungal elicitor and α-tocopherol phosphate but not α-tocopherol. Interestingly, α-tocopherol showed the transient inhibitory effect on the activation of stimuli-induced MAP Kinases in BY2 cells and tobacco plants, whereas ascorbate did not inhibit the activation of MAP kinases. The inhibitory activity test indicated that current application may indirectly affect the activity of MAP kinases. These results suggest that α-tocopherol can negatively regulate stimuliinduced signal transduction pathways via inactivation of MAP kinases. The purine-analogues have been tested and reported to be specific inhibitors of protein kinases mediated by structural-based selectivity in mammalian. Here, we tested C2, N6, N9-trisubstituted purines to determine basic relationship between their chemical structure and inhibitory activity using a particular plant MAP kinase. The modification of substitution in position C2 and N9 caused the increased inhibitory activity of 6-(benzylamino) purine analogue. In addition, 6-(isopentenylamino) purine analogues suggested that addition of a methyl group to position N9 caused at least 2-fold increased inhibitory activity compared with the addition of isopropyl group.Taken together, our study suggests that the selectivity and potency of inhibitors can be improved by structure modification. In addition, we have characterized the physiological function of Arabidopsis thaliana PLAT domain protein 1 (AtPDP1) in modulating the interaction of defense pathways mediated by biotic and abiotic factors. Interestingly, overexpression of AtPDP1 resulted in increasing susceptibility of virulent pathogens and necrotrophic fungus, and developing necrosis induced by unknown biotic factors. However, these overexperssion lines showed the significantly delayed senescence and higher level of phosystem II quantum yield compared with control plants against high salt stress. Our results strongly indicate that AtPDP1 positively regulate with salt tolerance, and enhances the sensitivity to biotic stresses. We propose that the AtPDP1 might be regulated with the complex pathways of interplay among various signaling during stress adaptation.