LRP1 modulates APP trafficking and APP metabolism within compartments of the secretory pathway. Increased AICD generation is ineffective in nuclear translocation and transcriptional activation

LRP1 modulates APP trafficking and metabolism within compartments of the secretory pathway The amyloid precursor protein (APP) is the parent protein to the amyloid beta peptide (Abeta) and is a central player in Alzheimer’s disease (AD) pathology. Abeta liberation depends on APP cleavage by beta- and gamma-secretases. To date, only a unilateral view of APP processing exists, excluding other proteins, which might be transported together and/or processed dependent on each other by the secretases described above. The low density lipoprotein receptor related protein 1 (LRP1) was shown to function as such a mediator of APP processing at multiple steps. Newly synthesized LRP1 can interact with APP, implying an interaction between these two proteins early in the secretory pathway. Therefore, we wanted to investigate whether LRP1 can mediate APP trafficking along the secretory pathway, and, if so, whether it affects APP processing. Indeed, we demonstrate that APP trafficking is strongly influenced by LRP1 transport through the endoplasmic reticulum (ER) and Golgi compartments. LRP1-constructs with ER- and Golgi-retention motifs (LRP-CT KKAA, LRP-CT KKFF) had the capacity to retard APP trafficking at the respective steps in the secretory pathway. Here, we provide evidence that APP metabolism occurs in close conjunction with LRP1 trafficking, highlighting a new role of lipoprotein receptors in neurodegenerative diseases. Increased AICD generation is ineffective in nuclear translocation and transcriptional activity A sequence of amyloid precursor protein (APP) cleavages gives rise to the APP intracellular domain (AICD) together with amyloid beta peptide (Abeta) and/or p3 fragment. One of the environmental factors identified favouring the accumulation of AICD appears to be a rise in intracellular pH. This accumulation is a result of an abrogated cleavage event and does not extend to other secretase substrates. AICD can activate the transcription of artificially expressed constructs and many downstream gene targets have been discussed. Here we further identified the metabolism and subcellular localization of the constructs used in this well documented gene reporter assay. We also co-examined the mechanistic lead up to the AICD accumulation and explored possible significances for its increased expression. We found that most of the AICD generated under pH neutralized conditions is likely that cleaved from C83. Furthermore, the AICD surplus is not transcriptionally active but rather remains membrane tethered and free in the cytosol where it interacts with Fe65. However, Fe65 is still essential in AICD mediated transcriptional transactivation although its exact role in this set of events is unclear.

Differential cell type-specific transcriptional regulation of the CYP1A1 gene

Cytochrome P450 1A1 (CYP1A1) monooxygenase plays an important role in the metabolism of environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and halogenated polycyclic aromatic hydrocarbons (HAHs). Oxidation of these compounds converts them to the metabolites that subsequently can be conjugated to hydrophilic endogenous entities e.g. glutathione. Derivates generated in this way are water soluble and can be excreted in bile or urine, which is a defense mechanism. Besides detoxification, metabolism by CYP1A1 may lead to deleterious effects since the highly reactive intermediate metabolites are able to react with DNA and thus cause mutagenic effects, as it is in the case of benzo(a) pyrene (B[a]P). CYP1A1 is normally not expressed or expressed at a very low level in the cells but it is inducible by many PAHs and HAHs e.g. by B[a]P or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Transcriptional activation of the CYP1A1 gene is mediated by aryl hydrocarbon receptor (AHR), a basic-helix-loop-helix (bHLH) transcription factor. In the absence of a ligand AHR stays predominantly in the cytoplasm. Ligand binding causes translocation of AHR to the nuclear compartment, its heterodimerization with another bHLH protein, the aryl hydrocarbon nuclear translocator (ARNT) and binding of the AHR/ARNT heterodimer to a DNA motif designated dioxin responsive element (DRE). This process leads to the transcriptional activation of the responsive genes containing DREs in their regulatory regions, e.g. that coding for CYP1A1. TCDD is the most potent known agonist of AHR. Since it is not metabolized by the activated enzymes, exposure to this compound leads to a persisting activation of AHR resulting in diverse toxic effects in the organism. To enlighten the molecular mechanisms that mediate the toxicity of xenobiotics like TCDD and related compounds, the AHR-dependent regulation of the CYP1A1 gene was investigated in two cell lines: human cervix carcinoma (HeLa) and mouse hepatoma (Hepa). Study of AHR activation and its consequence concerning expression of the CYP1A1 enzyme confirmed the TCDD-dependent formation of the AHR/ARNT complex on DRE leading to an increase of the CYP1A1 transcription in Hepa cells. In contrast, in HeLa cells formation of the AHR/ARNT heterodimer and binding of a protein complex containing AHR and ARNT to DRE occurred naturally in the absence of TCDD. Moreover, treatment with TCDD did not affect the AHR/ARNT dimer formation and binding of these proteins to DRE in these cells. Even though the constitutive complex on DRE exists in HeLa, transcription of the CYP1A1 gene was not increased. Furthermore, the CYP1A1 level in HeLa cells remained unchanged in the presence of TCDD suggesting repressional mechanism of the AHR complex function which may hinder the TCDD-dependent mechanisms in these cells. Similar to the native, the mouse CYP1A1-driven reporter constructs containing different regulatory elements were not inducible by TCDD in HeLa cells, which supported a presence of cell type specific trans-acting factor in HeLa cells able to repress both the native CYP1A1 and CYP1A1-driven reporter genes rather than species specific differences between CYP1A1 genes of human and rodent origin. The different regulation of the AHR-mediated transcription of CYP1A1 gene in Hepa and HeLa cells was further explored in order to elucidate two aspects of the AHR function: (I) mechanism involved in the activation of AHR in the absence of exogenous ligand and (II) factor that repress function of the exogenous ligand-independent AHR/ARNT complex. Since preliminary studies revealed that the activation of PKA causes an activation of AHR in Hepa cells in the absence of TCDD, the PKA-dependent signalling pathway was the proposed endogenous mechanism leading to the TCDD-independent activation of AHR in HeLa cells. Activation of PKA by forskolin or db-cAMP as well as inhibition of the kinase by H89 in both HeLa and Hepa cells did not lead to alterations in the AHR interaction with ARNT in the absence of TCDD and had no effect on binding of these proteins to DRE. Moreover, the modulators of PKA did not influence the CYP1A1 activity in these cells in the presence and in the absence of TCDD. Thus, an involvement of PKA in the regulation of the CYP1A1 Gen in HeLa cells was not evaluated in the course of this study. Repression of genes by transcription factors bound to their responsive elements in the absence of ligands has been described for nuclear receptors. These receptors interact with protein complex containing histone deacetylase (HDAC), enzyme responsible for the repressional effect. Thus, a participation of histone deacetylase in the transcriptional modulation of CYP1A1 gene by the constitutively DNA-bound AHR/ARNT complex was supposed. Inhibition of the HDAC activity by trichostatin A (TSA) or sodium butyrate (NaBu) led to an increase of the CYP1A1 transcription in the presence but not in the absence of TCDD in Hepa and HeLa cells. Since amount of the AHR and ARNT proteins remained unchanged upon treatment of the cells with TSA or NaBu, the transcriptional upregulation of CYP1A1 gene was not due to an increased expression of the regulatory proteins. These findings strongly suggest an involvement of HDAC in the repression of the CYP1A1 gene. Similar to the native human CYP1A1 also the mouse CYP1A1-driven reporter gene transfected into HeLa cells was repressed by histone deacetylase since the presence of TSA or NaBu led to an increase in the reporter activity. Induction of reporter gene did not require a presence of the promoter or negative regulatory regions of the CYP1A1 gene. A promoter-distal fragment containing three DREs together with surrounding sequences was sufficient to mediate the effects of the HDAC inhibitors suggesting that the AHR/ARNT binding to its specific DNA recognition site may be important for the CYP1A1 repression. Histone deacetylase is recruited to the specific genes by corepressors, proteins that bind to the transcription factors and interact with other members of the HDAC complex. Western blot analyses revealed a presence of HDAC1 and the corepressors mSin3A (mammalian homolog of yeast Sin3) and SMRT (silencing mediator for retinoid and thyroid hormone receptor) in both cell types, while the corepressor NCoR (nuclear receptor corepressor) was expressed exclusively in HeLa cells. Thus the high inducibility of CYP1A1 in Hepa cells may be due to the absence of NCoR in these cells in contrast to the non-responsive HeLa cells, where the presence of NCoR would support repression of the gene by histone deacetylase. This hypothesis was verified in reporter gene experiments where expression constructs coding for the particular members of the HDAC complex were cotransfected in Hepa cells together with the TCDD-inducible reporter constructs containing the CYP1A1 regulatory sequences. An overexpression of NCoR however did not decrease but instead led to a slight increase of the reporter gene activity in the cells. The expected inhibition was observed solely in the case of SMRT that slightly reduced constitutive and TCDD-induced reporter gene activity. A simultaneous expression of NCoR and SMRT shown no further effects and coexpression of HDAC1 with the two corepressors did not alter this situation. Thus, additional factors that are likely involved in the repression of CYP1A1 gene by HDAC complex remained to be identified. Taking together, characterisation of an exogenous ligand independent AHR/ARNT complex on DRE in HeLa cells that repress transcription of the CYP1A1 gene creates a model system enabling investigation of endogenous processes involved in the regulation of AHR function. This study implicates HDAC-mediated repression of CYP1A1 gene that contributes to the xenobiotic-induced expression in a tissue specific manner. Elucidation of these processes gains an insight into mechanisms leading to deleterious effects of TCDD and related compounds.

DNA-Array-Technologie

Es wurde ein genomischer DNA-Array der Modellpflanze Arabidopsis thaliana mit einer 13.800 EST-Klone umfassenden cDNA-Bibliothek entwickelt und in der Genexpressionsanalyse der pflanzlichen Pathogenabwehr eingesetzt. Mittels PCR-Amplifikation sind 13.000 PCR-Produkte der cDNA-Fragmente hergestellt worden, mit denen 66 genomische Arabidopsis-Arrays auf Nylon und Polypropylen als Trägermaterial hergestellt werden konnten. Die Validierung mit Fluoreszenz- und Radiaktivhybridisierung sowie der Vergleich von drei Normalisierungsmethoden führte zu reproduzierbaren Ergebnissen bei hohem Korrelationskoeffizienten. Die etablierte DNA-Array-Technologie wurde zur Genexpressionsanalyse der pathogeninduzierten Abwehrmechanismen der Pflanze Arabidopsis thaliana in den ersten 24 Stunden nach Infektion mit dem avirulenten Bakterium Pseudomonas syringae pv. tomato eingesetzt. In einer Auswahl von 75 Genen der Stoffwechselwege Glycolyse, Citrat-Cyclus, Pentosephosphat-Cyclus und Glyoxylatmetabolismus konnte für 25 % der Gene, im Shikimat-, Tryptophan- und Phenylpropanoidsyntheseweg für 60 % der Gene eine erhöhte Transkriptionsrate nachgewiesen werden. Die Ergebnisse dieser Arbeit stimmen mit experimentellen Daten verschiedener unabhängiger Studien zur pflanzlichen Pathogenantwort überein. Darüberhinaus sind erstmals Transkriptionsprofile von bisher auf Transkriptionsebene nicht untersuchten Genen erstellt worden. Diese Ergebnisse bestätigen die transkriptionelle Aktivierung ganzer Stoffwechselwege und gewähren erstmals einen Einblick in die koordinierte differentielle Transkription ganzer Stoffwechselwege während der Pathogenabwehr.

Untersuchungen zur Genexpression in Wurzeln der reblausresistenten Unterlagsrebsorte ’Börner’

Die reblausresistente Unterlagsrebsorte ’Börner’ reagiert auf einen Reblausbefall mit einer Hypersensitivitätsreaktion (HR), die sich in Form von Nekrosen an Blättern und Wurzeln zeigt. Im Rahmen dieser Dissertation wurde der Resistenzmechanismus mittels differenzieller Genexpressionsanalysen untersucht. Unter Anwendung der suppressiven subtraktiven Hybridisierung, der DNA-Microarraytechnik sowie der GeneFishingTM-Methode erfolgte ein Vergleich zwischen der Genexpression in hypersensitivem Wurzelgewebe und Normalgewebe der Unterlagsrebe ’Börner’. Neben der Reblaus induzierten HR wurde insbesondere auf die experimentelle Induktion durch das Pflanzenhormon Indol-3-Essigsäure (IES) zurückgegriffen. Damit sollten Kenntnisse über die Rolle der IES als auslösender Faktor der Resistenzreaktion gewonnen werden. Die Ergebnisse bestätigen die Annahme, dass die IES Pathogenabwehrreaktionen in ’Börner’ induziert. So konnten Hinweise auf die transkriptionelle Aktivierung Resistenz und HR assoziierter Proteine gefunden werden, wie z.B. Phytoalexine und pathogen-related (PR)-Proteine sowie Vertreter aus der hypersensitive-induced response-Familie. Es konnten weiterhin wertvolle Informationen im Hinblick auf die Transduktion des IES-Signals im Zusammenhang mit der Aktivierung von Resistenzreaktionen gewonnen werden. So wurden Hinweise auf die Beteiligung der Signalsubstanzen Ethylen, Salicylsäure, Jasmonsäure, Calcium sowie reaktiver Sauerstoffspezies gefunden. Es konnten zudem Anhaltspunkte für die Aktivierung des Auxin induzierten Ubiquitin/26S-Proteolyseweges und weiterer Signalkomponenten, wie z.B. Kinasen und Transkriptionsfaktoren, ermittelt werden. Auch auf die Beteiligung von Auxinrezeptoren konnte aufgrund der Resultate geschlossen werden. Damit war es im Rahmen der Dissertation möglich, potenzielle Signaltransduktionswege zu erarbeiten, die für weiterführende Untersuchungen des Reblausresistenzmechanismus von entscheidender Bedeutung sind.

On the cellular stress response to Staphylococcus aureus alpha-toxin

Staphylococcus aureus alpha-hemolysin was the first bacterial toxin recognized to form pores in the plasma membrane of eukaryotic cells. It is secreted as a water-soluble monomer that upon contact with target membranes forms an amphiphatic heptameric beta-barrel which perforates the bilayer. As a consequence, red cells undergo colloidosmotic lyses, while some nucleated cells may succumb to necrosis or programmed cell death. However, most cells are capable of repairing a limited number of membrane lesions, and then respond with productive transcriptional activation of NF-kB. In the present study, by using microarray and semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), data from a previously performed serial analysis of gene expression (SAGE) were extended and verified, revealing that immediate early genes (IEGs) such as c-fos, c-jun and egr-1 are strongly induced at 2-8 h after transient toxin treatment. Activating protein 1 (AP-1: c-Fos, c-Jun) binding activity was increased accordingly. As IEGs are activated by growth factors, these findings led to the discovery that -toxin promotes cell cycle progression of perforated cells in an EGFR-dependent fashion. Although the amount of c-fos mRNA rose rapidly after toxin treatment, c-Fos protein expression was observed only after a lag of about 3 h. Since translation consumes much ATP, which transiently drops after transient membrane perforation, the suspicion arised that membrane-perforation caused global, but temporary downregulation of translation. In fact, eIF2α became heavily phosphorylated minutes after cells had been confronted with the toxin, resulting in shutdown of protein synthesis before cellular ATP levels reached the nadir. GCN2 emerged as a candidate eIF2α kinase, since its expression rapidly increased in toxin-treated cells. Two hours after toxin treatment, GADD34 transcripts, encoding a protein that targets the catalytic subunit of protein phosphatase 1 (PP1) to the endoplasmic reticulum, were overexpressed. This was followed by dephosphorylation of eIF2α and resumption of protein synthesis. Addition of tautomycetin, a specific inhibitor of PP1, led to marked hyperphosphorylation of eIF2α and significantly reduced the drop of ATP-levels in toxin-treated cells. A novel link between two major stress-induced signalling pathways emerged when it was found that both translational arrest and restart were under the control of stress-activated protein kinase (SAPK) p38. The data provide an explanation for the indispensible role of p38 for defence against the archetypal threat of membrane perforation by agents that produce small transmembrane-pores.

The role of SENP1 in B cell development and differentiation

SUMOylation is a highly dynamic and reversible posttranslational protein modification closely related to ubiquitination. SUMOylation regulates a vast array of different cellular functions, such as cell cycle, nuclear transport, DNA damage response, proliferation and transcriptional activation. Several groups have shown in in vitro studies how important SUMOylation is for early B cell development and survival as well as for later plasma cell differentiation. This thesis focuses on the deSUMOylation protease SENP1 and its in vivo effects on B cell development and differentiation. For this a conditional SENP1 knockout mouse model was crossed to the CD19-Cre mouse strain to generate a B cell specific SENP1 knockout mouse.rnIn our conditional SENP1ff CD19-Cre mouse model we observed normal numbers of all B cell subsets in the bone marrow. However in the spleen we observed an impairment of B cell survival, based on a 50% reduction of the follicular B cell compartment, whereas the marginal zone B cell compartment was unchanged. T cell numbers were comparable to control mice. rnFurther, impairments of B cell survival in SENP1ff CD19-Cre mice were analysed after in vivo blocking of IL7R signalling. The αIL7R treatment in mature mice blocked new B cell formation in the bone marrow and increased apoptosis rates could be observed in splenic SENP1 KO B cells. Additionally, a higher turnover rate of B cells was measured by in vivo BrdU incorporation.rnSince it is known that the majority of transcription factors that are important for the maintenance of the germinal centre reaction or for induction of plasma cell development are SUMOylated, the question arose, how defective deSUMOylation will manifest itself in these processes. The majority of in vitro cultured splenic B cells, stimulated to undergo class switch recombination and plasma cell differentiation underwent activation induced cell death. However, the surviving cells increasingly differentiated into IgM expressing plasma cells. Class switch recombination to IgG1 was reduced. These observations stood in line with observation made in in vivo sheep red blood cell immunization experiments, which showed increased amounts of germinal centres and germinal centre B cells, as well as increased amounts of plasma cells differentiation in combination with decreased class switch to IgG1.rnThese results lead to the conclusion that SENP1 KO B cells increasingly undergo apoptosis, however, B cells that survive SENP1 deficiency are more prone to undergo plasma cell differentiation. Further, the precursors of these plasma cells either are not as capable of undergoing class switch recombination or they do switch to IgG1 and succumb to activation induced cell death. One possible explanation for both scenarios could be a defective DNA damage response mechanisms during class switch recombination, caused by impaired deSUMOylation. rn

Transcriptional regulation and functional characterization of the tumor suppressor genes Hugl-1 and Hugl-2

Cancer is a multi-step process in which both the activation of oncogenes and the inactivation of tumor suppressor genes alter the normal cellular programs to a state of proliferation and growth. The regulation of a number of tumor suppressor genes and the mechanism underlying the tumor suppression have been intensively studied. Hugl-1 and Hugl-2, the human homologues of Drosophila lgl are shown to be down-regulated in a variety of cancers including breast, colon, lung and melanoma, but the mechanism responsible for loss of expression is not yet known. The regulation of gene expression is influenced by factors inducing or repressing transcription. The present study was focused on the identification and characterization of the active promoters of Hugl-1 and Hugl-2. Further, the regulation of the promoter and functional consequences of this regulation by specific transcription factors was analyzed. Experiments to delineate the function of the mouse homologue of Hugl-2, mgl2 using transgenic mice model were performed. This study shows that the active promoter for both Hugl-1 and Hugl-2 is located 1000bp upstream of transcription start sites. The study also provides first insight into the regulation of Hugl-2 by an important EMT transcriptional regulator, Snail. Direct binding of Snail to four E-boxes present in Hugl-2 promoter region results in repression of Hugl-2 expression. Hugl-1 and Hugl-2 plays pivotal role in establishment and maintenance of cell polarity in a diversity of cell types and organisms. Loss of epithelial cell polarity is a prerequisite for cancer progression and metastasis and is an important step in inducing EMT in cells. Regulation of Hugl-2 by Snail suggests one of the initial events towards loss of epithelial cell polarity during Snail-mediated EMT. Another important finding of this study is the induction of Hugl-2 expression can reverse the Snail-driven EMT. Inducing Hugl-2 in Snail expressing cells results in the re-expression of epithelial markers E-cadherin and Cytokeratin-18. Further, Hugl-2 also reduces the rate of tumor growth, cell migration and induces the epithelial phenotype in 3D culture model in cells expressing Snail. Studies to gain insight into the signaling pathways involved in reversing Snail-mediated EMT revealed that induction of Hugl-2 expression interferes with the activation of extracellular receptor kinase, Erk. Functional aspects of mammalian lgl in vivo was investigated by establishing mgl2 conditional knockout mice. Though disruption of mgl2 gene in hepatic tissues did not alter the growth and development, ubiquitous disruption of mgl2 gene causes embryonic lethality which is evident by the fact that no mgl2-/- mice were born.