TY - JOUR A1 - Blättner, Sebastian A1 - Das, Sudip A1 - Paprotka, Kerstin A1 - Eilers, Ursula A1 - Krischke, Markus A1 - Kretschmer, Dorothee A1 - Remmele, Christian W. A1 - Dittrich, Marcus A1 - Müller, Tobias A1 - Schuelein-Voelk, Christina A1 - Hertlein, Tobias A1 - Mueller, Martin J. A1 - Huettel, Bruno A1 - Reinhardt, Richard A1 - Ohlsen, Knut A1 - Rudel, Thomas A1 - Fraunholz, Martin J. T1 - Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes JF - PLoS Pathogens N2 - Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection. KW - cell death KW - cytotoxicity KW - Staphylococcus aureus KW - host cells KW - neutrophils KW - macrophages KW - transposable elements KW - epithelial cells Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-180380 VL - 12 IS - 9 ER - TY - THES A1 - Clemen, Holger T1 - Analyse der Expression und möglicher signalinduzierender Eigenschaften des CD1d-Moleküls der Ratte T1 - Analysis of expression and potential signal inducing properties of rat CD1d-molecule N2 - Wie MHC Klasse I und II-Moleküle präsentieren CD1d-Moleküle dem TCR Antigene, allerdings Lipide und Glykolipide und nicht Proteinfragmente. Die Entdeckung der massiven TH1- und TH2-Zytokinproduktion von Typ I-NKT-Zellen nach CD1d-vermittelter Erkennung von α-Galactosylceramid, einem aus dem Meeresschwamm gewonnenen Glykosphingolipid, weckte großes Interesse an ihrem immunregulatorischen Potential und ihrem möglichen Nutzen für neue Immun- und Tumortherapien. Um die Funktion und die Bedeutung von CD1d besser zu verstehen, wurden in dieser Arbeit die Expressionslevel der lymphatischen Gewebe der Ratte und der Maus untersucht. Hierfür wurden die neu generierten monoklonalen Antikörper 232 und 58/4 verwendet, die die CD1d-Moleküle von Ratte und Maus binden und so den direkten Vergleich beider Spezies ermöglichen. Sowohl die isolierten Zellen des Thymus und der Milz als auch des Lymphknotens waren in der LEW- und F344-Ratte sowie in der BALB/c-Maus schwach bis stark CD1d positiv. In der LEW-Ratte und in der F344-Ratte wiesen jeweils ca. 18% der Milzzellen eine vergleichsweise erhöhte CD1d-Expression auf. Dabei handelte es sich in erster Linie um Marginalzonen-B-Zellen. Bestimmte Subpopulationen der Dendritischen Zellen und vermutlich Makrophagen stellten die restlichen CD1d stark positiven Populationen dar. Nur ca. 2% der isolierten Zellen der Lymphknoten der LEW-Ratte waren stark CD1d positiv, wohingegen der LEW-Thymus gemäß dem noch geringeren Anteil an APC kaum Zellen mit erhöhter CD1d-Expression enthielt. In der BALB/c-Maus war der Anteil CD1d stark positiver Milzzellen mit 4% deutlich geringer als in der LEW- oder F344-Milz. Abgesehen von MZ-B-Zellen konnten in der Maus kaum Populationen mit starker CD1d-Expression in den verschiedenen Färbungen festgestellt werden. Demnach stellt CD1d sowohl in der Ratte als auch in der Maus einen guten Marker für MZ-B-Zellen dar. Demgegenüber zeigten vereinzelt kleine Populationen der Milz, des Lymphknotens und des Thymus beider Spezies eine verminderte oder gar keine CD1d-Expression. Zur Analyse möglicher signalinduzierender Eigenschaften der verschiedenen Anti- CD1d-Antikörper wurden ihre Effekte auf rCD1d+ Transduktanten und primäre Zellen untersucht. 58/4 konnte im Gegensatz zu 232 spezifisch über Bindung an Ratten- CD1d Zelltod und Aggregatbildung in Tumor-B-Zellen des Menschen und der Maus, aber nicht in Tumor-T-Zellen, induzieren. Der zytoplasmatische Schwanz der CD1d-Moleküle scheint an der Aggregatbildung beteiligt zu sein. Die Bindung von 58/4 oder 232 führte in überlebenden rCD1d+ Raji-Zellen zu einer ähnlich starken Internalisierung der CD1d-Moleküle. Während nach 5-stündiger Inkubation mit 232 und erneuter CD1d-Färbung wieder die vorherige CD1d- Expression festgestellt wurde, konnte nach Inkubation mit 58/4 eine bleibende Herunterregulierung beobachtet werden. Folglich bewirkte 58/4 ein anderes bzw. stärkeres Signal in den Zellen als 232. Diese Beobachtungen stützen die Signaltransduktion als mögliche weitere Funktion der CD1d-Moleküle neben der Antigenpräsentation und definieren die monoklonalen Antikörper 232 und 58/4 als nützliche Werkzeuge für weitere Studien zur Analyse der molekularen Mechanismen der CD1d-vermittelten Signaltransduktion. Das Verständnis solcher Mechanismen bildet wiederum die Grundlage für die Entwicklung neuer Therapien z. B. zur Eliminierung CD1d exprimierender Tumore. N2 - CD1d-molecules present in contrast to MHC class I and II-molecules lipid antigens to T-cells. There are some observations showing strong CD1d-mediated effects on immune system, so that there is a growing interest in understanding these mechanisms in order to be able to regulate this influence. The expression of CD1d in rats hasnt been known yet. Since our anti-CD1d-antibodies recognize CD1d-molecules of rats as well as of mice, we could compare these species. Antigen-presenting cells, especially marginal-zone B-cells, showed in both the highest CD1d-expression, so that our anti-CD1d-antibodies can be used for their identification. Only a small part of the cells of thymus, spleen and lymphnodes showed a marginal or no CD1d-expression. The analysis of antibody-inducing effects on CD1d+ cells showed, that some of our antibodies can mediate a CD1d-specific cell-death similar to the well-known MHC II-mediated cell death. Survived cells react with an antibody-dependent rate of aggregation or down-regulation of CD1d indicating our antibodies can become a potential tool for further analysis of CD1d-mediated reactions in order to develop new therapies, for example for elimination of CD1d-expressing tumors. KW - Zelltod KW - Signaltransduktion KW - Immunmodulation KW - CD1d KW - CD1d KW - cell death KW - signal transduction KW - immune modulation Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-65242 ER - TY - JOUR A1 - Fuhr, Viktoria A1 - Heidenreich, Shanice A1 - Srivastava, Mugdha A1 - Riedel, Angela A1 - Düll, Johannes A1 - Gerhard-Hartmann, Elena A1 - Rosenwald, Andreas A1 - Rauert-Wunderlich, Hilka T1 - CD52 and OXPHOS-potential targets in ibrutinib-treated mantle cell lymphoma JF - Cell Death Discovery N2 - Altered features of tumor cells acquired across therapy can result in the survival of treatment-resistant clones that may cause minimal residual disease (MRD). Despite the efficacy of ibrutinib in treating relapsed/refractory mantle cell lymphoma, the obstacle of residual cells contributes to relapses of this mature B-cell neoplasm, and the disease remains incurable. RNA-seq analysis of an ibrutinib-sensitive mantle cell lymphoma cell line following ibrutinib incubation of up to 4 d, corroborated our previously postulated resistance mechanism of a metabolic switch to reliance on oxidative phosphorylation (OXPHOS) in surviving cells. Besides, we had shown that treatment-persisting cells were characterized by increased CD52 expression. Therefore, we hypothesized that combining ibrutinib with another agent targeting these potential escape mechanisms could minimize the risk of survival of ibrutinib-resistant cells. Concomitant use of ibrutinib with OXPHOS-inhibitor IACS-010759 increased toxicity compared to ibrutinib alone. Targeting CD52 was even more efficient, as addition of CD52 mAb in combination with human serum following ibrutinib pretreatment led to rapid complement-dependent-cytotoxicity in an ibrutinib-sensitive cell line. In primary mantle cell lymphoma cells, a higher toxic effect with CD52 mAb was obtained, when cells were pretreated with ibrutinib, but only in an ibrutinib-sensitive cohort. Given the challenge of treating multi-resistant mantle cell lymphoma patients, this work highlights the potential use of anti-CD52 therapy as consolidation after ibrutinib treatment in patients who responded to the BTK inhibitor to achieve MRD negativity and prolong progression-free survival. KW - cancer metabolism KW - cell death KW - target validation KW - targeted therapies Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-300817 SN - 2058-7716 VL - 8 ER - TY - JOUR A1 - Henriksson, Sofia A1 - Calderón-Montaño, José Manuel A1 - Solvie, Daniel A1 - Warpman Berglund, Ulrika A1 - Helleday, Thomas T1 - Overexpressed c-Myc sensitizes cells to TH1579, a mitotic arrest and oxidative DNA damage inducer JF - Biomolecules N2 - Previously, we reported that MTH1 inhibitors TH588 and TH1579 selectively induce oxidative damage and kill Ras-expressing or -transforming cancer cells, as compared to non-transforming immortalized or primary cells. While this explains the impressive anti-cancer properties of the compounds, the molecular mechanism remains elusive. Several oncogenes induce replication stress, resulting in under replicated DNA and replication continuing into mitosis, where TH588 and TH1579 treatment causes toxicity and incorporation of oxidative damage. Hence, we hypothesized that oncogene-induced replication stress explains the cancer selectivity. To test this, we overexpressed c-Myc in human epithelial kidney cells (HA1EB), resulting in increased proliferation, polyploidy and replication stress. TH588 and TH1579 selectively kill c-Myc overexpressing clones, enforcing the cancer cell selective killing of these compounds. Moreover, the toxicity of TH588 and TH1579 in c-Myc overexpressing cells is rescued by transcription, proteasome or CDK1 inhibitors, but not by nucleoside supplementation. We conclude that the molecular toxicological mechanisms of how TH588 and TH1579 kill c-Myc overexpressing cells have several components and involve MTH1-independent proteasomal degradation of c-Myc itself, c-Myc-driven transcription and CDK activation. KW - MTH1 KW - TH588 KW - TH1579 KW - c-Myc KW - replication stress KW - DNA damage KW - cell death KW - cancer Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-297547 SN - 2218-273X VL - 12 IS - 12 ER - TY - THES A1 - Hondke, Sylvia T1 - Elucidation of WISP3 function in human mesenchymal stem cells and chondrocytes T1 - Aufklärung der WISP3 Funktion in humanen mesenchymalen Stammzellen und Chondrozyten N2 - WISP3 is a member of the CCN family which comprises six members found in the 1990’s: Cysteine-rich,angiogenic inducer 61 (CYR61, CCN1), Connective tissue growth factor (CTGF, CCN2), Nephroblastoma overexpressed (NOV, CNN3) and the Wnt1 inducible signalling pathway protein 1-3 (WISP1-3, CCN4-6).They are involved in the adhesion, migration, mitogenesis, chemotaxis, proliferation, cell survival, angiogenesis, tumorigenesis, and wound healing by the interaction with different integrins and heparan sulfate proteoglycans. Until now the only member correlated to the musculoskeletal autosomal disease Progressive Pseudorheumatoid Dysplasia (PPD) is WISP3. PPD is characterised by normal embryonic development followed by cartilage degradation over time starting around the age of three to eight years. Animal studies in mice exhibited no differences between knock out or overexpression compared to wild type litter mates, thus were not able to reproduce the symptoms observed in PPD patients. Studies in vitro and in vivo revealed a role for WISP3 in antagonising BMP, IGF and Wnt signalling pathways. Since most of the knowledge of WISP3 was gained in epithelial cells, cancer cells or chondrocyte cell lines, we investigated the roll of WISP3 in primary human mesenchymal stem cells (hMSCs) as well as primary chondrocytes. WISP3 knock down was efficiently established with three short hairpin RNAs in both cell types, displaying a change of morphology followed by a reduction in cell number. Simultaneous treatment with recombinant WISP3 was not enough to rescue the observed phenotype nor increase the endogenous expression of WISP3. We concluded that WISP3 acts as an essential survival factor, where the loss resulted in the passing of cell cycle control points followed by apoptosis. Nevertheless, Annexin V-Cy3 staining and detection of active caspases by Western blot and immunofluorescence staining detected no clear evidence for apoptosis. Furthermore, the gene expression of the death receptors TRAILR1 and TRAILR2,important for the extrinsic activation of apoptosis, remained unchanged during WISP3 mRNA reduction. Autophagy as cause of cell death was also excluded, given that the autophagy marker LC3 A/B demonstrated to be uncleaved in WISP3-deficient hMSCs. To reveal correlated signalling pathways to WISP3 a whole genome expression analyses of WISP3-deficient hMSCs compared to a control (scramble) was performed. Microarray analyses exhibited differentially regulated genes involved in cell cycle control, adhesion, cytoskeleton and cell death. Cell death observed by WISP3 knock down in hMSCs and chondrocytes might be explained by the induction of necroptosis through the BMP/TAK1/RIPK1 signalling axis. Loss of WISP3 allows BMP to bind its receptor activating the Smad 2/3/4 complex which in turn can activate TAK1 as previously demonstrated in epithelial cells. TAK1 is able to block caspase-dependent apoptosis thereby triggering the assembly of the necrosome resulting in cell death by necroptosis. Together with its role in cell cycle control and extracellular matrix adhesion, as demonstrated in human mammary epithelial cells, the data supports the role of WISP3 as tumor suppressor and survival factor in cells of the musculoskeletal system as well as epithelial cells. N2 - WISP3 ist ein Mitglied der CCN-Familie, die aus sechs Familienmitgliedern besteht und in den 1990er Jahren endeckt wurde: Cysteine-rich, angiogenic inducer 61 (CYR61, CCN1), Connective tissue growth factor (CTGF, CCN2), Nephroblastoma overexpressed (NOV, CNN3) und den Wnt1 inducible signalling pathway protein 1-3 (WISP1-3, CCN4-6). Die CCN-Proteine sind durch ihre Interaktion mit verschiede- nen Integrinen und Heparansulfaten involviert in die Regulation der Adhäsion, der Migration, der Mi- togenese, der Chemotaxis, der Proliferation, des Zellüberlebens, der Angiogenese, der Tumorgenese und der Wundheilung. WISP3 ist momentan das einzige Mitglied, das direkt mit einer muskuloskelettalen Erkrankung, der Progressiven Pseudorheumatoiden Dysplasie (PPD), assoziiert wird. PPD ist charakter- isiert durch eine normale embryonale Entwicklung mit fortschreitender Knorpeldegeneration beginnend im Alter von drei bis acht Jahren. Tierversuche mit knock out oder Überexpression von WISP3 in Mäusen waren nicht in der Lage die Symptome der Erkrankung nachzustellen, da keine Unterschiede im Vergleich zu den Wurfgeschwistern beobachtbar waren. In vitro und in vivo Studien offenbarten eine antagonisierende Rolle für WISP3 im BMP, IGF und Wnt Signalweg. Da die meisten Informationen über WISP3 jedoch in Epithel- und Krebszellen sowie immortalisierten Chondrozytenzelllinien generiert wurden, untersuchten wir die Rolle von WISP3 in primären humanen mesenchymalen Stammzellen (hMSZs) und primären Chondrozyten. Der WISP3 knock down wurde mit drei short hairpin RNAs in beiden Zelltypen etabliert und wies eine veränderte Zellmorphologie sowie eine reduzierte Zellzahl auf. Knock down mit gleichzeitiger rekombi- nanter WISP3-Behandlung konnte den beobachteten Phänotyp sowie den Zellverlust nicht retten und auch eine Änderung der endogenen Genexpression von WISP3 war nicht zu detektieren. Schlussfolgernd muss WISP3 ein wichtiger Überlebensfaktor sein, dessen Verlust zur Überschreitung von Zellzyklus- Kontrollpunkten führt, was in Apoptose mündet. Apoptosenachweise wie Annexin V-Cy3 Färbung, Immunfluoreszenzfärbung und Western blot für aktive Caspasen lieferten keine positiven Beweise für diese Form des Zelltodes. Auch die Genexpression der Todesrezeptoren TRAILR1 und TRAILR2, wichtig für die extrinsische Aktivierung der Apoptose, zeigte kein verändertes Expressionsmuster in WISP3-defizienten hMSZs. Autophagie als Zelltod wurde ebenfalls ausgeschlossen, nachdem im West- ern Blot kein gespaltene Form des Autophagiemarkers LC3 A/B zu detektieren war. Um die Rolle von WISP3 beim Zelltod weiter zu entschlüsseln, wurden Genom-Expressionsanalysen von WISP3-defizienten hMSZs im Vergleich zu Kontroll-hMSZs angefertigt. Die Analysen ergaben unterschiedlich regulierte Gene vor allem in den Bereichen Zellzyklus-Regulation, Adhäsion, Zytoskelett und Zelltod. Der durch WISP3-Verlust ausgelöste Zelltod kann möglicherweise durch die Aktivierung der Nektroptose über den BMP/TAK1/RIPK1 Signalweg erklärt werden. Es ist bekannt, dass WISP3 BMP4 bindet und so dessen Bindung an den Rezeptor verhindert. Bei WISP3 Verlust bindet BMP4 an seinen Rezeptor und aktiviert den Smad 2/3/4 Komplex der wiederum TAK1 phosphoryliert, wie zuvor in Epithelzellen demonstriert. TAK1 ist in der Lage die Caspase-induzierte Apoptose zu blockieren und auf diese Weise die Bildung des Nekrosomes auszulösen, welches zum Zelluntergang durch Nekroptose führt. Zusammen mit seiner Rolle in der Zellzyklus-Kontrolle und der extrazellulären Matrixadhäsion, die in humanen Brustepithelialzellen nachgewiesen wurden, unterstützen diese Daten eine Rolle für WISP3 als Tumorsuppressor und Überlebensfaktor in Zellen des Epithel und des muskuloskelettalen Systems. KW - Knorpelzelle KW - PPD KW - mesenchymal stem cells KW - cell death KW - chondrocytes KW - Mesenchymzelle KW - Dysplasie KW - Genexpression KW - Werk Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-109641 ER - TY - JOUR A1 - Koch, Rebecca-Diana A1 - Hörner, Eva-Maria A1 - Münch, Nadine A1 - Maier, Elke A1 - Kozjak-Pavlovic, Vera T1 - Modulation of Host Cell Death and Lysis Are Required for the Release of Simkania negevensis JF - Frontiers in Cellular and Infection Microbiology N2 - Simkania negevensis is a Chlamydia-like bacterium and emerging pathogen of the respiratory tract. It is an obligate intracellular bacterium with a biphasic developmental cycle, which replicates in a wide range of host cells. The life cycle of S. negevensis has been shown to proceed for more than 12 days, but little is known about the mechanisms that mediate the cellular release of these bacteria. This study focuses on the investigation of host cell exit by S. negevensis and its connection to host cell death modulation. We show that Simkania-infected epithelial HeLa as well as macrophage-like THP-1 cells reduce in number during the course of infection. At the same time, the infectivity of the cell culture supernatant increases, starting at the day 3 for HeLa and day 4 for THP-1 cells and reaching maximum at day 5 post infection. This correlates with the ability of S. negevensis to block TNFα-, but not staurosporin-induced cell death up to 3 days post infection, after which cell death is boosted by the presence of bacteria. Mitochondrial permeabilization through Bax and Bak is not essential for host cell lysis and release of S. negevensis. The inhibition of caspases by Z-VAD-FMK, caspase 1 by Ac-YVAD-CMK, and proteases significantly reduces the number of released infectious particles. In addition, the inhibition of myosin II by blebbistatin also strongly affects Simkania release, pointing to a possible double mechanism of exit through host cell lysis and potentially extrusion. KW - exit KW - release KW - cell death KW - caspases Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-215158 SN - 2235-2988 VL - 10 ER - TY - JOUR A1 - Kucka, Kirstin A1 - Wajant, Harald T1 - Receptor Oligomerization and Its Relevance for Signaling by Receptors of the Tumor Necrosis Factor Receptor Superfamily JF - Frontiers in Cell and Developmental Biology N2 - With the exception of a few signaling incompetent decoy receptors, the receptors of the tumor necrosis factor receptor superfamily (TNFRSF) are signaling competent and engage in signaling pathways resulting in inflammation, proliferation, differentiation, and cell migration and also in cell death induction. TNFRSF receptors (TNFRs) become activated by ligands of the TNF superfamily (TNFSF). TNFSF ligands (TNFLs) occur as trimeric type II transmembrane proteins but often also as soluble ligand trimers released from the membrane-bound form by proteolysis. The signaling competent TNFRs are efficiently activated by the membrane-bound TNFLs. The latter recruit three TNFR molecules, but there is growing evidence that this is not sufficient to trigger all aspects of TNFR signaling; rather, the formed trimeric TNFL–TNFR complexes have to cluster secondarily in the cell-to-cell contact zone for full TNFR activation. With respect to their response to soluble ligand trimers, the signaling competent TNFRs can be subdivided into two groups. TNFRs of one group, designated as category I TNFRs, are robustly activated by soluble ligand trimers. The receptors of a second group (category II TNFRs), however, failed to become properly activated by soluble ligand trimers despite high affinity binding. The limited responsiveness of category II TNFRs to soluble TNFLs can be overcome by physical linkage of two or more soluble ligand trimers or, alternatively, by anchoring the soluble ligand molecules to the cell surface or extracellular matrix. This suggests that category II TNFRs have a limited ability to promote clustering of trimeric TNFL–TNFR complexes outside the context of cell–cell contacts. In this review, we will focus on three aspects on the relevance of receptor oligomerization for TNFR signaling: (i) the structural factors which promote clustering of free and liganded TNFRs, (ii) the signaling pathway specificity of the receptor oligomerization requirement, and (iii) the consequences for the design and development of TNFR agonists. KW - TNF receptor (TNFR) family KW - TNF ligand superfamily KW - NFκB KW - cell death KW - receptor cluster Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227180 SN - 2296-634X VL - 8 ER - TY - JOUR A1 - Lambour, Benjamin A1 - Glenz, René A1 - Forner, Carmen A1 - Krischke, Markus A1 - Mueller, Martin J. A1 - Fekete, Agnes A1 - Waller, Frank T1 - Sphingolipid long-chain base phosphate degradation can be a rate-limiting step in long-chain base homeostasis JF - Frontiers in Plant Science N2 - Sphingolipid long-chain bases (LCBs) are building blocks for membrane-localized sphingolipids, and are involved in signal transduction pathways in plants. Elevated LCB levels are associated with the induction of programmed cell death and pathogen-derived toxin-induced cell death. Therefore, levels of free LCBs can determine survival of plant cells. To elucidate the contribution of metabolic pathways regulating high LCB levels, we applied the deuterium-labeled LCB D-erythro-sphinganine-d7 (D7-d18:0), the first LCB in sphingolipid biosynthesis, to Arabidopsis leaves and quantified labeled LCBs, LCB phosphates (LCB-Ps), and 14 abundant ceramide (Cer) species over time. We show that LCB D7-d18:0 is rapidly converted into the LCBs d18:0P, t18:0, and t18:0P. Deuterium-labeled ceramides were less abundant, but increased over time, with the highest levels detected for Cer(d18:0/16:0), Cer(d18:0/24:0), Cer(t18:0/16:0), and Cer(t18:0/22:0). A more than 50-fold increase of LCB-P levels after leaf incubation in LCB D7-d18:0 indicated that degradation of LCBs via LCB-Ps is important, and we hypothesized that LCB-P degradation could be a rate-limiting step to reduce high levels of LCBs. To functionally test this hypothesis, we constructed a transgenic line with dihydrosphingosine-1-phosphate lyase 1 (DPL1) under control of an inducible promotor. Higher expression of DPL1 significantly reduced elevated LCB-P and LCB levels induced by Fumonisin B1, and rendered plants more resistant against this fungal toxin. Taken together, we provide quantitative data on the contribution of major enzymatic pathways to reduce high LCB levels, which can trigger cell death. Specifically, we provide functional evidence that DPL1 can be a rate-limiting step in regulating high LCB levels. KW - sphingolipid KW - long-chain base KW - plant sphingolipid metabolism KW - cell death KW - metabolic flux analysis KW - dihydrosphingosine-1-phosphate lyase KW - LC–MS/MS Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-277679 SN - 1664-462X VL - 13 ER - TY - THES A1 - Martens, Theresa Maria Christina T1 - Lobaplatin als Agens zur Induktion von Zelltod in triple-negativen Brustkrebszelllinien T1 - Lobaplatin as an agent for the induction of cell death in triple-negative breast cancer cells N2 - Das triple-negative Mammakarzinom ist Hormonrezeptor- und HER2 negativ und mit einer ungünstigen Prognose verbunden. Triple-negative Patientinnen sind unempfindlich gegen die endokrine Therapie und den HER-2/neu Antikörper Trastuzumab und deshalb auf die zytostatische Chemotherapie angewiesen. Lobaplatin ist ein Platinderivat mit wenigen Nebenwirkungen und guter Antitumorwirkung in vitro und deshalb möglicherweise auch für die Behandlung triple-negativer Tumoren geeignet. Für die vorliegende Arbeit haben wir zwei triple-negative und eine klassische Brustkrebszelllinie verwendet. Die beiden triple-negativen Zelllinien wurden anhand einer vorbeschriebenen Mutation identifiziert, um eine Verwechslung auszuschließen. Die Aktivität des p53 Gens wurde in allen drei Zelllinien überprüft, ein aktives p53 Gen jedoch nur in der hormonrezeptor-positiven MCF7 Zelllinie nachgewiesen. Ein Zusammenhang zwischen p53 Status und Ansprechen der Zellen auf Lobaplatin oder Cisplatin konnte nicht belegt werden. Wir haben die zytotoxische Wirkung von Lobaplatin und Cisplatin verglichen. Lobaplatin war in unseren Untersuchungen ähnlich wirksam wie Cisplatin und bei der Behandlung der MCF7 Zelllinie und einer Cisplatin-resistenten Ovarialkarzinomzelllinie sogar dem Cisplatin überlegen. Lobaplatin wies in diesen Zellen keine komplette Kreuzresistenz zu Cisplatin auf. In einem nächsten Schritt wurden unterschiedliche Zytostatika mit Lobaplatin kombiniert. Additive Synergien zwischen den einzelnen Substanzen konnten nicht bewiesen werden, jedoch war ein gutes Zusammenwirken von Lobaplatin und dem in der klinischen Erprobung befindlichen Todesliganden TRAIL zu erkennen. Mittels Nachweis von Spaltprodukten haben wir gezeigt, dass Lobaplatin und Cisplatin eine Aktivierung von Caspasen bewirken. Die aktivierte Caspase 3 konnte allerdings nicht dargestellt werden. Die Begriffe Apoptose und Nekroptose wurden diskutiert und eine Beteiligung beider Prozesse an der Wirkung von Lobaplatin und Cisplatin nachgewiesen. Veränderungen des Zellzyklus wurden untersucht. Die beiden Platinderivate führten zu einem Arrest in der G2-Phase und in höheren Konzentrationen teilweise zu einem G1-Arrest. Insgesamt sind an der zytotoxischen Wirkung von Lobaplatin und Cisplatin und der Entscheidung über Leben oder Tod viele wichtige Mechanismen in wechselnder Rangfolge beteiligt. In unseren Versuchen an zwei triple-negativen und einer klassischen Brustkrebszelllinie erweist sich Lobaplatin als ebenbürtiger Nachfahre des Cisplatin mit einer vergleichbaren Zytotoxizität, weniger schwerwiegenden Nebenwirkungen und der Fähigkeit, vorhandene Cisplatinresistenzen teilweise zu umgehen. Lobaplatin verspricht darüber hinaus in Kombination mit anderen Zytostatika interessante therapeutische Perspektiven, die bisher nur unzureichend erprobt sind. N2 - In this work Lobaplatin was investigated both as a single agent and in combination with other cytostatic drugs in in-vitro models of two triple-negative breast cancer cell lines and one oestrogen receptor-positive breast cancer cell line. KW - Brustkrebs KW - Chemotherapie KW - Platin KW - Zellkultur KW - Synergie KW - Triple-negativ KW - TRAIL KW - Breast cancer KW - triple-negative KW - cell death Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-71974 ER - TY - THES A1 - Niederlechner, Stefanie T1 - Assessment of the basic molecular mechanisms underlying L-glutamine's cytoprotective effects after intestinal injury T1 - Untersuchung und Auswertung der molekularen Mechanismen, die L-Glutamin's protektivem Zellmechanismus nach Intestinaler Verletzung zugrunde liegen N2 - Critical illness like sepsis, shock, and intestinal bowel disease are one of the leading causes of morbidity and mortality in the US and around the world. At present, studies to define new therapeutic interventions that can protect tissues and cells against injury and attenuate inflammation are fields of intense investigation. While research over the past decade has clearly identified GLN as a vital stress substrate facilitating cellular survival following injury, the initiation steps in GLN’s cytoprotective molecular mechanism still remain elusive. Previously published work suggested that stabilization of ECM proteins and activation of ECM receptor osmosignaling may play a central role in the orchestration of many cellular pathways following stress. Thus, I hypothesized that preservation of ECM protein and EGFR levels as well as ECM receptor signaling play key roles in the molecular mechanisms underlying GLN’s protection against thermal injury in the intestine. I was able to confirm via Western blotting and by using silencing RNA against FN, Ntn-1, EGFR, and their negative controls, that GLN-mediated preservation of FN, Ntn-1, and EGFR levels is critical in GLN’s protection against hyperthermia in IEC-6 cells. By using a selective FN-Integrin interaction inhibitor GRGDSP, its negative control peptide GRGESP, and Src-kinase inhibitor PP2, I showed that FN-Integrin signaling and Src-kinase activation are essential in GLN-mediated protection in the intestine. This applied to EGFR signaling as demonstrated using the EGFR tyrosine kinase inhibitor AG1478. In addition to GRGDSP and AG1478, ERK1/2 inhibitors PD98059 and UO126 as well as the p38MAPK inhibitor SB203580 revealed that GLN is protective by activating ERK1/2 and dephosphorylating p38MAPK via FN-Integrin and EGFR signaling. However, GLN-mediated PI3-K/Akt/Hsp70 activation seems to occur independently of FN-Integrin and EGFR signaling as indicated by Western blots as well as experiments using the PI3-K inhibitor LY294002, GRGDSP, and AG1478. The results showed that GLN activates cell survival signaling pathways via integrins as well as EGFRs after hyperthermia. Moreover, I found that GLN-mediated preservation of FN expression after HS is regulated via PI3-K signaling. Whether GLN-mediated PI3-K signaling happens simultaneously to FN-Integrin and EGFR signaling or whether PI3-K signaling coordinates FN-Integrin and EGFR signaling needs to be investigated in future studies. Further, experiments with PD98059 and GRGDSP revealed that ERK1/2 assists in mediating transactivation of HSF-1 following HS. This leads to increases in Hsp70 expression via FN-Integrin signaling, which is known to attenuate apoptosis after thermal injury. Fluorescence microscopy results indicated that HS and GLN regulate cell are size changes and the morphology of F-actin via FN-Integrin signaling. Experiments using GRGDSP and GRGESP showed that GLN enhances cellular survival via FN-Integrin signaling in a manner that does not require increased intracellular GLN concentrations (as quantified using LC-MS/MS). In summary, my thesis work gives new and potentially clinically relevant mechanistic insights into GLN-mediated molecular cell survival pathways. These results warrant clinical translation to assess if clinical outcome of critically ill patients suffering from gastrointestinal diseases can be improved by GLN treatment and/or by targeting the molecular pathways found in my studies. N2 - Kritische Erkrankungen wie Sepsis, Schock und entzündliche Darmerkrankungen sind eine der häufigsten Todesursachen in den Vereinigten Staaten und dem Rest der Welt. Von großer Bedeutung sind deshalb Studien, die darauf abzielen, neue therapeutische Ansätze zu finden, die Gewebe und Zellen vor Verletzungen schützen und Entzündungen verhindern. Im letzten Jahrzehnt hat sich herausgestellt, dass Glutamin Schutz gegen die pathophysiologischen und pathobiochemischen Entgleisungen während solcher Erkrankungen vermitteln kann. Trotzdem ist es bisher nur sehr unvollständig gelungen, die zugrundeliegenden molekularen Mechanismen, die für diese zellulären Schutzfunktionen von Glutamin verantwortlich sind, aufzuklären. In diesem Zusammenhang gab es erste Hinweise darauf, dass die Stabilisierung von extrazellulären Matrixproteinen und die Aktivierung der Signalwege extrazellulärer Matrixproteinrezeptoren eine wichtige Rolle spielen könnten. Hierauf basierend habe ich die Hypothesen aufgestellt, dass die Expression von extrazellulären Matrixproteingehalten und von epidermalen Wachstumsfaktorrezeptoren (EGFR), sowie die Aktivierung von Integrin- und EGFR-Signalwegen Schlüsselrollen in den protektiven Mechanismen von Glutamin spielen. Meine Arbeiten zeigten anhand von Western Blots und durch den Einsatz von Silencing RNA (gegen Fibronektin, Netrin-1, EGFR und deren Negativkontrollen), dass Glutamin extrazelluläre Matrixproteingehalte, wie Fibronektin und Netrin-1, und EGFR-Levels vor Hitzestress in Darmepithelzellen bewahrt, wodurch Apoptose verhindert wird. Außerdem konnte ich mit Hilfe des selektiven Inhibitors GRGDSP, seiner Negativkontrolle GRGESP und des Src-Kinase-Inhibitors PP2 nachweisen, dass auch die Aktivierung von Fibronektin-Integrin-Signalwegen und von Src-Kinasen an den Schutzmechanismen von Glutamin im Darm beteiligt sind. Wie mit Hilfe des spezifischen EGFR-Tyrosinkinase-Inhibitors AG1478 nachgewiesen werden konnte, trifft dies auch für die EGFR-Signalwege zu. Zusätzlich zu GRGDSP und AG1478 wurden auch die ERK1/2-Kinase-Inhibitoren PD98059 und UO126 und der p38MAPK-Inhibitor verwendet, um zu zeigen, dass Glutamin Darmepithelzellen durch die Aktivität von Fibronektin-Integrin- und EGFR-Signalwegen vor Hitzestress schützt, indem es EKR1/2 phosphoryliert und p38MAPK dephosphoryliert. PI-3K/Akt/Hsp70-Signalwege werden ebenfalls von Glutamin aktiviert, jedoch unabhängig von Fibronektin-Integrin- und EGFR-Signalwegen. Dies konnte anhand von Western Blot Experimenten und mit Hilfe von LY294002 (PI-3K-Inhibitor), GRGDSP und AG1478 bewiesen werden. Glutamin scheint die gleichen Signalwege sowohl mit Hilfe von Integrinen als auch von EGFR zu beeinflussen, um so den Zelltod in Darmepithelzellen nach Hitzestress zu verhindern. Desweiteren konnte gezeigt werden, dass Glutamin die Erhaltung von Fibronektinkonzentrationen nach Hitzestress durch den PI-3K-Signalweg reguliert. Ob Glutamin den PI-3K-Signalweg parallel zu den Integrin- und EGFR-Signalwegen aktiviert oder ob der von Glutamin ausgelöste PI-3K-Signalweg die Integrin- und EGFR-Signalwege steuert, muss noch weiter untersucht werden. Experimente mit PD98059 und GRGDSP zeigten, dass ERK1/2 in der Gegenwart von Glutamin den Transkriptionsfaktor HSF-1 durch den Fibronektin-Integrin-Signalweg nach Hitzestress transaktiviert. Dadurch nimmt die Expression von Hsp70, welches den Zelltod nach Hitzeschock verhindert, zu. Mit Hilfe der Fluoreszenzmikroskopie konnte gezeigt werden, dass Hitzestress und Glutamin die Zellgröße und die Aktinmorphologie von Darmepithelzellen durch Fibronektin-Integrin-Signalwege steuern können. Außerdem ergaben Versuche mit GRGDSP und GRGESP, dass Glutamin unabhänging von seinen intrazellulären Konzentrationen das Überleben von Darmepithelzellen durch die Aktivierung von Fibronektin-Integrin-Signalwegen erhöhen kann. Hierbei wurden die Glutaminkonzentrationen mit LC-MS/MS gemessen. Die Versuche im Rahmen meiner Dissertation konnten neue und potentiell klinisch relevante Signalwege der molekularen Schutzmechanismen von Glutamin aufdecken. Diese Ergebnisse können die Grundlage für translationale Studien darstellen, die weiter untersuchen, ob die Überlebensrate von Patienten mit entzündlichen Darmerkrankungen durch die Behandlung mit Glutamin oder durch gezielte Beeinflussung der hier untersuchten molekularen Mechanismen verbessert werden kann. KW - Glutamin KW - Integrine KW - Epidermaler Wachstumsfaktor-Rezeptor KW - Apoptosis KW - glutamine KW - integrins KW - epidermal growth factor receptor KW - cell death KW - cell survival KW - EGFR KW - ERK1/2 KW - p38MAPK KW - PI3-K KW - Akt KW - Apoptose Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-77399 ER - TY - JOUR A1 - Rauert-Wunderlich, Hilka A1 - Siegmund, Daniela A1 - Maier, Eduard A1 - Giner, Tina A1 - Bargou, Ralf C. A1 - Wajant, Harald A1 - Stühmer, Thorsten T1 - The IKK Inhibitor Bay 11-7082 Induces Cell Death Independent from Inhibition of Activation of NF kappa B Transcription Factors JF - PLoS ONE N2 - Multiple myeloma (MM) displays an NFκB activity-related gene expression signature and about 20% of primary MM samples harbor genetic alterations conducive to intrinsic NFκB signaling activation. The relevance of blocking the classical versus the alternative NFκB signaling pathway and the molecular execution mechanisms involved, however, are still poorly understood. Here, we comparatively tested NFκB activity abrogation through TPCA-1 (an IKK2 inhibitor), BAY 11-7082 (an IKK inhibitor poorly selective for IKK1 and IKK2), and MLN4924 (an NEDD8 activating enzyme (NAE)-inhibitor), and analyzed their anti-MM activity. Whereas TPCA-1 interfered selectively with activation of the classical NFκB pathway, the other two compounds inhibited classical and alternative NFκB signaling without significant discrimination. Noteworthy, whereas TPCA-1 and MLN4924 elicited rather mild anti-MM effects with slight to moderate cell death induction after 1 day BAY 11-7082 was uniformly highly toxic to MM cell lines and primary MM cells. Treatment with BAY 11-7082 induced rapid cell swelling and its initial effects were blocked by necrostatin-1 or the ROS scavenger BHA, but a lasting protective effect was not achieved even with additional blockade of caspases. Because MLN4924 inhibits the alternative NFκB pathway downstream of IKK1 at the level of p100 processing, the quite discordant effects between MLN4924 and BAY 11-7082 must thus be due to blockade of IKK1-mediated NFκB-independent necrosis-inhibitory functions or represent an off-target effect of BAY 11-7082. In accordance with the latter, we further observed that concomitant knockdown of IKK1 and IKK2 did not have any major short-term adverse effect on the viability of MM cells. KW - signal inhibition KW - necrotic cell death KW - cell viability testing KW - cell death KW - small interfering RNAs KW - HT29 cells KW - phosphorylation KW - multiple myeloma Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130140 VL - 8 IS - 3 ER - TY - JOUR A1 - Reddy, C. E. A1 - Albanito, L. A1 - De Marco, P. A1 - Aiello, D. A1 - Maggiolini, M. A1 - Napoli, A. A1 - Musti, A. M. T1 - Multisite phosphorylation of c-Jun at threonine 91/93/95 triggers the onset of c-Jun pro-apoptotic activity in cerebellar granule neurons JF - Cell Death & Disease N2 - Cerebellar granule cell (CGC) apoptosis by trophic/potassium (TK) deprivation is a model of election to study the interplay of pro-apoptotic and pro-survival signaling pathways in neuronal cell death. In this model, the c-Jun N-terminal kinase (JNK) induces pro-apoptotic genes through the c-Jun/activator protein 1 (AP-1) transcription factor. On the other side, a survival pathway initiated by lithium leads to repression of pro-apoptotic c-Jun/AP-1 target genes without interfering with JNK activity. Yet, the mechanism by which lithium inhibits c-Jun activity remains to be elucidated. Here, we used this model system to study the regulation and function of site-specific c-Jun phosphorylation at the S63 and T91/T93 JNK sites in neuronal cell death. We found that TK-deprivation led to c-Jun multiphosphorylation at all three JNK sites. However, immunofluorescence analysis of c-Jun phosphorylation at single cell level revealed that the S63 site was phosphorylated in all c-Jun-expressing cells, whereas the response of T91/T93 phosphorylation was more sensitive, mirroring the switch-like apoptotic response of CGCs. Conversely, lithium prevented T91T93 phosphorylation and cell death without affecting the S63 site, suggesting that T91T93 phosphorylation triggers c-Jun pro-apoptotic activity. Accordingly, a c-Jun mutant lacking the T95 priming site for T91/93 phosphorylation protected CGCs from apoptosis, whereas it was able to induce neurite outgrowth in PC12 cells. Vice versa, a c-Jun mutant bearing aspartate substitution of T95 overwhelmed lithium-mediate protection of CGCs from TK-deprivation, validating that inhibition of T91/T93/T95 phosphorylation underlies the effect of lithium on cell death. Mass spectrometry analysis confirmed multiphosphorylation of c-Jun at T91/T93/T95 in cells. Moreover, JNK phosphorylated recombinant c-Jun at T91/T93 in a T95-dependent manner. On the basis of our results, we propose that T91/T93/T95 multiphosphorylation of c-Jun functions as a sensitivity amplifier of the JNK cascade, setting the threshold for c-Jun pro-apoptotic activity in neuronal cells. KW - c-Jun KW - JNK KW - cell death KW - neurons KW - trophic/potassium deprivation KW - lithium Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-128793 VL - 4 IS - e852 ER - TY - THES A1 - Stelzner, Kathrin T1 - Identification of factors involved in Staphylococcus aureus- induced host cell death T1 - Identifizierung von Faktoren, die am Staphylococcus aureus-induzierten Wirtszelltod beteiligt sind N2 - Staphylococcus aureus is a Gram-positive commensal bacterium, that asymptomatically colonizes human skin and mucosal surfaces. Upon opportune conditions, such as immunodeficiency or breached barriers of the host, it can cause a plethora of infections ranging from local, superficial infections to life-threatening diseases. Despite being regarded as an extracellular pathogen, S. aureus can invade and survive within non-phagocytic and phagocytic cells. Eventually, the pathogen escapes from the host cell resulting in killing of the host cell, which is associated with tissue destruction and spread of infection. However, the exact molecular mechanisms underlying S. aureus-induced host cell death remain to be elucidated. In the present work, a genome-wide haploid genetic screen was performed to identify host cell genes crucial for S. aureus intracellular cytotoxicity. A mutant library of the haploid cell line HAP1 was infected with the pathogen and cells surviving the infection were selected. Twelve genes were identified, which were significantly enriched when compared to an infection with a non-cytotoxic S. aureus strain. Additionally, characteristics of regulated cell death pathways and the role of Ca2+ signaling in S. aureus-infected cells were investigated. Live cell imaging of Ca2+ reporter cell lines was used to analyze single cells. S. aureus-induced host cell death exhibited morphological features of apoptosis and activation of caspases was detected. Cellular H2O2 levels were elevated during S. aureus intracellular infection. Further, intracellular S. aureus provoked cytosolic Ca2+ overload in epithelial cells. This resulted from Ca2+ release from endoplasmic reticulum and Ca2+ influx via the plasma membrane and led to mitochondrial Ca2+ overload. The final step of S. aureus-induced cell death was plasma membrane permeabilization, a typical feature of necrotic cell death. In order to identify bacterial virulence factors implicated in S. aureus-induced host cell killing, the cytotoxicity of selected mutants was investigated. Intracellular S. aureus employs the bacterial cysteine protease staphopain A to activate an apoptosis-like cell death characterized by cell contraction and membrane bleb formation. Phagosomal escape represents a prerequisite staphopain A-induced cell death, whereas bacterial intracellular replication is dispensable. Moreover, staphopain A contributed to efficient colonization of the lung in a murine pneumonia model. In conclusion, this work identified at least two independent cell death pathways activated by intracellular S. aureus. While initially staphopain A mediates S. aureus-induced host cell killing, cytosolic Ca2+-overload follows later and leads to the final demise of the host cell. N2 - Staphylococcus aureus ist ein Gram-positives, kommensales Bakterium, welches menschliche Haut- und Schleimhautoberflächen asymptomatisch kolonisiert. Unter günstigen Bedingungen, wie z. B. Immunschwäche oder verletzten Barrieren des Wirtes, kann es eine Vielzahl von Infektionen verursachen, die von lokalen, oberflächlichen Infektionen bis hin zu lebensbedrohlichen Krankheiten reichen. Obwohl S. aureus als extrazellulärer Erreger angesehen wird, kann das Bakterium von nicht-phagozytischen und phagozytischen Zellen aufgenommen werden und dort überleben. Schließlich bricht das Pathogen aus der Wirtszelle aus und die damit einhergehende Tötung der Wirtszelle wird mit Gewebezerstörung und Ausbreitung der Infektion in Verbindung gebracht. Die genauen molekularen Mechanismen, die dem S. aureus induzierten Wirtszelltod zugrunde liegen, müssen jedoch noch geklärt werden. In dieser Arbeit wurde ein genomweiter haploid genetischer Screen durchgeführt, um Wirtszellgene zu identifizieren, die für die intrazelluläre Zytotoxizität von S. aureus entscheidend sind. Eine Mutantenbibliothek der haploiden Zelllinie HAP1 wurde mit dem Erreger infiziert und die Zellen, die die Infektion überlebten, wurden selektiert. Dabei wurden zwölf Gene identifiziert, die signifikant angereichert waren gegenüber einer Infektion mit einem nicht-zytotoxischen S. aureus Stamm. Des Weiteren wurden Eigenschaften regulierter Zelltod-Signalwege und die Rolle der Ca2+-Signalübertragung in S. aureus infizierten Zellen untersucht. Lebendzellbildgebung von Ca2+-Reporterzelllinien wurde zur Analyse von einzelnen Zellen eingesetzt. Der S. aureus induzierte Wirtszelltod wies morphologische Merkmale von Apoptose auf und die Aktivierung von Caspasen wurde nachgewiesen. Der zelluläre H2O2-Spiegel wurde durch die intrazelluläre Infektion mit S. aureus erhöht. Zusätzlich rief der intrazelluläre S. aureus eine zytosolische Ca2+-Überbelastung in Epithelzellen hervor. Dies resultierte aus der Ca2+-Freisetzung vom endoplasmatischen Retikulum und dem Einstrom von Ca2+ über die Plasmamembran und führte zu einer mitochondrialen Ca2+-Überbelastung. Der finale Schritt des durch S. aureus induzierten Zelltods war die Permeabilisierung der Plasmamembran, ein typisches Merkmal des nekrotischen Zelltods. Um bakterielle Virulenzfaktoren zu identifizieren, die am S. aureus-induzierten Wirtszelltod beteiligt sind, wurde die Zytotoxizität von ausgewählten Mutanten untersucht. Der intrazelluläre S. aureus nutzt die bakterielle Cysteinprotease Staphopain A, um einen Apoptose-artigen Zelltod zu aktivieren, der durch Zellkontraktion und Blasenbildung der Membran gekennzeichnet ist. Der phagosomale Ausbruch stellt eine Voraussetzung für den Staphopain A-induzierten Zelltod da, während die intrazelluläre Replikation der Bakterien nicht notwendig ist. Darüber hinaus trug Staphopain A zu einer effizienten Kolonisation der Lunge in einem murinen Pneumonie-Modell bei. Zusammenfassend lässt sich sagen, dass diese Arbeit mindestens zwei unabhängige Zelltod-Signalwege identifiziert hat, die durch den intrazellulären S. aureus aktiviert werden. Während zunächst Staphopain A den Tod der Wirtszelle einleitet, folgt später die zytosolische Ca2+-Überlastung und führt zum endgültigen Untergang der Wirtszelle. KW - Staphylococcus aureus KW - Zelltod KW - Wirtszelle KW - cell death KW - host cell Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-188991 N1 - Zusatzmaterial (Videos) befinden sich auch auf einer CD in der gedruckten Ausgabe ER - TY - JOUR A1 - Stelzner, Kathrin A1 - Winkler, Ann-Cathrin A1 - Liang, Chunguang A1 - Boyny, Aziza A1 - Ade, Carsten P. A1 - Dandekar, Thomas A1 - Fraunholz, Martin J. A1 - Rudel, Thomas T1 - Intracellular Staphylococcus aureus Perturbs the Host Cell Ca\(^{2+}\) Homeostasis To Promote Cell Death JF - mBio N2 - The opportunistic human pathogen Staphylococcus aureus causes serious infectious diseases that range from superficial skin and soft tissue infections to necrotizing pneumonia and sepsis. While classically regarded as an extracellular pathogen, S. aureus is able to invade and survive within human cells. Host cell exit is associated with cell death, tissue destruction, and the spread of infection. The exact molecular mechanism employed by S. aureus to escape the host cell is still unclear. In this study, we performed a genome-wide small hairpin RNA (shRNA) screen and identified the calcium signaling pathway as being involved in intracellular infection. S. aureus induced a massive cytosolic Ca\(^{2+}\) increase in epithelial host cells after invasion and intracellular replication of the pathogen. This was paralleled by a decrease in endoplasmic reticulum Ca\(^{2+}\) concentration. Additionally, calcium ions from the extracellular space contributed to the cytosolic Ca2+ increase. As a consequence, we observed that the cytoplasmic Ca\(^{2+}\) rise led to an increase in mitochondrial Ca\(^{2+}\) concentration, the activation of calpains and caspases, and eventually to cell lysis of S. aureus-infected cells. Our study therefore suggests that intracellular S. aureus disturbs the host cell Ca\(^{2+}\) homeostasis and induces cytoplasmic Ca\(^{2+}\) overload, which results in both apoptotic and necrotic cell death in parallel or succession. IMPORTANCE Despite being regarded as an extracellular bacterium, the pathogen Staphylococcus aureus can invade and survive within human cells. The intracellular niche is considered a hideout from the host immune system and antibiotic treatment and allows bacterial proliferation. Subsequently, the intracellular bacterium induces host cell death, which may facilitate the spread of infection and tissue destruction. So far, host cell factors exploited by intracellular S. aureus to promote cell death are only poorly characterized. We performed a genome-wide screen and found the calcium signaling pathway to play a role in S. aureus invasion and cytotoxicity. The intracellular bacterium induces a cytoplasmic and mitochondrial Ca\(^{2+}\) overload, which results in host cell death. Thus, this study first showed how an intracellular bacterium perturbs the host cell Ca\(^{2+}\) homeostasis." KW - Staphylococcus aureus KW - calcium signaling pathway KW - cell death KW - facultatively intracellular pathogens Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-231448 VL - 11 ER - TY - JOUR A1 - Stolpmann, K. A1 - Brinkmann, J. A1 - Salzmann, S. A1 - Genkinger, D. A1 - Fritsche, E. A1 - Hutzler, C. A1 - Wajant, H. A1 - Luch, A. A1 - Henkler, F. T1 - Activation of the aryl hydrocarbon receptor sensitises human keratinocytes for CD95L-and TRAIL-induced apoptosis JF - Cell Death & Disease N2 - In this study, we have analysed the apoptotic effects of the ubiquitous environmental toxin benzo[ a] pyrene (BP) in HaCaT cells and human keratinocytes. Although prolonged exposure to BP was not cytotoxic on its own, a strong enhancement of CD95 (Fas)-mediated apoptosis was observed with BP at concentrations activating the aryl hydrocarbon receptor (AhR). Importantly, the ultimately mutagenic BP-metabolite, that is, (+)-anti-BP-7,8-diol-9,10-epoxide (BPDE), failed to enhance CD95-mediated cell death, suggesting that the observed pro-apoptotic effect of BP is neither associated with DNA adducts nor DNA-damage related signalling. CD95-induced apoptosis was also enhanced by beta-naphtoflavone, a well-known agonist of the AhR that does not induce DNA damage, thus suggesting a crucial role for AhR activation. Consistently, BP failed to sensitise for CD95L-induced apoptosis in AhR knockdown HaCaT cells. Furthermore, inhibition of CYP1A1 and/or 1B1 expression did not affect the pro-apoptotic crosstalk. Exposure to BP did not increase expression of CD95, but led to augmented activation of caspase-8. Enhancement of apoptosis was also observed with the TRAIL death receptors that activate caspase-8 and apoptosis by similar mechanisms as CD95. Together, these observations indicate an interference of AhR signalling with the activity of receptor-associated signalling intermediates that are shared by CD95 and TRAIL receptors. Our data thus suggest that AhR agonists can enhance cytokine-mediated adversity upon dermal exposure. KW - CD95 KW - HaCaT cells KW - growth-factor receptor KW - cell death KW - mitochondrial dysfunction KW - mediated apoptosis KW - FAS KW - dermatitis KW - pathways KW - skin KW - progression KW - aryl hydrocarbon receptor (AhR) KW - apoptosis KW - benzo[a]pyrene KW - human keratinocytes Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133501 VL - 3 IS - e388 ER - TY - JOUR A1 - Wajant, Harald T1 - Molecular mode of action of TRAIL receptor agonists—common principles and their translational exploitation JF - Cancers N2 - Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptors TRAILR1/death receptor 4 (DR4) and TRAILR2/DR5 trigger cell death in many cancer cells but rarely exert cytotoxic activity on non-transformed cells. Against this background, a variety of recombinant TRAIL variants and anti-TRAIL death receptor antibodies have been developed and tested in preclinical and clinical studies. Despite promising results from mice tumor models, TRAIL death receptor targeting has failed so far in clinical studies to show satisfying anti-tumor efficacy. These disappointing results can largely be explained by two issues: First, tumor cells can acquire TRAIL resistance by several mechanisms defining a need for combination therapies with appropriate sensitizing drugs. Second, there is now growing preclinical evidence that soluble TRAIL variants but also bivalent anti-TRAIL death receptor antibodies typically require oligomerization or plasma membrane anchoring to achieve maximum activity. This review discusses the need for oligomerization and plasma membrane attachment for the activity of TRAIL death receptor agonists in view of what is known about the molecular mechanisms of how TRAIL death receptors trigger intracellular cell death signaling. In particular, it will be highlighted which consequences this has for the development of next generation TRAIL death receptor agonists and their potential clinical application. KW - antibody KW - antibody fusion proteins KW - apoptosis KW - cancer therapy KW - cell death KW - death receptors KW - TNF superfamily KW - TNF receptor superfamily KW - TRAIL Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-202416 VL - 11 IS - 7 ER - TY - JOUR A1 - Wajant, Harald T1 - Molecular mode of action of TRAIL receptor agonists—common principles and their translational exploitation JF - Cancers N2 - Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptors TRAILR1/death receptor 4 (DR4) and TRAILR2/DR5 trigger cell death in many cancer cells but rarely exert cytotoxic activity on non-transformed cells. Against this background, a variety of recombinant TRAIL variants and anti-TRAIL death receptor antibodies have been developed and tested in preclinical and clinical studies. Despite promising results from mice tumor models, TRAIL death receptor targeting has failed so far in clinical studies to show satisfying anti-tumor efficacy. These disappointing results can largely be explained by two issues: First, tumor cells can acquire TRAIL resistance by several mechanisms defining a need for combination therapies with appropriate sensitizing drugs. Second, there is now growing preclinical evidence that soluble TRAIL variants but also bivalent anti-TRAIL death receptor antibodies typically require oligomerization or plasma membrane anchoring to achieve maximum activity. This review discusses the need for oligomerization and plasma membrane attachment for the activity of TRAIL death receptor agonists in view of what is known about the molecular mechanisms of how TRAIL death receptors trigger intracellular cell death signaling. In particular, it will be highlighted which consequences this has for the development of next generation TRAIL death receptor agonists and their potential clinical application. KW - antibody KW - antibody fusion proteins KW - apoptosis KW - cancer therapy KW - cell death KW - death receptors KW - TNF superfamily KW - TNF receptor superfamily KW - TRAIL Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201833 N1 - Zugriff gesperrt. Zugriff auf den Volltext erhalten Sie unter https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-202416 VL - 11 IS - 7 ER - TY - JOUR A1 - Zaitseva, Olena A1 - Hoffmann, Annett A1 - Otto, Christoph A1 - Wajant, Harald T1 - Targeting fibroblast growth factor (FGF)-inducible 14 (Fn14) for tumor therapy JF - Frontiers in Pharmacology N2 - Fibroblast growth factor-inducible 14 (Fn14) is a member of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) and is activated by its ligand TNF-like weak inducer of apoptosis (TWEAK). The latter occurs as a homotrimeric molecule in a soluble and a membrane-bound form. Soluble TWEAK (sTWEAK) activates the weakly inflammatory alternative NF-κB pathway and sensitizes for TNF-induced cell death while membrane TWEAK (memTWEAK) triggers additionally robust activation of the classical NF-κB pathway and various MAP kinase cascades. Fn14 expression is limited in adult organisms but becomes strongly induced in non-hematopoietic cells by a variety of growth factors, cytokines and physical stressors (e.g., hypoxia, irradiation). Since all these Fn14-inducing factors are frequently also present in the tumor microenvironment, Fn14 is regularly found to be expressed by non-hematopoietic cells of the tumor microenvironment and most solid tumor cells. In general, there are three possibilities how the tumor-Fn14 linkage could be taken into consideration for tumor therapy. First, by exploitation of the cancer associated expression of Fn14 to direct cytotoxic activities (antibody-dependent cell-mediated cytotoxicity (ADCC), cytotoxic payloads, CAR T-cells) to the tumor, second by blockade of potential protumoral activities of the TWEAK/Fn14 system, and third, by stimulation of Fn14 which not only triggers proinflammtory activities but also sensitizes cells for apoptotic and necroptotic cell death. Based on a brief description of the biology of the TWEAK/Fn14 system and Fn14 signaling, we discuss the features of the most relevant Fn14-targeting biologicals and review the preclinical data obtained with these reagents. In particular, we address problems and limitations which became evident in the preclinical studies with Fn14-targeting biologicals and debate possibilities how they could be overcome. KW - agonistic antibodies KW - cell death KW - Fn14 KW - NFκB KW - TNF KW - TWEAK Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-290238 SN - 1663-9812 VL - 13 ER -