Institut für Pharmakologie und Toxikologie
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Fumonisin B1 (FB1) is a mycotoxin produced by various Fusarium species and constitutes a major contaminant of maize worldwide. A 2-year carcinogenicity study of the National Toxicology Program (NTP) in Fischer N344 rats showed that male rats were most susceptible to FB1-induced tumor formation in the kidney. Histopathologically, a rare and highly malignant tumor type originating from the proximal tubules of rat kidney with increased potential for invasion and metastasis was identified. However, mechanisms underlying the FB1-induced carcinogenesis in kidneys of male rats are still not clear. Previous studies have shown that FB1-mediated disruption of sphingolipid metabolism via inhibition of ceramide synthase is a primary key event in FB1 toxicity. The disruption of sphingolipid metabolism may cause time- and dose-related changes in the relative balance of various bioactive intermediates. Furthermore, the ability of FB1 to induce renal cell death and subsequent compensatory cell proliferation is well known, but it does not completely explain the invasive growth characteristics and exceptionally high metastatic potential of FB1-induced tumors. Considering the complexity of sphingolipid metabolism and the fact that various sphingolipids (e.g. ceramide, sphingoid bases and their respective 1-phosphates) act on opposing signaling pathways, it is hypothesized that the balance between individual sphingolipids and thus the overall cellular response to FB1 may shift with time and by continuing FB1 exposure, resulting in the disruption of specific cell signaling pathways, which may promote tumor formation in kidney. To identify early FB1-induced gene expression patterns in the kidney, which may be associated with sphingolipid-mediated signaling pathways in cancer, a short-term i.p. study on FB1 in male Sprague Dawley rats was performed and changes in gene expression were analyzed using a qRT-PCR array that comprises 84 relevant genes of 6 pathways pivotally involved in the formation of cancer. Furthermore, apoptosis and cell proliferation as well as changes in specific sphingolipids were investigated in FB1-treated kidneys. As shown by classical histopathology (H&E) and (immuno)-histochemical staining (TUNEL and BrdU), FB1 caused a time- and dose-dependent increase in tubular apoptosis in the cortex and OSOM of the kidney, which was compensated by the induction of proliferation in the affected areas. HPLC-MS/MS analysis of bioactive sphingolipids demonstrated that FB1 induced a marked elevation of the pro-apoptotic sphingoid bases sphinganine and sphingosine, which paralleled the time- and dose-dependent increase in renal tubular apoptosis. With prolonged exposure to FB1, increased metabolic conversion of the accumulated sphinganine to the sphinganine-1-phosphate, a second messenger with anti-apoptotic and proliferative properties, was observed in kidney. This finding was compliant with the increased regenerative cell proliferation in the cortex and OSOM. In addition to effects on sphingoid bases and their 1-phosphate metabolites, this study, for the first time, demonstrated reduced levels of specific ceramides in rat kidney after FB1 exposure. In particular, C16-ceramide, which is a widespread constituent of membrane-bound complex sphingolipids involved in cell adhesion, was time- and dose-dependently decreased after treatment with FB1. Besides its role as component of the cell membrane, C16-ceramide functions as a signaling molecule for the initiation of apoptosis in response to various stress stimuli. Under conditions of chronic FB1 exposure, a significant reduction in pro-apoptotic C16-ceramide together with markedly increased levels of anti-apoptotic and proliferation-promoting sphingoid base 1-phosphates may thus favor resistance to stress-induced apoptosis and facilitate the survival of abnormal cells with potential to initiate tumor formation. Our study also revealed that early exposure to FB1 resulted in increased expression of a plethora of genes involved in tumor initiation as well as tumor progression. While single FB1 exposure was demonstrated to predominately induce gene expression of proto-oncogenic transcription factors (e.g. Fos, Jun, Myc) and apoptotis-related genes (e.g. members of the tumor-necrosis factor family), repeated exposure resulted in marked upregulation of genes mediating cell survival and cell proliferation (e.g. Bcl-XL, Bcl-2, Nfκb1 and Egfr). Moreover, continued exposure to FB1 initiated increased expression of genes critically involved in tumor migration, adhesion, invasion and metastasis. A close correlation was established between gene expression changes in response to FB1 and known signaling pathways mediated by extracellular or intracellular action of sphingoid base 1-phosphates - bioactive lipids that were markedly increased after FB1 treatment. In particular, genes encoding components of the plasminogen activator system were abundantly upregulated. These mediate invasion and metastasis in response to So1P, and may hence particularly promote the formation of highly aggressive and invasive tumors in kidney as observed after chronic exposure to FB1. Thus, it is conceivable that upregulation of a majority of genes in response to FB1 may be a direct or indirect consequence of increased So1P signaling. Another aim of this study was to identify differences in the organ-specific susceptibility for tumor formation by comparing FB1-mediated effects on apoptosis, cell proliferation, sphingolipids, and selected cancer-related genes in kidney and liver. Collectively, the present results revealed that kidney and liver showed marked differences in several endpoints of FB1 toxicity, which seemed to be primarily associated with their different susceptibility to FB1-mediated alterations in sphingolipid metabolism. The strong correlation between histopathological lesions and alterations in sphingolipid metabolism as well as sphingoid base 1-phosphate accumulation and concomitant S1P receptor expression suggested that tumor formation and progression to highly malignant carcinomas seems to be rather favored in kidney compared to liver. However, genes mostly deregulated by FB1 treatment in kidney (PAI-1, Thbs1 and Itga2) were also found to be induced in liver. To verify FB1-induced gene expression in kidney, normal rat tubular epithelial (NRK-52E) cells were analyzed for FB1-induced expression changes of the same cancer-related genes as in vivo. The results of qRT-PCR analysis revealed that gene expression changes in NRK-52E cells after FB1 treatment strongly correlated with those found in rat kidney and paralleled the marked alterations in sphingolipid metabolism. Furthermore, a good correlation between FB1-induced expression changes of cancer-related genes obtained in vivo and in vitro and those known to be mediated by bioactive sphingoid base 1-phosphates in cancer was established. Moreover, experiments modeling the invasive behavior of NRK-52E cells showed that FB1 may enhance cell invasion, which also correlated with both the increase in invasion- and metastasis-associated genes and bioactive sphingoid base 1-phophates. Importantly, NRK-52E cells basally expressed the S1P receptors S1P2 and S1P3, which are known to be involved in tumor migration and invasion. Since these receptors were also identified as most abundant S1PRs in kidneys of male Sprague Dawley rats, they may present important mediators of gene expression and invasion in response to FB1 in vivo. In summary, FB1-mediated disruption of sphingolipid metabolism and subsequent time- and dose-related increase in intermediates, such as bioactive sphingoid base 1-phosphates, correlate with early changes in genes and signaling pathways that may mediate loss of growth control, replication, evasion of apoptosis, cell motility and invasion, and thus favor renal tumor formation in response to FB1. However, to clarify whether the obtained gene expression changes in cancer-related genes in kidney are specific to the biological action of sphingoid base 1-phosphates and their respective receptors, further mechanistic studies are necessary.
Der Fluoreszenz-Resonanz-Energie-Transfer ist ein Phänomen, welches erstmals 1948 von Theodor Förster beschrieben wurde. Mit der Entwicklung von Fluoreszenzproteinen konnten in Kombination mit Mikroskopietechniken Einblicke in zellbiologische Vorgänge gewonnen werden, die durch biochemische oder physiologische Experimente nicht möglich sind. Dabei spielt die hohe zeitliche und räumliche Auflösung eine wichtige Rolle. Auf dem Forschungsgebiet der GPCR, welche die größte Gruppe von Membranproteinen bei den Säugetieren darstellen, wurden insbesondere Erkenntnisse über Konformationsänderungen der Rezeptoren, die Kinetik der Rezeptoraktivierung und die Interaktion mit intrazellulären Signalproteinen gewonnen. Der µ-Opioidrezeptor gehört zur Familie der GPCR und stellt aufgrund seiner analgetischen Wirkungen eine wichtige pharmakologische Zielstruktur dar. Das Ziel dieser Arbeit war sowohl den Rezeptor als auch seine Signalwege mittels FRET-Mikroskopie zu untersuchen. Zunächst sollte ein intramolekularer FRET-Sensor des µ-Opioidrezeptors entwickelt werden, dazu wurden basierend auf den Kenntnissen über die Tertiärstruktur und dem Aufbau bereits bekannter GPCR-Sensoren verschiedene Rezeptorkonstrukte kloniert. Bei den Konstrukten wurden entweder zwei Fluoreszenzproteine oder ein Fluoreszenzprotein und ein Fluorophor-bindendes Tetracysteinmotiv kombiniert. Auch die Positionen der eingefügten Sequenzen wurden in den intrazellulären Domänen variiert, da der Rezeptor auf die Modifikationen mit beeinträchtigter Membranlokalisation reagierte. Durch die Optimierung wurden Rezeptoren konstruiert, die an der Zellmembran lokalisiert waren. Jedoch zeigte keines der Rezeptorkonstrukte Funktionalität im Hinblick auf die Rezeptoraktivierung. Im zweiten Teil wurden die pharmakologischen Effekte der Metabolite von Morphin am humanen µ-Opioidrezeptor systematisch analysiert. Dazu wurde die Fähigkeit der Metabolite, Gi-Proteine zu aktivieren und β-Arrestin2 zu rekrutieren, mittels FRET-basierter Messungen an lebenden Zellen untersucht. Außerdem wurde die Affinität der Metabolite zum humanen µ Opioidrezeptor anhand der Verdrängung eines radioaktiven Liganden analysiert. Meine Experimente identifizierten eine Gruppe mit stark agonistischen und eine mit schwach agonistischen Eigenschaften. Die starken Partialagonisten aktivieren den Rezeptor bereits bei nanomolaren Konzentrationen, während die schwachen Metabolite den Rezeptor erst bei Konzentrationen im mikromolaren Bereich aktivieren. Die Metabolite Normorphin, Morphin-6-Glucuronid und 6-Acetylmorphin zeigen geringere Potenz als Morphin bei der Gi-Aktivierung aber überraschenderweise höhere Potenz und Effizienz für die β-Arrestin-Rekrutierung. Dies deutet auf eine bevorzugte Aktivierung von β-Arrestin2 hin. Die aus diesen Studien gewonnenen Ergebnisse liefern Hinweise darauf, welche Metabolite bei der Signalverarbeitung am µ Opioidrezeptor in vivo beteiligt sind.
Pneumolysin, a protein toxin, represents one of the major virulence factors of Streptococcus pneumoniae. This pathogen causes bacterial meningitis with especially high disease rates in young children, elderly people and immunosuppressed patients. The protein toxin belongs to the family of cholesterol-dependent cytolysins, which require membrane cholesterol in order to bind and to be activated. Upon activation, monomers assemble in a circle and undergo conformational change. This conformational change leads to the formation of a pore, which eventually leads to cell lysis. This knowledge was obtained by studies that used a higher concentration compared to the concentration of pneumolysin found in the cerebrospinal fluid of meningitis patients. Thus, a much lower concentration of pneumolysin was used in this work in order to investigate effects of this toxin on primary mouse astrocytes. Previously, a small GTPase activation, possibly leading to cytoskeletal changes, was found in a human neuroblastoma cell line. This led to the hypothesis that pneumolysin can lead to similar cytoskeletal changes in primary cells. The aim of this work was to investigate and characterise the effects of pneumolysin on primary mouse astrocytes in terms of a possible pore formation, cellular trafficking and immunological responses. Firstly, the importance of pore-formation on cytoskeletal changes was to be investigated. In order to tackle this question, wild-type pneumolysin and two mutant variants were used. One variant was generated by exchanging one amino acid in the cholesterol recognising region, the second variant was generated by deleting two amino acids in a protein domain that is essential for oligomerisation. These variants should be incapable of forming a pore and were compared to the wild-type in terms of lytic capacities, membrane binding, membrane depolarisation, pore-formation in artificial membranes (planar lipid bilayer) and effects on the cytoskeleton. These investigations resulted in the finding that the pore-formation is required for inducing cell lysis, membrane depolarisation and cytoskeletal changes in astrocytes. The variants were not able to form a pore in planar lipid bilayer and did not cause cell lysis and membrane depolarisation. However, they bound to the cell membrane to the same extent as the wild-type toxin. Thus, the pore-formation, but not the membrane binding was the cause for these changes. Secondly, the effect of pneumolysin on cellular trafficking was investigated. Here, the variants showed no effect, but the wild-type led to an increase in overall endocytotic events and was itself internalised into the cell. In order to characterise a possible mechanism for internalisation, a GFP-tagged version of pneumolysin was used. Several fluorescence-labelled markers for different endocytotic pathways were used in a co-staining approach with pneumolysin. Furthermore, inhibitors for two key-players in classical endocytotic pathways, dynamin and myosin II, were used in order to investigate classical endocytotic pathways and their possible involvement in toxin internalisation. The second finding of this work is that pneumolysin is taken up into the cell via dynamin- and caveolin-independent pinocytosis, which could transfer the toxin to caveosomes. From there, the fate of the toxin remains unknown. Additionally, pneumolysin leads to an overall increase in endocytotic events. This observation led to the third aim of this work. If the toxin increases the overall rate of endocytosis, the question arises whether toxin internalisation favours bacterial tissue penetration of the host or whether it serves as a defence mechanism of the cell in order to degrade the protein. Thus, several proinflammatory cytokines were investigated, as previous studies describe an effect of pneumolysin on cytokine production. Surprisingly, only interleukin 6-production was increased after toxin-treatment and no effect of endocytotic inhibitors on the interleukin 6-production was observed. The conclusion from this finding is that pneumolysin leads to an increase of interleukin 6, which would not depend on the endocytotic uptake of pneumolysin. The production of interleukin 6 would enhance the production of acute phase proteins, T-cell activation, growth and differentiation. On the one hand, this activation could serve pathogen clearance from infected tissue. On the other hand, the production of interleukin 6 could promote a further penetration of pathogen into host tissue. This question should be further investigated.
Streptococcus pneumoniae is one of the major causes of bacterial meningitis, which mainly affects young infants in the developing countries of Africa, Asia (esp. India) and South America, and which has case fatality rates up to 50% in those regions. Bacterial meningitis comprises an infection of the meninges and the sub-meningeal cortex tissue of the brain, whereat the presence of pneumolysin (PLY), a major virulence factor of the pneumococcus, is prerequisite for the development of a severe outcome of the infection and associated tissue damage (e. g. apoptosis, brain edema, and ischemia). Pneumolysin belongs to the family of pore forming, cholesterol-dependent cytolysins (CDCs), bacterial protein toxins, which basically use membrane-cholesterol as receptor and oligomerize to big aggregates, which induce cell lysis and cell death by disturbance of membrane integrity. Multiple recent studies, including this work, have revealed a new picture of pneumolysin, whose cell-related properties go far beyond membrane binding, pore formation and the induction of cell death and inflammatory responses. For a long time, it has been known that bacteria harm the tissues of their hosts in order to promote their own survival and proliferation. Many bacterial toxins aim to rather hijack cells than to kill them, by interacting with cellular components, such as the cytoskeleton or other endogenous proteins. This study was able to uncover a novel capacity of pneumolysin to interact with components of the actin machinery and to promote rapid, actin-dependent cell shape changes in primary astrocytes. The toxin was applied in disease-relevant concentrations, which were verified to be sub-lytic. These amounts of toxin induced a rapid actin cortex collapse in horizontal direction towards the cell core, whereat membrane integrity was preserved, indicating an actin severing function of pneumolysin, and being consistent with cell shrinkage, displacement, and blebbing observed in live cell imaging experiments. In contrast to neuroblastoma cells, in which pneumolysin led to cytoskeleton remodeling and simultaneously to activation of Rac1 and RhoA, in primary astrocytes the cell shape changes were seen to be primarily independent of small GTPases. The level of activated Rac1 and RhoA did not increase at the early time points after toxin application, when the initial shape changes have been observed, but at later time points when the actin-dependent displacement of cells was slower and less severe, probably presenting the cell’s attempt to re-establish proper cytoskeleton function. A GUV (giant unilamellar vesicle) approach provided insight into the effects of pneumolysin in a biomimetic system, an environment, which is strictly biochemical, but still comprises cellular components, limited to the factors of interest (actin, Arp2/3, ATP, and Mg2+ on one side, and PLY on the other side). This approach was able to show that the wildtype-toxin, but not the Δ6 mutant (mutated in the unfolding domain, and thus non-porous), had the capacity to exhibit its functions through a membrane bilayer, meaning it was able to aggregate actin, which was located on the other side of the membrane, either via direct interaction with actin or in an Arp2/3 activating manner. Taking a closer look at these two factors with the help of several different imaging and biochemical approaches, this work unveiled the capacity of pneumolysin to bind and interact both with actin and Arp2 of the Arp2/3 complex. Pneumolysin was capable to slightly stabilize actin in an actin-pyrene polymerization assay. The same experimental setup was applied to show that the toxin had the capacity to lead to actin polymerization through activation of the Arp2/3 complex. This effect was additionally confirmed with the help of fluorescent microscopy of rhodamine (TRITC)-tagged actin. Strongest Arp2/3 activation, and actin nucleation/polymerization is achieved by the VCA domain of the WASP family proteins. However, addition of PLY to the Arp2/3–VCA system led to an enhanced actin nucleation, suggesting a synergistic activation function of pneumolysin. Hence, two different effects of pneumolysin on the actin cytoskeleton were observed. On the one hand an actin severing property, and on the other hand an actin stabilization property, both of which do not necessarily exclude each other. Actin remodeling is a common feature of bacterial virulence strategies. This is the first time, however, that these properties were assigned to a toxin of the CDC family. Cytoskeletal dysfunction in astrocytes leads to dysfunction and unregulated movement of these cells, which, in context of bacterial meningitis, can favor bacterial penetration and spreading in the brain tissue, and thus comprises an additional role of pneumolysin as a virulence factor of Streptococcus pneumonia in the context of brain infection.
It is shown by means of IR. spectroscopic methodsthat nigericin and monensin bave a cyclic conformation similar to that of their silver salts. Camplex fonnation constants with sodium and potassium ions follow the selectivity order determined by EMF. measurements on liquid membranes: nigericin: K\(^+\) >Rb\(^+\)> Na\(^+\)> Cs\(^+\) >Li\(^+\); monensin: Na\(^+\)> K\(^+\) >Li\(^+\)> Rb\(^+\)> Cs\(^+\). Transport experiments show that nigericin and monensin facilitate the diffusion of potassium ions across model membranes, although in electrolytic transport experiments the permeability is not affected.
The structure of monensin, C36H620 11 , has been deterrnined by X-ray analysis of its crystalline monohydrate (orthorhombic, a = 15.15, b = 23.61, c = 10.65 A, Z = 4, space group P212121). Phases were assigned by direct methods, malring use of the 'tangent formula'. Although the conformation of the free acid resembles that of the silver salt in being cyclic, there are differences in the hydrogen bonding pattern. These featurcs are discussed in relation to the cornplexation of metal ions by m.onensin.
The thermodynainic parameters ΔH0, ΔG0 and ΔS0 - and thereby the equilibrium constants - for the complexation of the carrier antibiotics nigericin and monensin with sodium and potassium ions in methanol at 25°C have been determined by microcalorimetry. Tbc results are discussed in terms of the nature of the interaction between ligands and cations.
Aryl hydrocarbon hydroxylase (AHH) has been measured in male rat Jiver nucJei and microsomes after treatment of adult animals with various inducers for up to 14 days. After daily i.p. injections of 3-methylcholanthrene (MC, 20 mg/kg) the nuclear activity increased to a maximum of 600 per cent of the control activity after 4 days whereas the microsomal activity was 400 per cent of control at the same date. After 12 days, both activities equilibrated at 400 per cent. A similar time course was found after a single i.p. injection of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD, 0.01 mg/kg) with an induction to .500 and 300 per cent for nuclei and microsomes, respectiveJy. after 2 days, and to 400 per cent for both after 12 days. PhenobarbitaJ (PB) was given continuously in the drinking water (I g/1) and induced the microsomal activity to 200 per cent after 8 days and 170 per cent after 14 days. The nuclear activity was only slightly induced to a constant Ievei of 130 per cent between day 8 and 14. Dieldrin did not significantly increase the microsomal activity after daiJy i.p. injections (20 mg/kg), but the nuclear activity raised to 200 per cent after 3 days and levelled down tocontrol valuesafter 12 days. Other inducers tested were benz[a)anthracene (BA), hexachlorobenzene (HCB} and 1,1.1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT). The induction pattern with BA was similar tothat of MC, a modeJ compound for the group of cytochrome P448 inducers. The induction by HCB and DDT resembled that by PB. a typical cytochrome P450 inducer.