No description available.

Ceramide, a sphingolipid, is an important lipid second messenger that is involved in regulating a number of cellular processes, including programmed cell death, cell growth and differentiation, as well as cellular responses to stress stimuli. Many of the biological effects of ceramide are linked to its ability to modulate the biophysical properties of membranes and cause clustering of signalling molecules in ceramide-rich domains, which allows for more efficient signal transmission in the cell. However, the specific roles of different ceramide species in these signaling pathways have yet to be clearly established. Assessing the effects of long N-acyl chain ceramides in cells involves some limitations due to their poor solubility and their low membrane permeability. Caging these molecules with photolabile protecting groups allows for their delivery into cells where photochemical uncaging of the biologically active compound can be achieved with spatial and temporal control. A series of coumarinyl-caged ceramides has been prepared in order to probe the biological effects of ceramide in cells. This unique series of compounds was used to investigate the dependence of these cellular effects on N-acyl chain length. Hereafter, I describe the photophysical and photochemical characterization of these novel caged ceramides, assess their uptake and measure the biological effects of the different ceramides which are generated photochemically in HeLa cells. The caged ceramides were shown to be taken up by the cells and to cause a decrease in viability, with UV irradiation, that can be detected after 24 hours of treatment. An investigation of the mechanism of cell death induced by coumarinyl-caged ceramides in HeLa cells revealed that cell death proceeds in a caspase-independent manner and involves the mitochondria. The role of the mitochondria in this cell death pathway, however, remains to be studied further. RIP1 kinase activity, which was also probed in the cells, was determined to not be implicated in cell death caused by photochemically generated ceramide. Intracellular ROS generation, however, was shown to occur in this system, but results primarily from UV irradiation of the free coumarin. Overall, the results from this study have provided insight into the signalling pathways triggered by treatment of HeLa cells with the bioactive lipid ceramide using coumarin photocages.

Universiẗat, Diss., 2001--München.

Acute kidney injury (AKI), resulting from chemotherapeutic agents such as cisplatin, remains an obstacle in the treatment of cancer. Cisplatin-induced AKI involves apoptotic and necrotic cell death, pathways regulated by sphingolipids such as ceramide and glucosylceramide. Results from this study indicate that C57BL/6J mice treated with cisplatin had increased ceramide and hexosylceramide levels in the renal cortex 72 h following cisplatin treatment. Pretreatment of mice with inhibitors of acid sphingomyelinase and de novo ceramide synthesis (amitriptyline and myriocin, respectively) prevented accumulation of ceramides and hexosylceramide in the renal cortex and protected from cisplatin-induced AKI. To determine the role of ceramide metabolism to hexosylceramides in kidney injury, we treated mice with a potent and highly specific inhibitor of glucosylceramide synthase, the enzyme responsible for catalyzing the glycosylation of ceramides to form glucosylceramides. Inhibition of glucosylceramide synthase attenuated the accumulation of the hexosylceramides and exacerbated ceramide accumulation in the renal cortex following treatment of mice with cisplatin. Increasing ceramides and decreasing glucosylceramides in the renal cortex sensitized mice to cisplatin-induced AKI according to markers of kidney function, kidney injury, inflammation, cell stress, and apoptosis. Under conditions of high ceramide generation, data suggest that metabolism of ceramides to glucosylceramides buffers kidney ceramides and helps attenuate kidney injury.-Dupre, T. V., M. A. Doll, P. P. Shah, C. N. Sharp, D. Siow, J. Megyesi, J. Shayman, A.Bielawska, J. Bielawski, L. J. Beverly, M. Hernandez-Corbacho, C. J. Clarke, A. J. Snider, R. G. Schnellmann, L. M. Obeid, Y. A. Hannun, and L. J. Siskind. Inhibiting glucosylceramide synthase exacerbates cisplatin-induced acute kidney injury.

Ceramide sind biologisch aktive Sphingolipide, die verschiedene zelluläre Signalwege regulieren, meist im Zusammenhang mit der Induktion von Apoptose oder der Regulation des Zellzyklus. Darüber hinaus wurde in der Literatur beschrieben, dass Ceramide die Zytoskelettdynamik unterschiedlicher Zelltypen beeinflussen, die Bedeutung von Ceramiden für die Funktion von T-Zellen wurde allerdings bisher wenig untersucht. In der vorliegenden Arbeit konnte gezeigt werden, dass die exogene Akkumulation von Ceramiden ebenso wie die Generierung von Ceramiden durch bSMase die Adhärenz von T-Zellen an FN bzw. ICAM-1 beeinträchtigt. Des Weiteren konnte eine verminderte T-Zell-Polarisierung auf FN sowie eine reduzierte Chemotaxis und Motilität ceramidmodifizierter T-Zellen in Antwort auf SDF-1 nachgewiesen werden. In Übereinstimmung mit der Unfähigkeit ceramidmodifizierter Zellen morphologisch zu polarisieren wird ferner die Relokalisation von Oberflächenmolekülen und intrazellulärer Proteine durch die Akkumulation von Ceramiden gestört. Überdies konnte in dieser Arbeit gezeigt werden, dass Ceramide mit dem Aktivierungsstatus von Akt und ERM-Proteinen interferieren, da eine verminderte stimulationsabhängige Phosphorylierung von Akt und ERM-Proteinen in ceramidmodifizierten Zellen nachgewiesen wurde. Ein wesentlicher Schritt im Verlauf der T-Zell-Aktivierung ist die Ausbildung einer immunologischen Synapse mit dendritischen Zellen. In dieser Arbeit konnte gezeigt werden, dass, obwohl ceramidreiche Membrandomänen von der Kontaktstelle ausgeschlossen werden, Konjugatfrequenz und Architektur der IS durch die Induktion von Ceramiden nicht beeinflusst werden, da eine normale Verteilung von CD3 und des MTOC beobachtet wurde. Allerdings wird die Funktionalität der Konjugate durch die Induktion von Ceramiden beeinträchtigt. Ceramidmodifizierte Zellen waren nur eingeschränkt in der Lage Orai1 und Stim1 zur Kontaktfläche mit DCs zu translozieren. In Übereinstimmung mit diesen Befunden wurde auch ein verminderter Calcium-Einstrom sowie eine verminderte Proliferation infolge der Akkumulation von Ceramiden detektiert. Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass Ceramide wesentliche Prozesse im Verlauf der T-Zell-Aktivierung beeinflussen, so dass die pathogeninduzierte Generierung von Ceramiden einen möglichen Mechanismus darstellt, die Funktion von T-Zellen zu beeinträchtigen. / Ceramides are sphingolipids regulating various signalling pathways mainly associated with the induction of apoptosis and cell cycle arrest. Besides their established role in these processes several lines of evidence suggest that ceramides also regulate cytoskeletal dynamics in non-hematopoietic cells, but their role in T lymphocyte function has not yet been addressed. In this study we show that accumulation of membrane ceramides affects several processes required for accurate T cell function. Treatment of T cells with bSMase or exogenously added ceramides interfered with T cell adhesion to FN and ICAM-1 as well as T cell polarisation, chemotaxis and motility in response to SDF-1. In line with the impairment to morphologically polarise, relocation of surface receptors as well as intracellular signalling molecules was also impaired upon ceramide treatment of T cells. Moreover, increase in cellular ceramide levels interfered with cellular signalling pathways as revealed by reduced phosphorylation levels of Akt and ERM proteins. T cell activation requires the formation of an IS with DCs. In this study we could show, that ceramides, although excluded from the DC/T cell interface, do not interfere with conjugate frequency or synapse organisation, since a normal distribution of CD3 and the MTOC was observed. Nevertheless, ceramide generation interfered with the translocation of Orai1 and Stim1 to the interface and in line with this observation a reduced calcium-influx in T cells was detected upon bSMase exposure. In addition to the decrease in cytosolic calcium levels ceramides also reduced the ability of T cells to proliferate in response to CD3/CD28 stimulation. Therefore the induction of ceramides by certain pathogens, including MV, might be a possible mechanism to interfere with essential processes required for T cell activation.

Introduction: Civilian trauma remains a significant health care problem in North American society. Hemorrhagic shock and resuscitation (S/R) have been shown to prime the immune system for an exaggerated response to subsequent otherwise innocuous inflammatory stimuli such as lipopolysaccharide (LPS), resulting in multiple organ failure or death. Using a rodent model of lung injury, we previously demonstrated that antecedent S/R leads to augmented LPS-induced lung injury by way of heightened NF-κB nuclear translocation, resulting in increased elaboration of pro-inflammatory cytokines in alveolar macrophages. Further studies revealed that oxidative stress generated during S/R is responsible for this priming phenomenon. Our group recently identified two significant alterations to LPS signaling under oxidative stress conditions in macrophages: 1) the rapid recruitment of the LPS receptor Toll-like receptor 4 (TLR4) to membrane lipid rafts, and 2) the reprogramming of LPS signaling to a Src-dependent pathway involving phosphatidylinositol 3-kinase (PI3K). Major Objective and Hypothesis: The objective of this thesis is to elucidate the molecular mechanisms underlying the augmented cellular responsiveness observed in macrophages following oxidative stress. The central hypothesis is that oxidative stress regulates LPS signaling by altering the activation and assembly of TLR4 receptor signaling components through generation of the lipid ceramide. Summary of Findings: In the first paper, we demonstrate that the antioxidant stilbazulenyl nitrone (STAZN), a novel second-generation azulenyl nitrone, is protective in a rodent two-hit model of lung injury involving hemorrhagic S/R and subsequent intra-tracheal LPS injection. Resultant oxidative stress and lung injury in vivo were significantly reduced by STAZN following S/R and LPS. In the second paper, we explore the mechanism underlying oxidant-induced surface up-regulation of TLR4 in macrophages. Using immunofluorescence microscopy and flow cytometry techniques, hydrogen peroxide in vitro and hemorrhagic S/R in vivo are shown to induce TLR4 translocation in macrophages in a ceramide and Src-dependent manner, and the enzyme acid sphingomyelinase (ASM) is shown to mediate ceramide generation. In the third paper, the role of ceramide in oxidant-induced macrophage priming for LPS signaling is investigated. Ceramide generation via ASM is shown to have a prominent upstream role in oxidant activation of the PI3K/Akt pathway via Src kinases in macrophages. Furthermore, oxidative stress is shown to reprogram LPS signaling to a ceramide dependent pathway. Conclusion: Together, these findings highlight the role of oxidative stress in mediating augmented cellular responsiveness following S/R, and describe the role of ceramide as a central upstream mediator of oxidant priming in macrophages. The hierarchy of signaling molecules and interactions described herein represent novel targets for modulating oxidative stress in the treatment of critical illness and organ injury.

Introduction: Civilian trauma remains a significant health care problem in North American society. Hemorrhagic shock and resuscitation (S/R) have been shown to prime the immune system for an exaggerated response to subsequent otherwise innocuous inflammatory stimuli such as lipopolysaccharide (LPS), resulting in multiple organ failure or death. Using a rodent model of lung injury, we previously demonstrated that antecedent S/R leads to augmented LPS-induced lung injury by way of heightened NF-κB nuclear translocation, resulting in increased elaboration of pro-inflammatory cytokines in alveolar macrophages. Further studies revealed that oxidative stress generated during S/R is responsible for this priming phenomenon. Our group recently identified two significant alterations to LPS signaling under oxidative stress conditions in macrophages: 1) the rapid recruitment of the LPS receptor Toll-like receptor 4 (TLR4) to membrane lipid rafts, and 2) the reprogramming of LPS signaling to a Src-dependent pathway involving phosphatidylinositol 3-kinase (PI3K). Major Objective and Hypothesis: The objective of this thesis is to elucidate the molecular mechanisms underlying the augmented cellular responsiveness observed in macrophages following oxidative stress. The central hypothesis is that oxidative stress regulates LPS signaling by altering the activation and assembly of TLR4 receptor signaling components through generation of the lipid ceramide. Summary of Findings: In the first paper, we demonstrate that the antioxidant stilbazulenyl nitrone (STAZN), a novel second-generation azulenyl nitrone, is protective in a rodent two-hit model of lung injury involving hemorrhagic S/R and subsequent intra-tracheal LPS injection. Resultant oxidative stress and lung injury in vivo were significantly reduced by STAZN following S/R and LPS. In the second paper, we explore the mechanism underlying oxidant-induced surface up-regulation of TLR4 in macrophages. Using immunofluorescence microscopy and flow cytometry techniques, hydrogen peroxide in vitro and hemorrhagic S/R in vivo are shown to induce TLR4 translocation in macrophages in a ceramide and Src-dependent manner, and the enzyme acid sphingomyelinase (ASM) is shown to mediate ceramide generation. In the third paper, the role of ceramide in oxidant-induced macrophage priming for LPS signaling is investigated. Ceramide generation via ASM is shown to have a prominent upstream role in oxidant activation of the PI3K/Akt pathway via Src kinases in macrophages. Furthermore, oxidative stress is shown to reprogram LPS signaling to a ceramide dependent pathway. Conclusion: Together, these findings highlight the role of oxidative stress in mediating augmented cellular responsiveness following S/R, and describe the role of ceramide as a central upstream mediator of oxidant priming in macrophages. The hierarchy of signaling molecules and interactions described herein represent novel targets for modulating oxidative stress in the treatment of critical illness and organ injury.

<p>Endozepine is an alternative name for the testis-specific isoform of the acyl-CoA binding protein (t-ACBP). Acyl-CoA binding proteins form a highly conserved family of proteins, which bind long chain fatty acid esters with nanomolar affinity. They are also known to be endogenous ligands to the --amino butyric acid (GABA) receptor in the central nervous system and to play a role in a wide variety of cellular functions such as vesicular trafficking, fatty acid biosynthesis and gene regulation. A role for endozepine in apoptosis was suggested through promoter gene trapping studies using CHO22 cells in which 90 % reduction in the expression of endozepine correlated with delayed mitochondrial permeabilization, a reduced activation of caspase-3 (an activator of apoptosis) and a consequent resistance to C2-ceramide induced apoptosis.</p> <p>Transduction studies using Tat-GFP-ELP fusion protein showed that endozepine restored the sensitivity of mutant CHO22 cells to C2-ceramide induced apoptosis. In this thesis, we have investigated two hypotheses for the involvement of endozepine in ceramide-induced apoptosis. The first hypothesis is that endozepine contributes to apoptosis through the transport of palmitoyl-CoA, a substrate required for the de novo synthesis of ceramide. The second hypothesis is that endozepine interacts directly with ceramide leading to interaction with peripheral benzodiazepine receptor and a subsequent opening of the mitochondria permeability transition pore, leading to apoptosis.</p>

Die Interaktion mit Gehirnendothelzellen stellt ein zentraler Schritt in der Infektionspathogenese von Neisseria meningitidis dar. In dieser Promotionsarbeit konnte gezeigt werden, dass die Infektion von menschlichen Gehirnendothelzellen mit N. meningitidis zu einer transienten Aktivierung der sauren Sphingomyelinase (ASM) gefolgt von einer vermehrten Ceramidproduktion führt. Als Antwort auf die Infektion mit N. meningitidis kommt es zu einer vermehrten Präsentation der ASM und von Ceramiden an der äusseren Seite der Plasmamembran und zu einer Ausbildung von großen Ceramid-reichen Membran-Domänen, welche mit cortical plaque assoziierten Proteinen kolokalisieren. Bei dieser N. meningitids vermittelten Aktivierung der ASM spielt das bakterielle Aussenmembranprotein Opc sowie die Aktivierung der Phosphatidylcholin-spezifische Phospholipase C über die Interaktion von Opc mit Heparansulfat-Proteoglykane eine entscheidende Rolle. Die pharmakologische oder genetische Inhibition der ASM Funktion führt zu einer geringeren Invasivität der Meningokokken ohne dabei die Adhärenz zu beeinflussen. Im Einklang mit diesen Ergebnissen steht die Beobachtung, dass die geringere Invasivität von ausgewählten Isolaten des ST-11/ST-8 Komplex in menschlichen Gehirnendothelzellen direkt mit ihrer eingeschränkter Fähigkeit korreliert, die ASM zu aktivieren bzw. eine Ceramidproduktion zu induzieren. Schlussfolgernd ist die ASM Aktivierung und eine nachfolgende Ceramidproduktion essenziell für die Internalisierung von Opc-exprimierende Meningokokken in Gehirnendothelzellen und bietet einen Erklärungsansatz für die unterschiedliche Invasivität von verschiedenen N. meningitidis Stämmen. / The interaction with brain endothelial cells is central to the pathogenicity of Neisseria meningitidis infections. Here, we show that N. meningitidis causes transient activation of acid sphingomyelinase (ASM) followed by ceramide release in brain endothelial cells. In response to N. meningitidis infection, ASM and ceramide are displayed at the outer leaflet of the cell membrane and condense into large membrane platforms which also colocalize with cortical plaque associated proteins. The outer membrane protein Opc and phosphatidylcholine-specific phospholipase C that is activated upon binding of the pathogen to heparan sulfate proteoglycans, are required for N. meningitidis-mediated ASM activation. Pharmacologic or genetic ablation of ASM abrogated meningococcal internalization without affecting bacterial adherence. In accordance, the restricted invasiveness of a defined set of pathogenic isolates of the ST-11/ST-8 clonal complex into brain endothelial cells directly correlated with their restricted ability to induce ASM and ceramide release. In conclusion, ASM activation and ceramide release are essential for internalization of Opc-expressing meningococci into brain endothelial cells, and this segregates with invasiveness of N. meningitidis strains.

Sphingolipids are important signaling molecules regulating cell growth, cell death and differentiation, thus making them important molecules in determining the fate of a cell and in the pathogenesis of chronic illnesses. The sphingolipid signaling pathway can be initiated by reactive oxygen species (ROS) and inflammatory molecules, both of which are believed to be upregulated in a state of obesity. The hypothesis tested in this dissertation is that due to the inflammatory state of obese animals, the sphingolipid pathway is altered, shifting the balance of pro- and anti-apoptotic proteins and contributing to the pathogenesis of diseases associated with an obese state. The specific aims were to compare, 1) key sphingolipid signaling enzymes; 2) levels of sphingolipid signaling molecules and 3) pro and anti-apoptotic protein levels, in hepatic and colonic tissues procured from lean and obese animals. Obese animals are susceptible to various diseases, including colon cancer and hepatic steatosis. To assess the effect of obesity on sphingolipid signaling, and to provide insight as to the pathogenesis of diseases in a state of obesity, liver and colon tissues from Zucker obese female rats (fa/fa) were compared to tissues from their lean counterparts (Fa/fa or Fa/Fa Zucker rats). Enzyme analyses included an assay of sphingomyelinase (SMase) activity and quantification of ceramidase and sphingosine kinase-1 (SK1) protein expression by western blot. Also, sphingomyelin (SM), ceramide, ceramide-1 phosphate (C1P), sphingosine and sphingosine-1-phosphate (S1P) levels were determined by high-performance liquid chromatography (HPLC) -tandem mass spectroscopy (MS). Representative apoptotic proteins, Bax and Bcl-2 were quantified by western blot. Obese liver demonstrates hepatic steatosis in the Zucker animal model. Among the major differences noted between obese and lean liver were significantly upregulated ceramidase, and downregulated SK1 and C1P levels (P<0.05), as well as a difference in ceramide and SM species composition. Bax was overexpressed while Bcl-2 level was lower in obese compared to lean liver (P<0.05). Taken together, the results indicate a shift toward higher apoptotic signaling in obese liver tissue and correspond with the diseased state of the steatotic liver. Analysis of the sphingolipid pathway in colon revealed upregulation of ceramidase and downregulation of SK1 (P<0.05), similar to liver tissue. C1P levels were lower (P<0.05) but no changes were observed for ceramide, SM or sphingosine levels. A trend toward higher SMase activity in obese colon was observed. Bax was overexpressed in obese colon tissue (P< 0.05), while Bcl-2 results were inconclusive. The liver expressed lower level of molecules associated with sphingolipid signaling than the colons. This study is first to demonstrate tissue-specific differences in the sphingolipid signaling pathway, regardless of genotype. Nevertheless, overall the genotype of Zucker model was found to be a factor altering the expression levels of various sphingolipid enzymes and metabolites in both colon and liver. The findings of the present research provide incentive to further understand the role and modulation of sphingolipid signaling pathway in causation and prevention of chronic diseases prevalent in obese state.