The G-protein-coupled receptor EDG6

1\. Einführung 1 2\. Material 15 3\. Methoden 25 4\. Ergebnisse 65 5\. Diskussion 115 6\. Zusammenfassung 133 7\. Summary 135 8\. Literatur 137 9\. Abkürzungsverzeichnis 149 10\. Danksagung 153In der vorliegenden Arbeit konnte gezeigt werden, daß der aus in vitro- differenzierten humanen und murinen dendritischen Zellen isolierte G-Protein- gekoppelte Rezeptor EDG6 auf mRNA-Ebene in hämatopoietischen und lymphatischen Geweben und Zellinien sowie in der Lunge exprimiert wird. Das Expressionsmuster ist in Mensch und Maus identisch und läßt auf eine mögliche Funktion des EDG6-Rezeptors im Immunsystem schließen. Das humane edg6-Gen ist auf Chromosom 19p13.3, das murine Homolog auf Chromosom 10 jeweils hinter dem Gna15/16-Gen lokalisiert. Auf Proteinebene konnte der humane und der murine Rezeptor in der richtigen Orientierung auf der Oberfläche EDG6-transfizierter Zellen nachgewiesen werden. Ferner sind drei monoklonale Antikörper hergestellt worden, die gegen den N-Terminus des murinen EDG6-Rezeptors gerichtet sind und deutliche Signale auf transfizierten Zellen zeigen, auf primären Zellen bislang jedoch keine eindeutigen Ergebnisse liefern. Die Herstellung verschiedener stabil EDG6 überexprimierender Zellinien ermöglichte Untersuchungen bezüglich des Liganden sowie der G-Protein Kopplung und der Signaltransduktion von EDG6. Als ein spezifischer Ligand konnte Sphingosin-1-phosphat mit der moderaten Bindungskonstante von 63nM ermittelt werden. Die Stimulierung von EDG6 führt zur Aktivierung von G alpha i- und G alpha12/13-Untereinheiten trimärer G-Proteine. Über die G alpha i-Untereinheiten werden anschließend die Mitogen-aktivierten Proteinkinasen ERK1/2 sowie die Phospholipase C aktiviert. Ferner führt die Stimulierung des EDG6-Rezeptors zu deutlichen Zytoskelettveränderungen, die sich in einer erhöhten Anzahl peripherer Streßfasern, abgerundeter Zellen und besonders langer Filopodien äußern und vermutlich über G alpha 12/13 und Rho-GTPase- vermittelte intrazelluläre Signalwege induziert werden. Eine gewisse Pertussistoxin-sensitive und Liganden-unabhängige Basalaktivität des EDG6-Rezeptors ist in EDG6 überexprimierenden Jurkat-Zellen festzustellen, die eine deutlich erhöhte Spontanmigration zeigen. Allerdings lassen die Migrationsexperimente nicht auf eine durch EDG6 induzierte gerichtete Migration schließen. Versuche bezüglich der Oberflächenexpression des EDG6-Rezeptors deuten auf eine komplexe zellspezifische Regulation des Oberflächentransports hin. In HEK293-Zellen konnte die Internalisierung des EDG6-Rezeptors nach Stimulierung mit Sphingosin-1-phosphat nachgewiesen werden, während EDG6 in CHO-K1-Zellen erst Minuten nach Sphingosin-1-phosphat-Stimulierung und in HeLa-Zellen Minuten nach Zugabe eines unbekannten Hitze-instabilen Serumproteins massiv an die Zelloberfläche transportiert wird. Diese Regulation scheint von einem freien EDG6-N-Terminus abhängig zu sein, da N-terminal Epitop markierte EDG6-Konstrukte nicht mehr dieser differentiellen Oberflächenregulation in HeLa-Zellen unterliegen und konstitutiv an der Zelloberfläche präsent sind. Um die in vivo-Funktion des EDG6-Rezeptors näher untersuchen zu können, wurde ein embryonaler Stammzellklon zur Generierung edg6-defizienter Mäuse hergestellt. Möglicherweise moduliert EDG6 nach Bindung von freigesetztem Sphingosin-1-phosphat in autokriner und parakriner Weise die Immunantwort in Folge einer Stimulierung des Immunsystems, die beispielsweise durch eine Infektion induziert werden könnte.The present study demonstrates that the G protein-coupled receptor EDG6 isolated from in vitro differentiated human and murine dendritic cells is expressed in hematopoietic and lymphoid tissues and cell lines as well as in lung. The mRNA expression pattern is identical in man and mouse and points to a putative function of the EDG6 receptor in the immune system. The human edg6 gene is encoded on chromosome 19p13.3, the murine homolog on chromosome 10. Both genes are located upstream of the of the Gna15/16 gene. The human and murine EDG6 receptors have been detected on the cell surface of transiently transfected cells in the correct orientation. Furthermore, three monoclonal antibodies that are directed against the N-terminus of the murine EDG6 receptor have been generated . They show distinct signals on transiently transfected cell lines, but they lack any specific signal on primary cells so far. The generation of stably human EDG6 overexpressing cell lines has enabled the investigation of the ligand, the G protein-coupling, and the signal transduction of EDG6. Sphingosine 1-phosphate could be identified as a specific ligand for EDG6 with a moderate binding constant of 63nM. Stimulation of EDG6 leads to the activation of G alpha i and G alpha 12/13 subunits of trimeric G proteins. The mitogen-activated protein-kinases ERK1/2 as well as the phospholipase C are subsequently activated in a G alpha i-dependent manner. Moreover, stimulation of the EDG6 receptor leads to distinct cytoskeleton rearrangements resulting in increasing amounts of peripheral stress fibers, cell rounding and very long filopodia. These effects are induced presumably by G alpha 12/13 and Rho GTPase-driven signaling pathways. A certain pertussis toxin-sensitive and ligand-independent basal activity of the EDG6 receptor is demonstrable in stably human EDG6 overexpressing Jurkat cells that show a higher degree of spontaneous migration events. However, these experiments do not suggest an EDG6-induced migration towards a sphingosine 1-phosphate stimulus. Experiments concerning the surface expression of EDG6 indicate a complex cell- specific regulation of the surface transportation. In HEK293 cells the internalization of the EDG6 receptor has been shown after stimulation with sphingosine 1-phosphate. In CHO-K1 cells EDG6 is transported towards the cell surface only several minutes after stimulation with sphingosine 1-phosphate, and in HeLa cells the addition of an unknown heat instable serum protein is critical for the surface transportation of EDG6. The mentioned regulation seems to be dependent on a free EDG6 N-terminus because N-terminal epitope- tagged EDG6 constructs lack the differential surface regulation in HeLa cells. They are constitutively present on the cell surface. To further investigate the in vivo function of the EDG6 receptor, an embryonic stem cell clone has been generated to establish edg6-deficient mice. EDG6 possibly modulates the immunological response in an autocrine or paracrine fashion after binding free sphingosine 1-phosphate. This event may happen after an immunological challenge like an infection

Evaluating Sphingosine and its Analogues as Potential Alternatives for Aggressive Lymphoma Treatment

Ceramide (Cer) and sphingosine (Sph) interfere with critical cellular functions relevant for cancer progression and cell survival. While Cer has already been investigated as a potential drug target for lymphoma treatment, information about the potency of sphingosine is scarce. The aim of this study therefore was to evaluate Sph and its synthetic stereoisomer L-threo- sphingosine (Lt-Sph) as potential treatment options for aggressive lymphomas. Methods: Diffuse large B cell lymphoma (DLBCL) cell lines were incubated with Sph and Lt-Sph and consequently analysed by flow cytometry (FACS), enzyme- linked immunosorbent assay (ELISA), liquid chromatography coupled to triple- quadrupole mass spectrometry (LC/MS/MS), electron microscopy, and Western blot. Results: Sph induced cell death and blocked cell growth independently of S1P receptors in different DLBCL cell lines. Three different modes of Sph- mediated cell death were observed: Apoptosis, autophagy, and protein kinase C (PKC) inhibition. Generation of pro-apoptotic Cer accounted only for a minor portion of the apoptotic rate. Conclusion: Sph and its analogues could evolve as alternative treatment options for aggressive lymphomas via PKC inhibition, apoptosis, and autophagy. These physiological responses induced by different intracellular signalling cascades (phosphorylation of JNK, PARP cleavage, LC3-II accumulation) identify Sph and analogues as potent cell death inducing agents

Restless-Legs-Syndrom und subjektives Wohlbefinden

In der COR-Studie wurden Mitglieder deutscher und schweizer Selbsthilfegruppen sowie Patienten aus dem ARELESS-Register über einen Zeitraum von 3 Jahren befragt. Die Fragebögen enthielten validierte Skalen zur Erhebung des RLS-Schweregrades (IRLS), der Depressivität (CESD), der Lebensqualität (SF-36) und der Schlafqualität (PSQI). Der RLS-Schweregrad zeigte in allen Skalen durchgehend einen starken negativen Einfluss auf die Domänen des Wohlbefinden. Der Einfluss des RLS-Schweregrades nahm für alle Skalen über die drei Jahre hinweg zu. Die Betrachtung der Veränderungen der einzelnen Skalen über den Studienzeitraum zeigte ebenfalls durchgehend einen hohen Einfluss der Veränderung des RLS-Schweregrades. Nicht nur das Vorhandensein des RLS an sich, sondern auch das Ausmaß der Erkrankung spielt eine entscheidende Rolle in Bezug auf das Wohlbefinden der Patienten. Zur Besserung des Wohlbefindens sollte der RLS-Schweregrad durch eine frühzeitige Diagnose und Therapie gesenkt werden

Deficiency of Sphingosine-1-phosphate Lyase Impairs Lysosomal Metabolism of the Amyloid Precursor Protein

Progressive accumulation of the amyloid β protein in extracellular plaques is a neuropathological hallmark of Alzheimer disease. Amyloid β is generated during sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. In addition to the proteolytic processing by secretases, APP is also metabolized by lysosomal proteases. Here, we show that accumulation of intracellular sphingosine-1-phosphate (S1P) impairs the metabolism of APP. Cells lacking functional S1P-lyase, which degrades intracellular S1P, strongly accumulate full-length APP and its potentially amyloidogenic C-terminal fragments (CTFs) as compared with cells expressing the functional enzyme. By cell biological and biochemical methods, we demonstrate that intracellular inhibition of S1P-lyase impairs the degradation of APP and CTFs in lysosomal compartments and also decreases the activity of γ-secretase. Interestingly, the strong accumulation of APP and CTFs in S1P-lyase-deficient cells was reversed by selective mobilization of Ca(2+) from the endoplasmic reticulum or lysosomes. Intracellular accumulation of S1P also impairs maturation of cathepsin D and degradation of Lamp-2, indicating a general impairment of lysosomal activity. Together, these data demonstrate that S1P-lyase plays a critical role in the regulation of lysosomal activity and the metabolism of APP

Activation of Sphingomyelinase-Ceramide-Pathway in COVID-19 Purposes Its Inhibition for Therapeutic Strategies

Effective treatment strategies for severe coronavirus disease (COVID-19) remain scarce. Hydrolysis of membrane-embedded, inert sphingomyelin by stress responsive sphingomyelinases is a hallmark of adaptive responses and cellular repair. As demonstrated in experimental and observational clinical studies, the transient and stress-triggered release of a sphingomyelinase, SMPD1, into circulation and subsequent ceramide generation provides a promising target for FDA-approved drugs. Here, we report the activation of sphingomyelinase-ceramide pathway in 23 intensive care patients with severe COVID-19. We observed an increase of circulating activity of sphingomyelinase with subsequent derangement of sphingolipids in serum lipoproteins and from red blood cells (RBC). Consistent with increased ceramide levels derived from the inert membrane constituent sphingomyelin, increased activity of acid sphingomyelinase (ASM) accurately distinguished the patient cohort undergoing intensive care from healthy controls. Positive correlational analyses with biomarkers of severe clinical phenotype support the concept of an essential pathophysiological role of ASM in the course of SARS-CoV-2 infection as well as of a promising role for functional inhibition with anti-inflammatory agents in SARS-CoV-2 infection as also proposed in independent observational studies. We conclude that large-sized multicenter, interventional trials are now needed to evaluate the potential benefit of functional inhibition of this sphingomyelinase in critically ill patients with COVID-19

Long-Chain and Very Long-Chain Ceramides Mediate Doxorubicin-Induced Toxicity and Fibrosis

Doxorubicin (Dox) is a chemotherapeutic agent with cardiotoxicity associated with profibrotic effects. Dox increases ceramide levels with pro-inflammatory effects, cell death, and fibrosis. The purpose of our study was to identify the underlying ceramide signaling pathways. We aimed to characterize the downstream effects on cell survival, metabolism, and fibrosis. Human fibroblasts (hFSF) were treated with 0.7 µM of Dox or transgenically overexpressed ceramide synthase 2 (FLAG-CerS2). Furthermore, cells were pre-treated with MitoTempo (MT) (2 h, 20 µM) or Fumonisin B1 (FuB) (4 h, 100 µM). Protein expression was measured by Western blot or immunofluorescence (IF). Ceramide levels were determined with mass spectroscopy (MS). Visualizations were conducted using laser scanning microscopy (LSM) or electron microscopy. Mitochondrial activity was measured using seahorse analysis. Dox and CerS2 overexpression increased CerS2 protein expression. Coherently, ceramides were elevated with the highest peak for C24:0. Ceramide- induced mitochondrial ROS production was reduced with MT or FuB preincubation. Mitochondrial homeostasis was reduced and accompanied by reduced ATP production. Our data show that the increase in pro-inflammatory ceramides is an essential contributor to Dox side-effects. The accumulation of ceramides resulted in a lipotoxic shift and subsequently mitochondrial structural and functional damage, which was partially reversible following inhibition of ceramide synthesis

Targeting defective sphingosine kinase 1 in Niemann–Pick type C disease with an activator mitigates cholesterol accumulation

Niemann–Pick type C (NPC) disease is a lysosomal storage disorder arising from mutations in the cholesterol-trafficking protein NPC1 (95%) or NPC2 (5%). These mutations result in accumulation of low-density lipoprotein-derived cholesterol in late endosomes/lysosomes, disruption of endocytic trafficking, and stalled autophagic flux. Additionally, NPC disease results in sphingolipid accumulation, yet it is unique among the sphingolipidoses because of the absence of mutations in the enzymes responsible for sphingolipid degradation. In this work, we examined the cause for sphingosine and sphingolipid accumulation in multiple cellular models of NPC disease and observed that the activity of sphingosine kinase 1 (SphK1), one of the two isoenzymes that phosphorylate sphingoid bases, was markedly reduced in both NPC1 mutant and NPC1 knockout cells. Conversely, SphK1 inhibition with the isotype-specific inhibitor SK1-I in WT cells induced accumulation of cholesterol and reduced cholesterol esterification. Of note, a novel SphK1 activator (SK1-A) that we have characterized decreased sphingoid base and complex sphingolipid accumulation and ameliorated autophagic defects in both NPC1 mutant and NPC1 knockout cells. Remarkably, in these cells, SK1-A also reduced cholesterol accumulation and increased cholesterol ester formation. Our results indicate that a SphK1 activator rescues aberrant cholesterol and sphingolipid storage and trafficking in NPC1 mutant cells. These observations highlight a previously unknown link between SphK1 activity, NPC1, and cholesterol trafficking and metabolism.Fil: Newton, Jason. Virginia Commonwealth University School of Medicine; Estados UnidosFil: Palladino, Elisa N.D.. Virginia Commonwealth University School of Medicine; Estados UnidosFil: Weigel, Cynthia. Virginia Commonwealth University School of Medicine; Estados UnidosFil: Maceyka, Michael. Virginia Commonwealth University School of Medicine; Estados UnidosFil: Gräler, Markus H.. Universitätsklinikum Jena; AlemaniaFil: Senkal, Can E.. Virginia Commonwealth University School of Medicine; Estados UnidosFil: Enriz, Ricardo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Marvanova, Pavlina. Veterinární univerzita Brno; República ChecaFil: Jampilek, Josef. Univerzita Komenského v Bratislave; EslovaquiaFil: Lima, Santiago. Virginia Commonwealth University; Estados UnidosFil: Milstien, Sheldon. Virginia Commonwealth University School of Medicine; Estados UnidosFil: Spiegel, Sarah. Virginia Commonwealth University School of Medicine; Estados Unido

Defects of High-Density Lipoproteins in Coronary Artery Disease Caused by Low Sphingosine-1-Phosphate Content Correction by Sphingosine-1-Phosphate—Loading

AbstractBackgroundSphingosine-1-phosphate (S1P) is a constituent of high-density lipoproteins (HDL) that contributes to their beneficial effects. We have shown decreased HDL-S1P in coronary artery disease (CAD) but its functional relevance remains unclear.ObjectivesThis study investigated the functional consequences of reduced HDL-S1P content in CAD and tested if increasing it may improve or restore HDL function.MethodsHuman HDL from healthy and CAD subjects, as well as mouse HDL, were isolated by ultracentrifugation. HDL-S1P-dependent activation of cell-signaling pathways and induction of vasodilation were examined in vitro and in isolated arteries using native and S1P-loaded HDL, S1P receptor antagonists, and S1P-blocking antibodies.ResultsHDL-S1P-dependent signaling was clearly impaired and S1P content reduced in CAD-HDL as compared to healthy HDL. Both healthy and CAD-HDL could be efficiently and equally well loaded with S1P from cellular donors and plasma. S1P-loading greatly improved HDL signaling and vasodilatory potential in pre-contracted arteries and completely corrected the defects inherent to CAD-HDL. HDL-S1P content and uptake was reduced by oxidation and was lower in HDL3 than HDL2. Loading with S1P in vitro and in vivo fully replenished the virtually absent S1P content of apolipoprotein M-deficient HDL and restored their defective signaling. Infusion of erythrocyte-associated C17-S1P in mice led to its rapid and complete uptake by HDL providing a means to directly S1P-load HDL in vivo.ConclusionsReduced HDL-S1P content contributes to HDL dysfunction in CAD. It can be efficiently increased by S1P-loading in vitro and in vivo, providing a novel approach to correcting HDL dysfunction in CAD