TY - JOUR A1 - Al Fadel, Frdoos A1 - Fayyaz, Susann A1 - Japtok, Lukasz A1 - Kleuser, Burkhard T1 - Involvement of Sphingosine 1-Phosphate in Palmitate-Induced Non-Alcoholic Fatty Liver Disease JF - Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry and pharmacology N2 - Background/Aims: Ectopic lipid accumulation in hepatocytes has been identified as a risk factor for the progression of liver fibrosis and is strongly associated with obesity. In particular, the saturated fatty acid palmitate is involved in initiation of liver fibrosis via formation of secondary metabolites by hepatocytes that in turn activate hepatic stellate cells (HSCs) in a paracrine manner Methods: a-smooth muscle actin-expression (alpha-SMA) as a marker of liver fibrosis was investigated via western blot analysis and immunofluorescence microscopy in HSCs (LX-2). Sphingolipid metabolism and the generation of the bioactive secondary metabolite sphingosine I-phosphate (SIP) in response to palmitate were analyzed by LC-MS/MS in hepatocytes (HepG2). To identify the molecular mechanism involved in the progression of liver fibrosis real-time PCR analysis and pharmacological modulation of SIP receptors were performed. Results: Palmitate oversupply increased intra- and extracellular SIP-concentrations in hepatocytes. Conditioned medium from HepG2 cells initiated fibrosis by enhancing alpha-SMA-expression in LX-2 in a S1P-dependent manner In accordance, fibrotic response in the presence of SIP was also observed in HSCs. Pharmacological inhibition of SIP receptors demonstrated that S1P(3) is the crucial receptor subtype involved in this process. Conclusion: SIP is synthesized in hepatocytes in response to palmitate and released into the extracellular environment leading to an activation of HSCs via the S1P(3) receptor (C) 2016 The Author(s) Published by S. Karger AG, Basel KW - Palmitate KW - Liver fibrosis KW - Sphingosine 1-phosphate KW - Hepatic stellate cells KW - Hepatocytes KW - alpha-SMA Y1 - 2016 U6 - https://doi.org/10.1159/000453213 SN - 1015-8987 SN - 1421-9778 VL - 40 SP - 1637 EP - 1645 PB - Karger CY - Basel ER - TY - JOUR A1 - Bernacchioni, Caterina A1 - Ghini, Veronica A1 - Cencetti, Francesca A1 - Japtok, Lukasz A1 - Donati, Chiara A1 - Bruni, Paola A1 - Turano, Paola T1 - NMR metabolomics highlights sphingosine kinase-1 as a new molecular switch in the orchestration of aberrant metabolic phenotype in cancer cells JF - Molecular oncology / Federation of European Biochemical Societies N2 - Strong experimental evidence in animal and cellular models supports a pivotal role of sphingosine kinase-1 (SK1) in oncogenesis. In many human cancers, SK1 levels are upregulated and these increases are linked to poor prognosis in patients. Here, by employing untargeted NMR- based metabolomic profiling combined with functional validations, we report the crucial role of SK1 in the metabolic shift known as the Warburg effect in A2780 ovarian cancer cells. Indeed, expression of SK1 induced a high glycolytic rate, characterized by increased levels of lactate along with increased expression of the proton/monocarboxylate symporter MCT1, and decreased oxidative metabolism, associated with the accumulation of intermediates of the tricarboxylic acid cycle and reduction in CO2 production. Additionally, SK1-expressing cells displayed a significant increase in glucose uptake paralleled by GLUT3 transporter upregulation. The role of SK1 is not limited to the induction of aerobic glycolysis, affecting metabolic pathways that appear to support the biosynthesis of macromolecules. These findings highlight the role of SK1 signaling axis in cancer metabolic reprogramming, pointing out innovative strategies for cancer therapies. KW - NMR-based metabolomics KW - ovarian cancer KW - sphingosine kinase-1 KW - Warburg effect Y1 - 2017 U6 - https://doi.org/10.1002/1878-0261.12048 SN - 1878-0261 VL - 11 SP - 517 EP - 533 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Bruno, Gennaro A1 - Cencetti, Francesca A1 - Pertici, Irene A1 - Japtok, Lukasz A1 - Bernacchioni, Caterina A1 - Donati, Chiara A1 - Bruni, Paola T1 - CTGF/CCN2 exerts profibrotic action in myoblasts via the up-regulation of sphingosine kinase-1/S1P(3) signaling axis: Implications in the action mechanism of TGF beta JF - Biochimica et biophysica acta : Molecular and cell biology of lipids N2 - The matricellular protein connective tissue growth factor (CTGF/CCN2) is recognized as key player in the onset of fibrosis in various tissues, including skeletal muscle. In many circumstances, CTGF has been shown to be induced by transforming growth factor beta (TGF beta) and accounting, at least in part, for its biological action. In this study it was verified that in cultured myoblasts CTGF/CCN2 causes their transdifferentiation into myofibroblasts by up-regulating the expression of fibrosis marker proteins alpha-smooth muscle actin and transgelin. Interestingly, it was also found that the profibrotic effect exerted by CTGF/CCN2 was mediated by the sphingosine kinase (SK)-1/S1P(3) signaling axis specifically induced by the treatment with the profibrotic cue. Following CTGF/CCN2-induced up-regulation, S1P(3) became the SIP receptor subtype expressed at the highest degree, at least at mRNA level, and was thus capable of readdressing the sphingosine 1-phosphate signaling towards fibrosis rather than myogenic differentiation. Another interesting finding is that CTGF/CCN2 silencing prevented the TGF beta-dependent up-regulation of SKI/S1P(3) signaling axis and strongly reduced the profibrotic effect exerted by TGF beta, pointing at a crucial role of endogenous CTGF/CCN2 generated following TGF beta challenge in the transmission of at least part of its profibrotic effect These results provide new insights into the molecular mechanism by which CTGF/CCN2 drives its biological action and strengthen the concept that SK1/S1P(3) axis plays a critical role in the onset of fibrotic cell phenotype. (C) 2014 Elsevier B.V. All rights reserved. KW - Sphingosine kinase KW - S1P(3) receptor KW - Connective tissue growth factor KW - Myoblasts KW - Transforming growth factor beta Y1 - 2015 U6 - https://doi.org/10.1016/j.bbalip.2014.11.011 SN - 1388-1981 SN - 0006-3002 VL - 1851 IS - 2 SP - 194 EP - 202 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Carpinteiro, Alexander A1 - Becker, Katrin Anne A1 - Japtok, Lukasz A1 - Hessler, Gabriele A1 - Keitsch, Simone A1 - Pozgajova, Miroslava A1 - Schmid, Kurt W. A1 - Adams, Constantin A1 - Müller, Stefan A1 - Kleuser, Burkhard A1 - Edwards, Michael J. A1 - Grassme, Heike A1 - Helfrich, Iris A1 - Gulbins, Erich T1 - Regulation of hematogenous tumor metastasis by acid sphingomyelinase JF - EMBO molecular medicine N2 - Metastatic dissemination of cancer cells is the ultimate hallmark of malignancy and accounts for approximately 90% of human cancer deaths. We investigated the role of acid sphingomyelinase (Asm) in the hematogenous metastasis of melanoma cells. Intravenous injection of B16F10 melanoma cells into wild-type mice resulted in multiple lung metastases, while Asm-deficient mice (Smpd1(-/-) mice) were protected from pulmonary tumor spread. Transplanting wild-type platelets into Asm-deficient mice reinstated tumor metastasis. Likewise, Asm-deficient mice were protected from hematogenous MT/ret melanoma metastasis to the spleen in a mouse model of spontaneous tumor metastasis. Human and mouse melanoma cells triggered activation and release of platelet secretory Asm, in turn leading to ceramide formation, clustering, and activation of 51 integrins on melanoma cells finally leading to adhesion of the tumor cells. Clustering of integrins by applying purified Asm or C-16 ceramide to B16F10 melanoma cells before intravenous injection restored trapping of tumor cells in the lung in Asm-deficient mice. This effect was revertable by arginine-glycine-aspartic acid peptides, which are known inhibitors of integrins, and by antibodies neutralizing 1 integrins. These findings indicate that melanoma cells employ platelet-derived Asm for adhesion and metastasis. KW - acid sphingomyelinase KW - ceramide KW - integrins KW - platelets KW - tumor-metastasis Y1 - 2015 SN - 1757-4676 SN - 1757-4684 VL - 7 IS - 6 SP - 714 EP - 734 PB - Wiley-Blackwell CY - Hoboken ER - TY - JOUR A1 - Cencetti, Francesca A1 - Bruno, Gennaro A1 - Bernacchioni, Caterina A1 - Japtok, Lukasz A1 - Puliti, Elisa A1 - Donati, Chiara A1 - Bruni, Paola T1 - Sphingosine 1-phosphate lyase blockade elicits myogenic differentiation of murine myoblasts acting via Spns2/S1P(2) receptor axis JF - Biochimica et biophysica acta : Molecular and cell biology of lipids N2 - The bioactive sphingolipid sphingosine 1-phosphate (S1P) has emerged in the last three decades as main regulator of key cellular processes including cell proliferation, survival, migration and differentiation. A crucial role for this sphingolipid has been recognized in skeletal muscle cell biology both in vitro and in vivo. S1P lyase (SPL) is responsible for the irreversible degradation of S1P and together with sphingosine kinases, the S1P producing enzymes, regulates cellular S1P levels. In this study is clearly showed that the blockade of SPL by pharmacological or RNA interference approaches induces myogenic differentiation of C2C12 myoblasts. Moreover, down-regulation of the specific S1P transporter spinster homolog 2 (Spns2) abrogates myogenic differentiation brought about by SPL inhibition or down-regulation, pointing at a role of extracellular S1P in the pro-myogenic action induced by SPL blockade. Furthermore, also S1P(2) receptor down-regulation was found to abrogate the pro-myogenic effect of SPL blockade. These results provide further proof that inside-out S1P signaling is critically implicated in skeletal muscle biology and provide support to the concept that the specific targeting of SPL could represent an exploitable strategy to treat skeletal muscle disorders. KW - Sphingosine 1-phosphate KW - Myogenic differentiation Y1 - 2020 U6 - https://doi.org/10.1016/j.bbalip.2020.158759 SN - 1388-1981 SN - 1879-2618 VL - 1865 IS - 9 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Collenburg, Lena A1 - Walter, Tim A1 - Burgert, Anne A1 - Mueller, Nora A1 - Seibel, Juergen A1 - Japtok, Lukasz A1 - Kleuser, Burkhard A1 - Sauer, Markus A1 - Schneider-Schaulies, Sibylle T1 - A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells JF - The journal of immunology N2 - Sphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C-6-ceramides were incorporated into and localized within plasma membrane microdomains and proximal vesicles in T cells. They segregated into clusters after TCR, and especially CD28 ligation, indicating efficient sorting into plasma membrane domains associated with T cell activation; this was abolished upon sphingomyelinase inhibition. Importantly, T cell activation was not abrogated upon incorporation of the compound, which was efficiently excluded from the immune synapse center as has previously been seen in Ab-based studies using fixed cells. Therefore, the functionalized ceramides are novel, highly potent tools to study the subcellular redistribution of ceramides in the course of T cell activation. Moreover, they will certainly also be generally applicable to studies addressing rapid stimulation-mediated ceramide release in living cells. Y1 - 2016 U6 - https://doi.org/10.4049/jimmunol.1502447 SN - 0022-1767 SN - 1550-6606 VL - 196 SP - 3951 EP - 3962 PB - American Assoc. of Immunologists CY - Bethesda ER - TY - JOUR A1 - Derakhshani, Shaghayegh A1 - Kurz, Andreas A1 - Japtok, Lukasz A1 - Schumacher, Fabian A1 - Pilgram, Lisa A1 - Steinke, Maria A1 - Kleuser, Burkhard A1 - Sauer, Markus A1 - Schneider-Schaulies, Sibylle A1 - Avota, Elita T1 - Measles Virus Infection Fosters Dendritic Cell Motility in a 3D Environment to Enhance Transmission to Target Cells in the Respiratory Epithelium JF - Frontiers in immunology N2 - Transmission of measles virus (MV) from dendritic to airway epithelial cells is considered as crucial to viral spread late in infection. Therefore, pathways and effectors governing this process are promising targets for intervention. To identify these, we established a 3D respiratory tract model where MV transmission by infected dendritic cells (DCs) relied on the presence of nectin-4 on H358 lung epithelial cells. Access to recipient cells is an important prerequisite for transmission, and we therefore analyzed migration of MV-exposed DC cultures within the model. Surprisingly, enhanced motility toward the epithelial layer was observed for MV-infected DCs as compared to their uninfected siblings. This occurred independently of factors released from H358 cells indicating that MV infection triggered cytoskeletal remodeling associated with DC polarization enforced velocity. Accordingly, the latter was also observed for MV-infected DCs in collagen matrices and was particularly sensitive to ROCK inhibition indicating infected DCs preferentially employed the amoeboid migration mode. This was also implicated by loss of podosomes and reduced filopodial activity both of which were retained in MV-exposed uninfected DCs. Evidently, sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) as produced in response to virus-infection in DCs contributed to enhanced velocity because this was abrogated upon inhibition of sphingosine kinase activity. These findings indicate that MV infection promotes a push-and-squeeze fast amoeboid migration mode via the SphK/S1P system characterized by loss of filopodia and podosome dissolution. Consequently, this enables rapid trafficking of virus toward epithelial cells during viral exit. KW - dendritic cell KW - cell migration KW - measles virus KW - 3D tissue model KW - sphingosine-1-phosphate Y1 - 2019 U6 - https://doi.org/10.3389/fimmu.2019.01294 SN - 1664-3224 VL - 10 PB - Frontiers Research Foundation CY - Lausanne ER - TY - JOUR A1 - Fayyaz, Susann A1 - Henkel, Janin A1 - Japtok, Lukasz A1 - Krämer, Stephanie A1 - Damm, Georg A1 - Seehofer, Daniel A1 - Püschel, Gerhard Paul A1 - Kleuser, Burkhard T1 - Involvement of sphingosine 1-phosphate in palmitate-induced insulin resistance of hepatocytes via the S1P(2) receptor subtype JF - Diabetologia : journal of the European Association for the Study of Diabetes (EASD) N2 - Enhanced plasma levels of NEFA have been shown to induce hepatic insulin resistance, which contributes to the development of type 2 diabetes. Indeed, sphingolipids can be formed via a de novo pathway from the saturated fatty acid palmitate and the amino acid serine. Besides ceramides, sphingosine 1-phosphate (S1P) has been identified as a major bioactive lipid mediator. Therefore, our aim was to investigate the generation and function of S1P in hepatic insulin resistance. The incorporation of palmitate into sphingolipids was performed by rapid-resolution liquid chromatography-MS/MS in primary human and rat hepatocytes. The influence of S1P and the involvement of S1P receptors in hepatic insulin resistance was examined in human and rat hepatocytes, as well as in New Zealand obese (NZO) mice. Palmitate induced an impressive formation of extra- and intracellular S1P in rat and human hepatocytes. An elevation of hepatic S1P levels was observed in NZO mice fed a high-fat diet. Once generated, S1P was able, similarly to palmitate, to counteract insulin signalling. The inhibitory effect of S1P was abolished in the presence of the S1P(2) receptor antagonist JTE-013 both in vitro and in vivo. In agreement with this, the immunomodulator FTY720-phosphate, which binds to all S1P receptors except S1P(2), was not able to inhibit insulin signalling. These data indicate that palmitate is metabolised by hepatocytes to S1P, which acts via stimulation of the S1P(2) receptor to impair insulin signalling. In particular, S1P(2) inhibition could be considered as a novel therapeutic target for the treatment of insulin resistance. KW - FTY720 KW - Insulin signalling KW - Palmitate KW - S1P receptors KW - Sphingolipids KW - Sphingosine 1-phosphate Y1 - 2014 U6 - https://doi.org/10.1007/s00125-013-3123-6 SN - 0012-186X SN - 1432-0428 VL - 57 IS - 2 SP - 373 EP - 382 PB - Springer CY - New York ER - TY - JOUR A1 - Fayyaz, Susann A1 - Japtok, Lukasz A1 - Schumacher, Fabian A1 - Wigger, Dominik A1 - Schulz, Tim Julius A1 - Haubold, Kathrin A1 - Gulbins, Erich A1 - Völler, Heinz A1 - Kleuser, Burkhard T1 - Lysophosphatidic acid inhibits insulin signaling in primary rat hepatocytes via the LPA(3) receptor subtype and is increased in obesity JF - Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry and pharmacology N2 - Background/Aims: Obesity is a main risk factor for the development of hepatic insulin resistance and it is accompanied by adipocyte hypertrophy and an elevated expression of different adipokines such as autotaxin (ATX). ATX converts lysophosphatidylcholine to lysophosphatidic acid (LPA) and acts as the main producer of extracellular LPA. This bioactive lipid regulates a broad range of physiological and pathological responses by activation of LPA receptors (LPA1-6). Methods: The activation of phosphatidylinositide 3-kinases (PI3K) signaling (Akt and GSK-3ß) was analyzed via western blotting in primary rat hepatocytes. Incorporation of glucose into glycogen was measured by using radio labeled glucose. Real-time PCR analysis and pharmacological modulation of LPA receptors were performed. Human plasma LPA levels of obese (BMI > 30, n = 18) and normal weight individuals (BMI 18.5-25, n = 14) were analyzed by liquid chromatography tandem-mass spectrometry (LC-MS/MS). Results: Pretreatment of primary hepatocytes with LPA resulted in an inhibition of insulin-mediated Gck expression, PI3K activation and glycogen synthesis. Pharmacological approaches revealed that the LPA3-receptor subtype is responsible for the inhibitory effect of LPA on insulin signaling. Moreover, human plasma LPA concentrations (16: 0 LPA) of obese participants (BMI > 30) are significantly elevated in comparison to normal weight individuals (BMI 18.5-25). Conclusion: LPA is able to interrupt insulin signaling in primary rat hepatocytes via the LPA3 receptor subtype. Moreover, the bioactive lipid LPA (16: 0) is increased in obesity. KW - Lysophosphatidic acid KW - Insulin signaling KW - Adipose tissue KW - Autotaxin KW - Hepatic insulin resistance KW - LPA(3) receptor subtype Y1 - 2017 U6 - https://doi.org/10.1159/000480470 SN - 1015-8987 SN - 1421-9778 VL - 43 SP - 445 EP - 456 PB - Karger CY - Basel ER - TY - JOUR A1 - Folkesson, Maggie A1 - Vorkapic, Emina A1 - Gulbins, Erich A1 - Japtok, Lukasz A1 - Kleuser, Burkhard A1 - Welander, Martin A1 - Länne, Toste A1 - Wågsäter, Dick T1 - Inflammatory cells, ceramides, and expression of proteases in perivascular adipose tissue adjacent to human abdominal aortic aneurysms JF - Journal of vascular surgery N2 - Background: Abdominal aortic aneurysm (AAA) is a deadly irreversible weakening and distension of the abdominal aortic wall. The pathogenesis of AAA remains poorly understood. Investigation into the physical and molecular characteristics of perivascular adipose tissue (PVAT) adjacent to AAA has not been done before and is the purpose of this study. Methods and Results: Human aortae, periaortic PVAT, and fat surrounding peripheral arteries were collected from patients undergoing elective surgical repair of AAA. Control aortas were obtained from recently deceased healthy organ donors with no known arterial disease. Aorta and PVAT was found in AAA to larger extent compared with control aortas. Immunohistochemistry revealed neutrophils, macrophages, mast cells, and T-cells surrounding necrotic adipocytes. Gene expression analysis showed that neutrophils, mast cells, and T-cells were found to be increased in PVAT compared with AAA as well as cathepsin K and S. The concentration of ceramides in PVAT was determined using mass spectrometry and correlated with content of T-cells in the PVAT. Conclusions: Our results suggest a role for abnormal necrotic, inflamed, proteolytic adipose tissue to the adjacent aneurysmal aortic wall in ongoing vascular damage. Y1 - 2016 U6 - https://doi.org/10.1016/j.jvs.2015.12.056 SN - 0741-5214 VL - 65 IS - 4 SP - 1171 EP - 1179 PB - Elsevier CY - New York ER -