Publications by authors named "Sandra Trautmann"

32 Publications

Sapropterin (BH4) Aggravates Autoimmune Encephalomyelitis in Mice.

Neurotherapeutics 2021 Apr 12. Epub 2021 Apr 12.

Institute of Clinical Pharmacology, Medical Faculty, Goethe-University, Frankfurt, Germany.

Depletion of the enzyme cofactor, tetrahydrobiopterin (BH4), in T-cells was shown to prevent their proliferation upon receptor stimulation in models of allergic inflammation in mice, suggesting that BH4 drives autoimmunity. Hence, the clinically available BH4 drug (sapropterin) might increase the risk of autoimmune diseases. The present study assessed the implications for multiple sclerosis (MS) as an exemplary CNS autoimmune disease. Plasma levels of biopterin were persistently low in MS patients and tended to be lower with high Expanded Disability Status Scale (EDSS). Instead, the bypass product, neopterin, was increased. The deregulation suggested that BH4 replenishment might further drive the immune response or beneficially restore the BH4 balances. To answer this question, mice were treated with sapropterin in immunization-evoked autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. Sapropterin-treated mice had higher EAE disease scores associated with higher numbers of T-cells infiltrating the spinal cord, but normal T-cell subpopulations in spleen and blood. Mechanistically, sapropterin treatment was associated with increased plasma levels of long-chain ceramides and low levels of the poly-unsaturated fatty acid, linolenic acid (FA18:3). These lipid changes are known to contribute to disruptions of the blood-brain barrier in EAE mice. Indeed, RNA data analyses revealed upregulations of genes involved in ceramide synthesis in brain endothelial cells of EAE mice (LASS6/CERS6, LASS3/CERS3, UGCG, ELOVL6, and ELOVL4). The results support the view that BH4 fortifies autoimmune CNS disease, mechanistically involving lipid deregulations that are known to contribute to the EAE pathology.
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http://dx.doi.org/10.1007/s13311-021-01043-4DOI Listing
April 2021

The sphingosine kinase 1 activator, K6PC-5, attenuates Ebola virus infection.

iScience 2021 Apr 5;24(4):102266. Epub 2021 Mar 5.

Institute of General Pharmacology and Toxicology, University Hospital Frankfurt, Goethe University Frankfurt, Frankfurt am Main 60590, Germany.

Ebola virus (EBOV) is responsible for outbreaks with case fatality rates of up to 90% and for an epidemic in West Africa with more than ten thousand deaths. EBOV glycoprotein (EBOV-GP) is the only viral surface protein and is responsible for viral entry into cells. Here, by employing pseudotyped EBOV-GP viral particles, we uncover a critical role for sphingolipids in inhibiting viral entry. Sphingosine kinase 1 (SphK1) catalyzes the phosphorylation of sphingosine to sphingosine 1-phosphate (S1P). The administration of the SphK1 activator, K6PC-5, or S1P, or the overexpression of SphK1 consistently exhibited striking inhibitory effects in EBOV-GP-driven entry in diverse cell lines. Finally, K6PC-5 markedly reduced the EBOV titer in infected cells and the de novo production of viral proteins. These data present K6PC-5 as an efficient tool to inhibit EBOV infection in endothelial cells and suggest further studies to evaluate its systemic effects.
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http://dx.doi.org/10.1016/j.isci.2021.102266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005759PMC
April 2021

Autophagy activation, lipotoxicity and lysosomal membrane permeabilization synergize to promote pimozide- and loperamide-induced glioma cell death.

Autophagy 2021 Jan 19:1-20. Epub 2021 Jan 19.

Experimental Neurosurgery, Goethe University Hospital Frankfurt/Main , Frankfurt, Germany.

Increasing evidence suggests that induction of lethal macroautophagy/autophagy carries potential significance for the treatment of glioblastoma (GBM). In continuation of previous work, we demonstrate that pimozide and loperamide trigger an ATG5- and ATG7 (autophagy related 5 and 7)-dependent type of cell death that is significantly reduced with cathepsin inhibitors and the lipid reactive oxygen species (ROS) scavenger α-tocopherol in MZ-54 GBM cells. Global proteomic analysis after treatment with both drugs also revealed an increase of proteins related to lipid and cholesterol metabolic processes. These changes were accompanied by a massive accumulation of cholesterol and other lipids in the lysosomal compartment, indicative of impaired lipid transport/degradation. In line with these observations, pimozide and loperamide treatment were associated with a pronounced increase of bioactive sphingolipids including ceramides, glucosylceramides and sphingoid bases measured by targeted lipidomic analysis. Furthermore, pimozide and loperamide inhibited the activity of SMPD1/ASM (sphingomyelin phosphodiesterase 1) and promoted induction of lysosomal membrane permeabilization (LMP), as well as release of CTSB (cathepsin B) into the cytosol in MZ-54 wild-type (WT) cells. Whereas LMP and cell death were significantly attenuated in and knockout (KO) cells, both events were enhanced by depletion of the lysophagy receptor VCP (valosin containing protein), supporting a pro-survival function of lysophagy under these conditions. Collectively, our data suggest that pimozide and loperamide-driven autophagy and lipotoxicity synergize to induce LMP and cell death. The results also support the notion that simultaneous overactivation of autophagy and induction of LMP represents a promising approach for the treatment of GBM. : ACD: autophagic cell death; AKT1: AKT serine/threonine kinase 1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG14: autophagy related 14; CERS1: ceramide synthase 1; CTSB: cathepsin B; CYBB/NOX2: cytochrome b-245 beta chain; ER: endoplasmatic reticulum; FBS: fetal bovine serum; GBM: glioblastoma; GO: gene ontology; HTR7/5-HT7: 5-hydroxytryptamine receptor 7; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAP: LC3-associated phagocytosis; LMP: lysosomal membrane permeabilization; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; RB1CC1: RB1 inducible coiled-coil 1; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SMPD1/ASM: sphingomyelin phosphodiesterase 1; VCP/p97: valosin containing protein; WT: wild-type.
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http://dx.doi.org/10.1080/15548627.2021.1874208DOI Listing
January 2021

C6 Ceramide (d18:1/6:0) as a Novel Treatment of Cutaneous T Cell Lymphoma.

Cancers (Basel) 2021 Jan 13;13(2). Epub 2021 Jan 13.

Department of Dermatology, Venerology and Allergology, Goethe University Hospital, 60590 Frankfurt am Main, Germany.

Cutaneous T cell lymphomas (CTCLs) represent a heterogeneous group of T cell lymphomas that primarily affect the skin. The most frequent forms of CTCL are mycosis fungoides and Sézary syndrome. Both are characterized by frequent recurrence, developing chronic conditions and high mortality with a lack of a curative treatment. In this study, we evaluated the effect of short-chain, cell-permeable C6 Ceramide (C6Cer) on CTCL cell lines and keratinocytes. C6Cer significantly reduced cell viability of CTCL cell lines and induced cell death via apoptosis and necrosis. In contrast, primary human keratinocytes and HaCaT keratinocytes were less affected by C6Cer. Both keratinocyte cell lines showed higher expressions of ceramide catabolizing enzymes and HaCaT keratinocytes were able to metabolize C6Cer faster and more efficiently than CTCL cell lines, which might explain the observed protective effects. Along with other existing skin-directed therapies, C6Cer could be a novel well-tolerated drug for the topical treatment of CTCL.
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http://dx.doi.org/10.3390/cancers13020270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828274PMC
January 2021

Validation of highly selective sphingosine kinase 2 inhibitors SLM6031434 and HWG-35D as effective anti-fibrotic treatment options in a mouse model of tubulointerstitial fibrosis.

Cell Signal 2021 Mar 8;79:109881. Epub 2020 Dec 8.

Pharmazentrum Frankfurt/ZAFES, Institute of General Pharmacology and Toxicology, Universitätsklinikum and Goethe-Universität Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany.

Renal fibrosis is characterized by chronic inflammation and excessive accumulation of extracellular matrix and progressively leads to functional insufficiency and even total loss of kidney function. In this study we investigated the anti-fibrotic potential of two highly selective and potent SK2 inhibitors, SLM6031434 and HWG-35D, in unilateral ureter obstruction (UUO), a model for progressive renal fibrosis, in mice. In both cases, treatment with SLM6031434 or HWG-35D resulted in an attenuated fibrotic response to UUO in comparison to vehicle-treated mice as demonstrated by reduced collagen accumulation and a decreased expression of collagen-1 (Col1), fibronectin-1 (FN-1), connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA). Similar to our previous study in Sphk2 mice, we found an increased protein expression of Smad7, a negative regulator of the pro-fibrotic TGFβ/Smad signalling cascade, accompanied by a strong accumulation of sphingosine in SK2 inhibitor-treated kidneys. Treatment of primary renal fibroblasts with SLM6031434 or HWG-35D dose-dependently increased Smad7 expression and ameliorated the expression of Col1, FN-1 and CTGF. In summary, these data prove the anti-fibrotic potential of SK2 inhibition in a mouse model of renal fibrosis, thereby validating SK2 as pharmacological target for the treatment of fibrosis in chronic kidney disease.
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http://dx.doi.org/10.1016/j.cellsig.2020.109881DOI Listing
March 2021

Consistent alteration of chain length-specific ceramides in human and mouse fibrotic kidneys.

Biochim Biophys Acta Mol Cell Biol Lipids 2021 01 1;1866(1):158821. Epub 2020 Oct 1.

Institute of General Pharmacology and Toxicology, University Hospital, Goethe University Frankfurt am Main, Germany.

Background: Several studies revealed alterations of single sphingolipid species, such as chain length-specific ceramides, in plasma and serum of patients with kidney diseases. Here, we investigated whether such alterations occur in kidney tissue from patients and mice suffering from renal fibrosis, the common endpoint of chronic kidney diseases.

Methods: Human fibrotic kidney samples were collected from nephrectomy specimens with hydronephrosis and/or pyelonephritis. Healthy parts from tumor nephrectomies served as nonfibrotic controls. Mouse fibrotic kidney samples were collected from male C57BL/6J mice treated with an adenine-rich diet for 14 days or were subjected to 7 days of unilateral ureteral obstruction (UUO). Kidneys of untreated mice and contralateral kidneys (UUO) served as respective controls. Sphingolipid levels were detected by LC-MS/MS. Fibrotic markers were analyzed by TaqMan® analysis and immunohistology.

Results: Very long-chain ceramides Cer d18:1/24:0 and Cer d18:1/24:1 were significantly downregulated in both fibrotic human kidney cortex and fibrotic murine kidney compared to respective control samples. These effects correlate with upregulation of COL1α1, COL3α1 and αSMA expression in fibrotic human kidney cortex and fibrotic mouse kidney.

Conclusion: We have shown that very long-chain ceramides Cer d18:1/24:0 and Cer d18:1/24:1 are consistently downregulated in fibrotic kidney samples from human and mouse. Our findings support the use of in vivo murine models as appropriate translational means to understand the involvement of ceramides in human kidney diseases. In addition, our study raises interesting questions about the possible manipulation of ceramide metabolism to prevent progression of fibrosis and the use of ceramides as potential biomarkers of chronic kidney disease.
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http://dx.doi.org/10.1016/j.bbalip.2020.158821DOI Listing
January 2021

Dissecting G-Mediated Plasma Membrane Translocation of Sphingosine Kinase-1.

Cells 2020 09 29;9(10). Epub 2020 Sep 29.

Institut für Allgemeine Pharmakologie und Toxikologie, Universitätsklinikum, Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany.

Diverse extracellular signals induce plasma membrane translocation of sphingosine kinase-1 (SphK1), thereby enabling inside-out signaling of sphingosine-1-phosphate. We have shown before that G-coupled receptors and constitutively active Gα specifically induced a rapid and long-lasting SphK1 translocation, independently of canonical G/phospholipase C (PLC) signaling. Here, we further characterized G regulation of SphK1. SphK1 translocation by the M receptor in HEK-293 cells was delayed by expression of catalytically inactive G-protein-coupled receptor kinase-2, p63Rho guanine nucleotide exchange factor (p63RhoGEF), and catalytically inactive PLCβ, but accelerated by wild-type PLCβ and the PLCδ PH domain. Both wild-type SphK1 and catalytically inactive SphK1-G82D reduced M receptor-stimulated inositol phosphate production, suggesting competition at Gα. Embryonic fibroblasts from Gα double-deficient mice were used to show that amino acids W263 and T257 of Gα, which interact directly with PLCβ and p63RhoGEF, were important for bradykinin B receptor-induced SphK1 translocation. Finally, an AIXXPL motif was identified in vertebrate SphK1 (positions 100-105 in human SphK1a), which resembles the Gα binding motif, ALXXPI, in PLCβ and p63RhoGEF. After M receptor stimulation, SphK1-A100E-I101E and SphK1-P104A-L105A translocated in only 25% and 56% of cells, respectively, and translocation efficiency was significantly reduced. The data suggest that both the AIXXPL motif and currently unknown consequences of PLCβ/PLCδ(PH) expression are important for regulation of SphK1 by G.
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http://dx.doi.org/10.3390/cells9102201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599897PMC
September 2020

A Sphingosine 1-Phosphate Gradient Is Linked to the Cerebral Recruitment of T Helper and Regulatory T Helper Cells during Acute Ischemic Stroke.

Int J Mol Sci 2020 Aug 28;21(17). Epub 2020 Aug 28.

Institute of General Pharmacology and Toxicology, Pharmazentrum Frankfurt, Goethe University Frankfurt, 60528 Frankfurt am Main, Germany.

Emerging evidence suggests a complex relationship between sphingosine 1-phosphate (S1P) signaling and stroke. Here, we show the kinetics of S1P in the acute phase of ischemic stroke and highlight accompanying changes in immune cells and S1P receptors (S1P). Using a C57BL/6 mouse model of middle cerebral artery occlusion (MCAO), we assessed S1P concentrations in the brain, plasma, and spleen. We found a steep S1P gradient from the spleen towards the brain. Results obtained by qPCR suggested that cells expressing the S1P type 1 (S1P) were the predominant population deserting the spleen. Here, we report the cerebral recruitment of T helper (T) and regulatory T (T) cells to the ipsilateral hemisphere, which was associated with differential regulation of cerebral S1P expression patterns in the brain after MCAO. This study provides insight that the S1P-S1P axis facilitates splenic T cell egress and is linked to the cerebral recruitment of S1P T and T cells. Further insights by which means the S1P-S1P-axis orchestrates neuronal positioning may offer new therapeutic perspectives after ischemic stroke.
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http://dx.doi.org/10.3390/ijms21176242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7503682PMC
August 2020

High Glucosylceramides and Low Anandamide Contribute to Sensory Loss and Pain in Parkinson's Disease.

Mov Disord 2020 10 11;35(10):1822-1833. Epub 2020 Jul 11.

Institute of Clinical Pharmacology, Goethe-University, Medical Faculty, Frankfurt, Germany.

Background: Parkinson's disease (PD) causes chronic pain in two-thirds of patients, in part originating from sensory neuropathies. The aim of the present study was to describe the phenotype of PD-associated sensory neuropathy and to evaluate its associations with lipid allostasis, the latter motivated by recent genetic studies associating mutations of glucocerebrosidase with PD onset and severity. Glucocerebrosidase catalyzes the metabolism of glucosylceramides.

Methods: We used quantitative sensory tests, pain ratings, and questionnaires and analyzed plasma levels of multiple bioactive lipid species using targeted lipidomic analyses. The study comprised 2 sets of patients and healthy controls: the first 128 Israeli PD patients and 224 young German healthy controls for exploration, the second 50/50 German PD patients and matched healthy controls for deeper analyses.

Results: The data showed a 70% prevalence of PD pain and sensory neuropathies with a predominant phenotype of thermal sensory loss plus mechanical hypersensitivity. Multivariate analyses of lipids revealed major differences between PD patients and healthy controls, mainly originating from glucosylceramides and endocannabinoids. Glucosylceramides were increased, whereas anandamide and lysophosphatidic acid 20:4 were reduced, stronger in patients with ongoing pain and with a linear relationship with pain intensity and sensory losses, particularly for glucosylceramide 18:1 and glucosylceramide 24:1.

Conclusions: Our data suggest that PD-associated sensory neuropathies and PD pain are in part caused by accumulations of glucosylceramides, raising the intriguing possibility of reducing PD pain and sensory loss by glucocerebrosidase substituting or refolding approaches. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
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http://dx.doi.org/10.1002/mds.28186DOI Listing
October 2020

Ceramide Synthase 5 Deficiency Aggravates Dextran Sodium Sulfate-Induced Colitis and Colon Carcinogenesis and Impairs T-Cell Activation.

Cancers (Basel) 2020 Jul 1;12(7). Epub 2020 Jul 1.

Institute of Clinical Pharmacology, Faculty of Medicine, Goethe-University Frankfurt, 60590 Frankfurt, Germany.

Ceramide synthase 5 is one of six enzymes that catalyze the production of ceramides from sphingosine or sphinganine. Ceramides are important components of cell membranes and act as signaling molecules. Previously it has been shown that ceramide synthase 6 and 2 influence colitis in several animal models with sometimes opposite effects. Here, we investigated the disease course of dextran sodium sulfate-induced acute colitis and azoxymethane/dextran sodium sulfate-induced colitis-associated colon cancer in mice with global ceramide synthase 5 knockout (CerS5-ko) or with ceramide synthase 5 knockout restricted to the colon epithelium (CerS5fl/fl VilCre). We monitored disease development and analyzed colon barrier function as well as the immune cell status in these mice. CerS5-ko mice but not CerS5fl/fl-VilCre mice were more susceptible to acute and chronic inflammation. However, the cell barrier function of colon epithelial cells was not disturbed by downregulation of ceramide synthase 5. Instead, untreated CerS5-ko mice displayed reduced numbers of CD3 immune cells in the spleen, colon, and blood, especially of intraepithelial CD8 T-cells, which was not obvious in CerS5fl/fl Vil Cre mice. Reduced T-cell number in colon tissue of CerS5-ko mice was accompanied by a reduced expression of IL-1β, IFNγ, and IL-4. In vitro investigations revealed that knockdown of ceramide synthase 5 in T-cells impaired T-cell activation. In summary, we show that CerS5-ko mice were more susceptible to dextran sodium sulfate-induced colitis and azoxymethane/dextran sodium sulfate-induced colitis-associated colon cancer. A reduced number of T-cells in the colon epithelium that was already the case in untreated CerS5-ko mice might have contributed to this effect.
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http://dx.doi.org/10.3390/cancers12071753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409364PMC
July 2020

UGCG overexpression leads to increased glycolysis and increased oxidative phosphorylation of breast cancer cells.

Sci Rep 2020 05 18;10(1):8182. Epub 2020 May 18.

Pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Johann Wolfgang Goethe University, Theodor Stern-Kai 7, 60590, Frankfurt am Main, Germany.

The only enzyme in the glycosphingolipid (GSL) metabolic pathway, which produces glucosylceramide (GlcCer) de novo is UDP-glucose ceramide glucosyltransferase (UGCG). UGCG is linked to pro-cancerous processes such as multidrug resistance development and increased proliferation in several cancer types. Previously, we showed an UGCG-dependent glutamine metabolism adaption to nutrient-poor environment of breast cancer cells. This adaption includes reinforced oxidative stress response and fueling the tricarboxylic acid (TCA) cycle by increased glutamine oxidation. In the current study, we investigated glycolytic and oxidative metabolic phenotypes following UGCG overexpression (OE). UGCG overexpressing MCF-7 cells underwent a metabolic shift from quiescent/aerobic to energetic metabolism by increasing both glycolysis and oxidative glucose metabolism. The energetic metabolic phenotype was not associated with increased mitochondrial mass, however, markers of mitochondrial turnover were increased. UGCG OE altered sphingolipid composition of the endoplasmic reticulum (ER)/mitochondria fractions that may contribute to increased mitochondrial turnover and increased cell metabolism. Our data indicate that GSL are closely connected to cell energy metabolism and this finding might contribute to development of novel therapeutic strategies for cancer treatment.
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http://dx.doi.org/10.1038/s41598-020-65182-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7234995PMC
May 2020

In-vitro safety and off-target profile of the anti-parasitic arylmethylaminosteroid 1o.

Sci Rep 2020 05 5;10(1):7534. Epub 2020 May 5.

Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Branch for Translational Medicine and Pharmacology (TMP), Theodor-Stern-Kai 7, 60596, Frankfurt/Main, Germany.

Parasite-mediated diseases like malaria and schistosomiasis are growing health problems worldwide and novel drug candidates are urgently needed. In this study, the in-vitro safety profile of steroid compound 1o (sc1o), effective against the parasites Plasmodium falciparum and Schistosoma mansoni with an IC value of 5 nM, was characterized. We assessed viability/proliferation, apoptosis and cell cycle tests to determine the cytotoxic profile of sc1o in cancer cells. The mutagenic potential was determined with the AMES test. To identify off-target effects we investigated whether sc1o interacts with safety-relevant molecules such as cytochrome P450 (CYP) enzymes, phosphodiesterases (PDE), histone deacteylases (HDAC) and human ether-a-go-go related gene (hERG). Furthermore, to predict the potential bioavailability of sc1o, its effect on Caco-2 cell barrier integrity, by measurement of the transepithelial electrical resistance (TEER), was determined. Sc1o at 25 µM reduced cell viability, probably through cell-cycle arrest, but did not induce apoptosis in cancer cells. No adverse off-target effects nor mutagenic potential of sc1o were observed. Furthermore, sc1o did not disturb the integrity of the cell barrier, but exhibited low membrane permeability, apparently due to cell adherence. In conclusion, sc1o up to 10 µM showed a good in-vitro safety profile.
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http://dx.doi.org/10.1038/s41598-020-64382-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7200784PMC
May 2020

The WD40 repeat protein, WDR36, orchestrates sphingosine kinase-1 recruitment and phospholipase C-β activation by G-coupled receptors.

Biochim Biophys Acta Mol Cell Biol Lipids 2020 07 31;1865(7):158704. Epub 2020 Mar 31.

Institut für Allgemeine Pharmakologie und Toxikologie, Universitätsklinikum, Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. Electronic address:

Sphingosine kinases (SphK) catalyse the formation of sphingosine-1-phosphate (S1P) and play important roles in the cardiovascular, nervous and immune systems. We have shown before that G-coupled receptors induce a rapid and long-lasting translocation of SphK1 to the plasma membrane and cross-activation of S1P receptors. Here, we further addressed G regulation of SphK1 by analysing the influence of the WD40 repeat protein, WDR36. WDR36 has been described as a scaffold tethering Gα to phospholipase C (PLC)-β and the thromboxane A receptor-β (TPβ receptor). Overexpression of WDR36 in HEK-293 cells enhanced TPβ receptor-induced inositol phosphate production, as reported (Cartier et al. 2011), but significantly attenuated inositol phosphate production induced by muscarinic M and bradykinin B receptors. In agreement with its effect on PLCβ, WDR36 augmented TPβ receptor-induced [Ca] increases. Surprisingly, WDR36 also augmented M receptor-induced [Ca] increases, which was due to increased Ca mobilization while the Ca content of thapsigargin-sensitive stores remained unaltered. Interestingly, overexpression of WDR36 significantly delayed SphK1 translocation by G-coupled M, B and H receptors in HEK-293 cells, while TPβ receptor-induced SphK1 translocation was generally slow and not altered by WDR36 in these cells. Finally, in C2C12 myoblasts, overexpression of WDR36 delayed SphK1 translocation induced by B receptors. It is concluded that WDR36 reduces signalling of G-coupled receptors other than TPβ towards PLC and SphK1, most likely by scavenging Gα and PLCβ. Our results support a role of WDR36 in orchestration of G signalling complexes, and might help to functionally unravel its genetic association with asthma and allergy.
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http://dx.doi.org/10.1016/j.bbalip.2020.158704DOI Listing
July 2020

S1P d20:1, an endogenous modulator of S1P d18:1/S1P -dependent signaling.

FASEB J 2020 03 15;34(3):3932-3942. Epub 2020 Jan 15.

Department of Neurology, University Hospital, Goethe University Frankfurt, Frankfurt am Main, Germany.

Sphingosine 1-phosphate (S1P) signaling influences numerous cell biological mechanisms such as differentiation, proliferation, survival, migration, and angiogenesis. Intriguingly, our current knowledge is based solely on the role of S1P with an 18-carbon long-chain base length, S1P d18:1. Depending on the composition of the first and rate-limiting enzyme of the sphingolipid de novo metabolism, the serine palmitoyltransferase, other chain lengths have been described in vivo. While cells are also able to produce S1P d20:1, its abundance and function remains elusive so far. Our experiments are highlighting the role of S1P d20:1 in the mouse central nervous system (CNS) and human glioblastoma. We show here that S1P d20:1 and its precursors are detectable in both healthy mouse CNS-tissue and human glioblastoma. On the functional level, we focused our work on one particular, well-characterized pathway, the induction of cyclooxygenase (COX)-2 expression via the S1P receptor 2 (S1P ). Intriguingly, S1P d20:1 only fairly induces COX-2 expression and can block the S1P d18:1-induced COX-2 expression mediated via S1P activation in the human glioblastoma cell line LN229. This data indicates that S1P d20:1 might act as an endogenous modulator of S1P signaling via a partial agonism at the S1P receptor. While our findings might stimulate further research on the relevance of long-chain base lengths in sphingolipid signaling, the metabolism of S1P d20:1 has to be considered as an integral part of S1P signaling pathways in vivo.
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http://dx.doi.org/10.1096/fj.201902391RDOI Listing
March 2020

Implementation of lipidomics in clinical routine: Can fluoride/citrate blood sampling tubes improve preanalytical stability?

Talanta 2020 Mar 26;209:120593. Epub 2019 Nov 26.

Pharmazentrum Frankfurt/ ZAFES, Institute of Clinical Pharmacology, Goethe University, Frankfurt, Germany. Electronic address:

The impact of preanalytical sample handling on lipid stability has been assessed in human plasma using targeted LC-MS/MS quantification of endocannabinoids, sphingolipids and LPA, complemented by non-targeted lipidomics screening with LC-QTOFMS. The study involved incubation of whole blood and plasma from healthy volunteers at room temperature or in ice water for time periods ranging from 20 min to 24 h. The impact of two different anticoagulants, K3EDTA and sodium fluoride/citrate, on lipid stability was evaluated. It was found that the concentrations determined for several endogenous lipids vary when whole blood and plasma samples are processed at room temperature, whereas the concentrations of most lipids were stable for 4 h in ice water. Surprisingly, the detected amounts of endocannabinoids 1- and 2-arachidonoyl glycerol and arachidonoyl ethanolamide increased markedly by 60, 95, and 30% in K3EDTA whole blood after storage in ice water for only 20 min. When using sodium fluoride/citrate blood collection tubes, the stability of several lipids, including that of the endocannabinoids, was improved. Accordingly, it is absolutely necessary to keep the blood sampling and plasma processing time below 1 h to avoid ex-vivo formation of endocannabinoids. It is worth mentioning that baseline lipid levels differ when using K3EDTA or sodium fluoride/citrate blood sampling tubes, which emphasizes the importance of traceability of reported plasma concentrations to the used anticoagulant.
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http://dx.doi.org/10.1016/j.talanta.2019.120593DOI Listing
March 2020

Inhibiting eicosanoid degradation exerts antifibrotic effects in a pulmonary fibrosis mouse model and human tissue.

J Allergy Clin Immunol 2020 03 5;145(3):818-833.e11. Epub 2019 Dec 5.

Division of Pharmacology, Otto Loewi Research Center, Medical University of Graz, Graz, Austria. Electronic address:

Background: Idiopathic pulmonary fibrosis (IPF) is a disease with high 5-year mortality and few therapeutic options. Prostaglandin (PG) E exhibits antifibrotic properties and is reduced in bronchoalveolar lavage from patients with IPF. 15-Prostaglandin dehydrogenase (15-PGDH) is the key enzyme in PGE metabolism under the control of TGF-β and microRNA 218.

Objective: We sought to investigate the expression of 15-PGDH in IPF and the therapeutic potential of a specific inhibitor of this enzyme in a mouse model and human tissue.

Methods: In vitro studies, including fibrocyte differentiation, regulation of 15-PGDH, RT-PCR, and Western blot, were performed using peripheral blood from healthy donors and patients with IPF and A549 cells. Immunohistochemistry, immunofluorescence, 15-PGDH activity assays, and in situ hybridization as well as ex vivo IPF tissue culture experiments were done using healthy donor and IPF lungs. Therapeutic effects of 15-PGDH inhibition were studied in the bleomycin mouse model of pulmonary fibrosis.

Results: We demonstrate that 15-PGDH shows areas of increased expression in patients with IPF. Inhibition of this enzyme increases PGE levels and reduces collagen production in IPF precision cut lung slices and in the bleomycin model. Inhibitor-treated mice show amelioration of lung function, decreased alveolar epithelial cell apoptosis, and fibroblast proliferation. Pulmonary fibrocyte accumulation is also decreased by inhibitor treatment in mice, similar to PGE that inhibits fibrocyte differentiation from blood of healthy donors and patients with IPF. Finally, microRNA 218-5p, which is downregulated in patients with IPF, suppressed 15-PGDH expression in vivo and in vitro.

Conclusions: These findings highlight the role of 15-PGDH in IPF and suggest 15-PGDH inhibition as a promising therapeutic approach.
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http://dx.doi.org/10.1016/j.jaci.2019.11.032DOI Listing
March 2020

Blood ceramides as novel markers for renal impairment in systemic lupus erythematosus.

Prostaglandins Other Lipid Mediat 2019 10 10;144:106348. Epub 2019 Jul 10.

Department of General Pharmacology and Toxicology, Goethe University Hospital and Goethe University Frankfurt, Frankfurt am Main, Germany. Electronic address:

Background: Lupus nephritis (LN) is the most common organ manifestation in systemic lupus erythematosus (SLE) and associated with a poor prognosis. Still, a noninvasive but reliable method to diagnose LN has not been established. Thus, we evaluated whether blood sphingolipids could serve as valid biomarkers for renal injury.

Methods: In this cross-sectional study, 82 participants were divided into three groups: 36 healthy controls and 17 SLE patients without renal injury (both: estimated glomerular filtration rate (eGFR) ≥ 80 ml/min/1.73 m and albumin/creatinine ≤ 30 mg/g) and 29  LN patients. LN patients were identified by renal biopsies and impaired renal function (eGFR < 80 ml/min/1.73 m and albumin/creatinine ratio > 30 mg/g). Venous blood was collected from all participants and sphingolipid levels in plasma and serum were measured by LC-MS/MS.

Results: Most interesting, concentrations of some specific ceramides, C16ceramide (Cer), C18Cer, C20Cer and C24:1Cer, were elevated in both, plasma and serum samples of patients suffering from biopsy-proven LN and impaired renal function, compared to healthy controls as well as SLE patients without renal injury. C24:1dhCer levels were elevated in plasma and serum samples from LN patients compared to SLE patients. Sphingosine levels were higher in plasma and serum of LN patients compared to healthy controls, but not compared to SLE patients. Sphinganine concentrations were significantly elevated in serum samples from LN patients compared to healthy controls and SLE. S1P and SA1P levels were higher in plasma samples of SLE and LN patients compared to healthy controls. Subsequent ROC analyses of plasma and serum data of the most altered ceramide species (C16Cer, C18Cer, C20Cer, C24:1Cer) between LN patients and SLE patients display a high diagnostic differentiation with significant AUCs especially for C24:1Cer serum levels. Further, C24:1Cer serum levels were not affected by glucocorticoid treatment and did not correlate with other renal markers, such as serum creatinine, eGFR and albumin/creatinine ratio.

Conclusion: Our data reveal that chain-length specific ceramides in blood, most likely C24:1Cer levels in serum, could act as potent biomarkers for renal impairment in patients suffering from SLE.
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http://dx.doi.org/10.1016/j.prostaglandins.2019.106348DOI Listing
October 2019

The Lipid Status in Patients with Ulcerative Colitis: Sphingolipids are Disease-Dependent Regulated.

J Clin Med 2019 Jul 4;8(7). Epub 2019 Jul 4.

Institute of Clinical Pharmacology, Goethe University Hospital, Theodor-Stern-Kai 7, 60590 Frankfurt/Main, Germany.

The factors that contribute to the development of ulcerative colitis (UC), are still not fully identified. Disruption of the colon barrier is one of the first events leading to invasion of bacteria and activation of the immune system. The colon barrier is strongly influenced by sphingolipids. Sphingolipids impact cell-cell contacts and function as second messengers. We collected blood and colon tissue samples from UC patients and healthy controls and investigated the sphingolipids and other lipids by LC-MS/MS or LC-QTOFMS. The expression of enzymes of the sphingolipid pathway were determined by RT-PCR and immunohistochemistry. In inflamed colon tissue, the de novo-synthesis of sphingolipids is reduced, whereas lactosylceramides are increased. Reduction of dihydroceramides was due to posttranslational inhibition rather than altered serine palmitoyl transferase or ceramide synthase expression in inflamed colon tissue. Furthermore, in human plasma from UC-patients, several sphinglipids change significantly in comparison to healthy controls. Beside sphingolipids free fatty acids, lysophosphatidylcholines and triglycerides changed significantly in the blood of colitis patients dependent on the disease severity. Our data indicate that detraction of the sphingolipid de novo synthesis in colon tissue might be an important trigger for UC. Several lipids changed significantly in the blood, which might be used as biomarkers for disease control; however, diet-related variabilities need to be considered.
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http://dx.doi.org/10.3390/jcm8070971DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6678307PMC
July 2019

GPER1 influences cellular homeostasis and cytostatic drug resistance via influencing long chain ceramide synthesis in breast cancer cells.

Int J Biochem Cell Biol 2019 07 11;112:95-106. Epub 2019 May 11.

pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Johann Wolfgang Goethe University, Theodor Stern-Kai 7, 60590 Frankfurt am Main, Germany.

The G protein-coupled estrogen receptor 1 (GPER1) is involved in the regulation of physiological processes such as cellular growth and proliferation, but also in pathophysiological processes such as tumor development. The role of GPER1 in breast cancer is contradictory. Therefore, we investigated the influence of GPER1 overexpression on cellular processes in MCF-7 breast cancer cells. GPER1 overexpression leads to a cell cycle arrest in the G1 phase, induction of autophagy and reduced proliferation. Reduced proliferation was accompanied by a reduced basal respiration and reduced glycolysis rate in GPER1 overexpressing cells. This is presumably ascribable to mitophagy induction following GPER1 overexpression. However, GPER1 overexpressing cells were less sensitive against doxorubicin as compared to control cells. In previous work we showed the effect of transient GPER1 overexpression on the synthesis of several ceramide synthases (CerS) thereby influencing the sphingolipid pathway. Therefore, we investigated CerS expression and sphingolipid level in stable GPER1 overexpressing and control cells. Stable GPER1 overexpression strongly reduced CerS4, CerS5 and CerS6 promoter activity and CerS5 and CerS6 mRNA expression, whereas CerS2 mRNA expression was upregulated. The GPER1 effect on CerS5 promoter is mediated by GSK-3β signaling. In addition, other enzymes of the sphingolipid pathway were upregulated. Our study provides new insights into the role of GPER1 and the activated sphingolipid pathways and how GPER1 may influence cellular processes such as cancer cell survival following chemotherapy. Further studies are needed to investigate the molecular mechanisms leading to these cellular effects. Finding new therapeutic targets for modulating specifically GPER1 in breast tumors may improve endocrine breast cancer therapy.
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http://dx.doi.org/10.1016/j.biocel.2019.05.002DOI Listing
July 2019

Targeted lipidomics reveal derangement of ceramides in major depression and bipolar disorder.

Metabolism 2019 06 5;95:65-76. Epub 2019 Apr 5.

Institute of Clinical Pharmacology, Goethe-University Hospital Frankfurt, Germany. Electronic address:

Changes of sphingolipid metabolism were suggested to contribute to the patho-etiology of major depression (MD) and bipolar disorder (BD). In a pilot study we assessed if lipid allostasis manifested in pathological plasma concentrations of bioactive lipids i.e. endocannabinoids, sphingolipids, ceramides, and lysophosphatidic acids.

Methods: Targeted and untargeted lipidomic analyses were performed according to GLP guidelines in 67 patients with unipolar or bipolar disorders (20-67 years, 36 male, 31 female) and 405 healthy controls (18-79 years, 142 m, 263 f), who were matched according to gender, age and body mass index. Multivariate analyses were used to identify major components, which accounted for the variance between groups and were able to predict group membership.

Results: Differences between MD and BP patients versus controls mainly originated from ceramides and their hexosyl-metabolites (C16Cer, C18Cer, C20Cer, C22Cer, C24Cer and C24:1Cer; C24:1GluCer, C24LacCer), which were strongly increased, particularly in male patients. Ceramide levels were neither associated with the current episode, nor with the therapeutic improvement of the Montgomery Åsberg Depression Rating Scale (MARDS). However, long-chain ceramides were linearly associated with age, stronger in patients than controls, and with high plasma levels of diacyl- and triacylglycerols. Patients receiving antidepressants had higher ceramide levels than patients not taking these drugs. There was no such association with lithium or antipsychotics except for olanzapine.

Conclusion: Our data suggest that high plasma ceramides in patients with major depression and bipolar disorder are indicative of a high metabolic burden, likely aggravated by certain medications.
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http://dx.doi.org/10.1016/j.metabol.2019.04.002DOI Listing
June 2019

Diurnal regulation of sphingolipids in blood.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 03 15;1864(3):304-311. Epub 2018 Dec 15.

Department of General Pharmacology and Toxicology, Goethe University Hospital, Frankfurt am Main, Germany.

Key homeostatic functions are regulated in a diurnal manner and a miss-alignment of such rhythms is believed to contribute to the pathophysiology of several diseases. Signaling sphingolipids (SLs) in plasma such as sphingosine 1-phosphate control lymphocytic trafficking, vascular reactivity and platelet activity, physiological functions all of which display a diurnal rhythm themselves. However, the rhythmicity of SL metabolism in plasma and its potential causes have not been sufficiently investigated so far. Therefore, we analyzed blood of mice and healthy adult human subjects by targeted tandem mass-spectrometry at different time points. In order to investigate the influence of the synchronizing hormone melatonin, we compared melatonin proficient C3H/HeN wildtype mice (C3H) with melatonin receptor-1/2 double knockout mice (MT1/2-/-) and melatonin deficient C57BL/6J mice. We found a strong upregulation of plasma S1P with the beginning of the light period in C3H but not in MT1/2-/- or C57BL/6J mice. Accordingly, our study revealed an upregulation of sphingosine 1-phosphate (S1P d18:1) and sphinganine 1-phosphate (S1P d18:0) with the beginning of the light period in humans. Furthermore, plasma S1P d18:1 and S1P d18:0 were inversely correlated with the respective concentrations in platelets, pointing to a possible involvement of platelet SL metabolism. In humans, the diurnal rhythm of SLs was not associated with changes of SL-binding proteins or counts of cellular SL sources. Overall, this study indicates a physiological rhythmicity of plasma and platelet SL metabolism, likely mediated by melatonin, with potentially important implications for physiological diurnal rhythms and the regulation of SL metabolism and its functions.
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http://dx.doi.org/10.1016/j.bbalip.2018.12.001DOI Listing
March 2019

Vitamin D effects on sphingosine 1-phosphate signaling and metabolism in monocytes from type 2 diabetes patients and controls.

J Steroid Biochem Mol Biol 2019 02 15;186:130-135. Epub 2018 Oct 15.

Department of Internal Medicine I, Division of Endocrinology, Diabetes and Metabolism, Goethe University Hospital, Frankfurt am Main, Germany.

Elevated sphingosine 1-phopshate (S1P) concentration was observed in type 2 diabetes mellitus (T2D). On the other side, 1α,25-dihydroxyvitamin D (1,25(OH)D) can influence the formation of sphingosine 1-phopshate (S1P) and the expression of S1P receptors, which are known to be involved in T2D. In order to evaluate mechanisms for the antiinflammatory potential of 1,25(OH)D, we investigated whether 1,25(OH)D alters S1P signaling and metabolism in human CD14 monocytes. Primary monocytes isolated from healthy controls (HC) and T2D patients were treated for 24 h with 10 nM 1,25(OH)D in the absence or presence of 500 IU/ml interleukin-(IL)-1β. Thereafter, sphingosine kinase (SPHK)1, SPHK2 and S1P receptor 1-5 (S1P) mRNA expression levels were measured by TaqMan analyses. Sphingolipid levels in cell supernatant were determined by high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS). 1,25(OH)D treatment downregulated S1P and S1P mRNA expression compared to untreated monocytes of HC and T2D patients. In contrast, SPHK1, S1P and S1P mRNA expression levels were upregulated by 1,25(OH)D treatment compared to the respective controls. Furthermore, reduced S1P and increased S1P and S1P mRNA expression levels upon treatment with 1,25(OH)D occurred in the presence of IL-1β. Additionally, S1P levels in cell supernatants were decreased in monocytes from HC and T2D patients by 1,25(OH)D with or without IL-1β costimulation. The levels of sphingosine in cell supernatants were not influenced by 1,25(OH)D Overall, our results demonstrate for the first time that 1,25(OH)D treatment can influence S1P receptor and SPHK expression and S1P levels in primary monocytes of both HC and subjects with T2D. These findings justify further investigations into the sphingolipid metabolism and potential benefits of vitamin D treatment in diabetes.
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http://dx.doi.org/10.1016/j.jsbmb.2018.10.005DOI Listing
February 2019

Chemosensitivity of human colon cancer cells is influenced by a p53-dependent enhancement of ceramide synthase 5 and induction of autophagy.

Biochim Biophys Acta Mol Cell Biol Lipids 2018 10 27;1863(10):1214-1227. Epub 2018 Jul 27.

Institute of Clinical Pharmacology, Faculty of Medicine, Goethe-University Frankfurt, Germany. Electronic address:

Resistance against chemotherapy is a life-threatening complication in colon cancer therapy. To increase response rate, new additional targets that contribute to chemoresistance are still needed to be explored. Ceramides, which belong to the group of sphingolipids, are well-known regulators of cell death and survival, respectively. Here, we show that in human wild-type () p53 HCT-116 colon cancer cells treatment with oxaliplatin or 5-fluorouracil (5-FU) leads to a strong increase in ceramide synthase 5 (CerS5) expression and C-ceramide levels, which was not shown in HCT-116 lacking p53 expression (HCT-116 p53). The increase in CerS5 expression occurs by stabilizing CerS5 mRNA at the 3'-UTR. By contrast, in the p53-deficient cells CerS2 expression and CerS2-related C- and C-ceramide levels were elevated which is possibly related to enhanced polyadenylation of the CerS2 transcript in these cells. Stable knockdown of CerS5 expression using CerS5-targeting shRNA led to an increased sensitivity of HCT-116 p53 cells, but not of p53 cells, to oxaliplatin and 5-FU. Enhanced sensitivity was accompanied by an inhibition of autophagy and inhibition of mitochondrial respiration in these cells. However, knockdown of CerS2 had no significant effects on chemosensitivity of both cell lines. In conclusion, in p53 colon cancer cells chemosensitivity against oxaliplatin or 5-FU could be enhanced by downregulation of CerS5 expression leading to reduced autophagy and mitochondrial respiration.
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http://dx.doi.org/10.1016/j.bbalip.2018.07.011DOI Listing
October 2018

Preanalytical Biases in the Measurement of Human Blood Sphingolipids.

Int J Mol Sci 2018 May 7;19(5). Epub 2018 May 7.

Department of General Pharmacology and Toxicology, Goethe University Hospital Frankfurt, 60590 Frankfurt am Main, Germany.

Dysregulation of blood sphingolipids is an emerging topic in clinical science. The objective of this study was to determine preanalytical biases that typically occur in clinical and translational studies and that influence measured blood sphingolipid levels. Therefore, we collected blood samples from four healthy male volunteers to investigate the effect of storage conditions (time, temperature, long-term storage, freeze⁻thaw cycles), blood drawing (venous or arterial sampling, prolonged venous compression), and sample preparation (centrifugation, freezing) on sphingolipid levels measured by LC-MS/MS. Our data show that sphingosine 1-phosphate (S1P) and sphinganine 1-phosphate (SA1P) were upregulated in whole blood samples in a time- and temperature-dependent manner. Increased centrifugation at higher speeds led to lower amounts of S1P and SA1P. All other preanalytical biases did not significantly alter the amounts of S1P and SA1P. Further, in almost all settings, we did not detect differences in (dihydro)ceramide levels. In summary, besides time-, temperature-, and centrifugation-dependent changes in S1P and SA1P levels, sphingolipids in blood remained stable under practically relevant preanalytical conditions.
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http://dx.doi.org/10.3390/ijms19051390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983773PMC
May 2018

UDP-glucose ceramide glucosyltransferase activates AKT, promoted proliferation, and doxorubicin resistance in breast cancer cells.

Cell Mol Life Sci 2018 Sep 17;75(18):3393-3410. Epub 2018 Mar 17.

pharmazentrum frankfurt/ZAFES, Institute of Clinical Pharmacology, Johann Wolfgang Goethe University, House 74, Theodor Stern-Kai 7, 60590, Frankfurt am Main, Germany.

The UDP-glucose ceramide glucosyltransferase (UGCG) is a key enzyme in the synthesis of glycosylated sphingolipids, since this enzyme generates the precursor for all complex glycosphingolipids (GSL), the GlcCer. The UGCG has been associated with several cancer-related processes such as maintaining cancer stem cell properties or multidrug resistance induction. The precise mechanisms underlying these processes are unknown. Here, we investigated the molecular mechanisms occurring after UGCG overexpression in breast cancer cells. We observed alterations of several cellular properties such as morphological changes, which enhanced proliferation and doxorubicin resistance in UGCG overexpressing MCF-7 cells. These cellular effects seem to be mediated by an altered composition of glycosphingolipid-enriched microdomains (GEMs), especially an accumulation of globotriaosylceramide (Gb3) and glucosylceramide (GlcCer), which leads to an activation of Akt and ERK1/2. The induction of the Akt and ERK1/2 signaling pathway results in an increased gene expression of multidrug resistance protein 1 (MDR1) and anti-apoptotic genes and a decrease of pro-apoptotic gene expression. Inhibition of the protein kinase C (PKC) and phosphoinositide 3 kinase (PI3K) reduced MDR1 gene expression. This study discloses how changes in UGCG expression impact several cellular signaling pathways in breast cancer cells resulting in enhanced proliferation and multidrug resistance.
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http://dx.doi.org/10.1007/s00018-018-2799-7DOI Listing
September 2018

Sphingosine-1-Phosphate Receptor 5 Modulates Early-Stage Processes during Fibrogenesis in a Mouse Model of Systemic Sclerosis: A Pilot Study.

Front Immunol 2017 29;8:1242. Epub 2017 Sep 29.

pharmazentrum frankfurt/ZAFES, Institute of Pharmacology and Toxicology, Hospital of the Goethe University, Frankfurt, Germany.

Systemic sclerosis (SSc) is a rare multi-organ autoimmune disease characterized by progressive skin fibrosis. Inflammation, type 2 immunity, and fibrogenic processes are involved in disease development and may be affected by sphingolipids. However, details about early-stage pathophysiological mechanisms and implicated mediators remain elusive. The sphingolipid sphingosine-1-phosphate (S1P) is elevated in the sera of SSc patients, and its receptor S1P5 is expressed in skin tissue. Nevertheless, almost nothing is known about the dermatological contribution of S1P5 to inflammatory and pro-fibrotic processes leading to the pathological changes seen in SSc. In this study, we observed a novel effect of S1P5 on the inflammatory processes during low-dose bleomycin (BLM)-induced fibrogenesis in murine skin. By comparing 2-week-treated skin areas of wild-type (WT) and S1P5-deficient mice, we found that S1P5 is important for the transcriptional upregulation of the Th2 characteristic transcription factor under treatment-induced inflammatory conditions, while (Th1) and (Treg) mRNA expression was regulated independently of S1P5. Additionally, treatment caused a regulation of and mRNA as well as a regulation of long-chain ceramide profiles, which both differ significantly between the genotypes. Despite S1P5-dependent differences regarding inflammatory processes, similar macroscopic evidence of fibrosis was detected in the skin histology of WT and S1P5-deficient mice after 4 weeks of subcutaneous BLM treatment. However, at the earlier 2-week point in time, the mRNA data of and indicate a pro-fibrotic S1P5 contribution in the applied SSc mouse model. In conclusion, we propose that S1P5 plays a role as a novel modulator during the early phase of BLM-caused fibrogenesis in murine skin. An immediate relationship between dermal S1P5 expression and fibrotic processes leading to skin alterations, such as formative for SSc pathogenesis, is indicated but should be studied more profound in further investigations. Therefore, this study is an initial step in understanding the role of S1P5-mediated effects during early stages of fibrogenesis, which may encourage the ongoing search for new therapeutic options for SSc patients.
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http://dx.doi.org/10.3389/fimmu.2017.01242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626866PMC
September 2017

The Role of PGE in Alveolar Epithelial and Lung Microvascular Endothelial Crosstalk.

Sci Rep 2017 08 11;7(1):7923. Epub 2017 Aug 11.

Institute of Experimental and Clinical Pharmacology, Medical University of Graz, Graz, Austria.

Disruption of the blood-air barrier, which is formed by lung microvascular endothelial and alveolar epithelial cells, is a hallmark of acute lung injury. It was shown that alveolar epithelial cells release an unidentified soluble factor that enhances the barrier function of lung microvascular endothelial cells. In this study we reveal that primarily prostaglandin (PG) E accounts for this endothelial barrier-promoting activity. Conditioned media from alveolar epithelial cells (primary ATI-like cells) collected from BALB/c mice and A549 cells increased the electrical resistance of pulmonary human microvascular endothelial cells, respectively. This effect was reversed by pretreating alveolar epithelial cells with a cyclooxygenase-2 inhibitor or by blockade of EP4 receptors on endothelial cells, and in A549 cells also by blocking the sphingosine-1-phosphate receptor. Cyclooxygenase-2 was constitutively expressed in A549 cells and in primary ATI-like cells, and was upregulated by lipopolysaccharide treatment. This was accompanied by enhanced PGE secretion into conditioned media. Therefore, we conclude that epithelium-derived PGE is a key regulator of endothelial barrier integrity via EP4 receptors under physiologic and inflammatory conditions. Given that pharmacologic treatment options are still unavailable for diseases with compromised air-blood barrier, like acute lung injury, our data thus support the therapeutic potential of selective EP4 receptor agonists.
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http://dx.doi.org/10.1038/s41598-017-08228-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5554158PMC
August 2017

Vitamin D Supplementation Enhances C18(dihydro)ceramide Levels in Type 2 Diabetes Patients.

Int J Mol Sci 2017 Jul 15;18(7). Epub 2017 Jul 15.

Department of Internal Medicine I, Division of Endocrinology, Diabetes and Metabolism, Goethe University Hospital, 60590 Frankfurt am Main, Germany.

Sphingolipids are characterized by a broad range of bioactive properties. Particularly, the development of insulin resistance, a major pathophysiological hallmark of Type 2 Diabetes mellitus (T2D), has been linked to ceramide signaling. Since vitamin D supplementation may slow down T2D progression by improving glucose concentrations and insulin sensitivity, we investigated whether vitamin D supplementation impacts on plasma sphingolipid levels in T2D patients. Thus, plasma samples of 59 patients with non-insulin-requiring T2D from a placebo-controlled, randomized, and double-blind study were retrospectively analyzed. Once per week, patients received either 20 drops of Vigantol oil, corresponding to a daily dose of 1904 IU/d vitamin D (verum: = 31), or a placebo oil consisting of medium chain triglycerides (placebo: = 28). Blood samples were taken from all of the participants at three different time points: 1) at the beginning of the study (baseline), 2) after 6 months supplementation, and 3) after an additional 6 months of follow-up. Plasma sphingolipids were measured by high-performance liquid chromatography tandem mass spectrometry. At baseline and 6 months follow-up, no significant differences in plasma sphingolipid species were detected between the placebo and verum groups. After 6 months, vitamin D supplementation significantly enhanced plasma C18dihydroceramide (dhCer; -stearoyl-sphinganine (d18:0/18:0)) and C18ceramide (Cer; -stearoyl-sphingosine (d18:1/18:0)) levels were observed in the verum group compared to the placebo group. This was accompanied by significantly higher 25-hydroxyvitamin D₃ (25(OH)D₃) blood levels in patients receiving vitamin D compared to the placebo group. Taken together, vitamin D supplementation induced changes of the C18 chain-length-specific dhCer and Cer plasma levels in patients with T2D. The regulation of sphingolipid signaling by vitamin D may thus unravel a novel mechanism by which vitamin D can influence glucose utilization and insulin action. Whether this acts favorably or unfavorably for the progression of T2D needs to be clarified.
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http://dx.doi.org/10.3390/ijms18071532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5536020PMC
July 2017

Ceramide synthase 2 deficiency aggravates AOM-DSS-induced colitis in mice: role of colon barrier integrity.

Cell Mol Life Sci 2017 08 12;74(16):3039-3055. Epub 2017 Apr 12.

Institute of Clinical Pharmacology, Faculty of Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590, Frankfurt am Main, Germany.

Loss of intestinal barrier functions is a hallmark of inflammatory bowel disease like ulcerative colitis. The molecular mechanisms are not well understood, but likely involve dysregulation of membrane composition, fluidity, and permeability, which are all essentially regulated by sphingolipids, including ceramides of different chain length and saturation. Here, we used a loss-of-function model (CerS2 and CerS2 mice) to investigate the impact of ceramide synthase 2, a key enzyme in the generation of very long-chain ceramides, in the dextran sodium salt (DSS) evoked model of UC. CerS2 mice developed more severe disease than CerS2 mice in acute DSS and chronic AOM/DSS colitis. Deletion of CerS2 strongly reduced very long-chain ceramides (Cer24:0, 24:1) but concomitantly increased long-chain ceramides and sphinganine in plasma and colon tissue. In naive CerS2 mice, the expression of tight junction proteins including ZO-1 was almost completely lost in the colon epithelium, leading to increased membrane permeability. This could also be observed in vitro in CerS2 depleted Caco-2 cells. The increase in membrane permeability in CerS2 mice did not manifest with apparent clinical symptoms in naive mice, but with slight inflammatory signs such as an increase in monocytes and IL-10. AOM/DSS and DSS treatment alone led to a further deterioration of membrane integrity and to severe clinical symptoms of the disease. This was associated with stronger upregulation of cytokines in CerS2 mice and increased infiltration of the colon wall by immune cells, particularly monocytes, CD4 and Th17 T-cells, and an increase in tumor burden. In conclusion, CerS2 is crucial for the maintenance of colon barrier function and epithelial integrity. CerS2 knockdown, and associated changes in several sphingolipids such as a drop in very long-chain ceramides/(dh)-ceramides, an increase in long-chain ceramides/(dh)-ceramides, and sphinganine in the colon, may weaken endogenous defense against the endogenous microbiome.
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http://dx.doi.org/10.1007/s00018-017-2518-9DOI Listing
August 2017

Interleukin 17 inhibits progenitor cells in rheumatoid arthritis cartilage.

Eur J Immunol 2016 Feb 14;46(2):440-5. Epub 2015 Dec 14.

Department of Nephrology and Rheumatology, University Medical Center, Goettingen, Germany.

Mesenchymal stem cells are known to exert immunomodulatory effects in inflammatory diseases. Immuneregulatory cells lead to progressive joint destruction in rheumatoid arthritis (RA). Proinflammatory cytokines, such as tumour necrosis factor α (TNF-α) and interleukins (ILs) are the main players. Here, we studied progenitor cells from RA cartilage (RA-CPCs) that are positive for IL-17 receptors to determinate the effects of inflammation on their chondrogenic potenial. IL-17A/F reduced the chondrogenic potential of these cells via the upregulation of RUNX2 protein and enhanced IL-6 protein and MMP3 mRNA levels. Blocking antibodies against IL-17 positively influenced their repair potential. Furthermore, treating the RA-CPCs with the anti-human IL-17 antibody secukinumab or the anti-TNF-α antibody adalimumab reduced the proinflammatory IL-6 protein level and positively influenced the secretion of anti-inflammatory IL-10 protein. Additionally, adalimumab and secukinumab in particular reduced RUNX2 protein to promote chondrogenesis. The amelioration of inflammation, particularly via IL-17 antagonism, might be a new therapeutic approach for enhancing intrinsic cartilage repair mechanisms in RA patients.
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http://dx.doi.org/10.1002/eji.201545910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5064647PMC
February 2016