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The sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that is now appreciated as key regulatory factor for various cellular functions in the kidney, including matrix remodeling. It is generated by two sphingosine kinases (Sphk), Sphk1 and Sphk2, which are ubiquitously expressed, but have distinct enzymatic activities and subcellular localizations. In this study, we have investigated the role of Sphk2 in podocyte function and its contribution to diabetic nephropathy. We show that streptozotocin (STZ)-induced nephropathy and albuminuria in mice is prevented by genetic depletion of Sphk2. This protection correlated with an increased protein expression of the transcription factor Wilm's tumor suppressor gene 1 (WT1) and its target gene nephrin, and a reduced macrophage infiltration in immunohistochemical renal sections of STZ-treated Sphk2-/- mice compared to STZ-treated wildtype mice. To investigate changes on the cellular level, we used an immortalized human podocyte cell line and generated a stable knockdown of Sphk2 (Sphk2-kd) by a lentiviral transduction method. These Sphk2-kd cells accumulated sphingosine as a consequence of the knockdown, and showed enhanced nephrin and WT1 mRNA and protein expressions similar to the finding in Sphk2 knockout mice. Treatment of wildtype podocytes with the highly selective Sphk2 inhibitor SLM6031434 caused a similar upregulation of nephrin and WT1 expression. Furthermore, exposing cells to the profibrotic mediator transforming growth factor β (TGFβ) resulted on the one side in reduced nephrin and WT1 expression, but on the other side, in upregulation of various profibrotic marker proteins, including connective tissue growth factor (CTGF), fibronectin (FN) and plasminogen activator inhibitor (PAI) 1. All these effects were reverted by Sphk2-kd and SLM6031434. Mechanistically, the protection by Sphk2-kd may depend on accumulated sphingosine and inhibited PKC activity, since treatment of cells with exogenous sphingosine not only reduced the phosphorylation pattern of PKC substrates, but also increased WT1 protein expression. Moreover, the selective stable knockdown of PKCδ increased WT1 expression, suggesting the involvement of this PKC isoenzyme in WT1 regulation. The glucocorticoid dexamethasone, which is a treatment option in many glomerular diseases and is known to mediate a nephroprotection, not only downregulated Sphk2 and enhanced cellular sphingosine, but also enhanced WT1 and nephrin expressions, thus, suggesting that parts of the nephroprotective effect of dexamethasone is mediated by Sphk2 downregulation. Altogether, our data demonstrated that loss of Sphk2 is protective in diabetes-induced podocytopathy and can prevent proteinuria, which is a hallmark of many glomerular diseases. Thus, Sphk2 could serve as a new attractive pharmacological target to treat proteinuric kidney diseases.

Breast cancer is one of the most common and devastating malignancies among women worldwide. Recent evidence suggests that malignant progression is also driven by processes involving the sphingolipid molecule sphingosine 1-phosphate (S1P) and its binding to cognate receptor subtypes on the cell surface. To investigate the effect of this interaction on the metastatic phenotype, we used the breast cancer cell line MDA-MB-231 and the sublines 4175 and 1833 derived from lung and bone metastases in nude mice, respectively. In both metastatic cell lines expression of the S1P3 receptor was strongly upregulated compared to the parental cells and correlated with higher S1P-induced intracellular calcium ([Ca2+]i), higher cyclooxygenase (COX)-2 and microsomal prostaglandin (PG) E2 synthase expression, and consequently with increased PGE2 synthesis. PGE2 synthesis was decreased by antagonists and siRNA against S1P3 and S1P2. Moreover, in parental MDA-MB-231 cells overexpression of S1P3 by cDNA transfection also increased PGE2 synthesis, but only after treatment with the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine, indicating reversible silencing of the COX-2 promoter. Functionally, the metastatic sublines showed enhanced migration and Matrigel invasion in adapted Boyden chamber assays, which further increased by S1P stimulation. This response was abrogated by either S1P3 antagonism, COX-2 inhibition or PGE2 receptor 2 (EP2) and 4 (EP4) antagonism, but not by S1P2 antagonism. Our data demonstrate that in breast cancer cells overexpression of S1P3 and its activation by S1P has pro-inflammatory and pro-metastatic potential by inducing COX-2 expression and PGE2 signaling via EP2 and EP4.

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.

The immunomodulatory FTY720 (fingolimod) is presently approved for the treatment of relapsing-remitting multiple sclerosis. It is a prodrug that acts by modulating sphingosine 1-phosphate (S1P) receptor signaling. In this study, we have developed and characterized two novel oxazolo-oxazole derivatives of FTY720, ST-968 and the oxy analog ST-1071, which require no preceding activating phosphorylation, and proved to be active in intact cells and triggered S1P(1) and S1P(3), but not S1P(2), receptor internalization as a result of receptor activation. Functionally, ST-968 and ST-1071 acted similar to FTY720 to abrogate S1P-triggered chemotaxis of mouse splenocytes, mouse T cells and human U937 cells, and reduced TNFa- and LPS-stimulated endothelial cell permeability. The compounds also reduced TNF alpha-induced ICAM-1 and VCAM-1 mRNA expression, but restored TNF alpha-mediated downregulation of PECAM-1 mRNA expression. In an in vivo setting, the application of ST-968 or ST-1071 to mice resulted in a reduction of blood lymphocytes and significantly reduced the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice comparable to FTY720 either by prophylactic or therapeutic treatment. In parallel to the reduced clinical symptoms, infiltration of immune cells in the brain was strongly reduced, and in isolated tissues of brain and spinal cord, the mRNA and protein expressions of ICAM-1 and VCAM-1, as well as of matrix metalloproteinase-9 were reduced by all compounds, whereas PECAM-1 and tissue inhibitor of metalloproteinase TIMP-1 were upregulated. In summary, the data suggest that these novel butterfly derivatives of FTY720 could have considerable implication for future therapies of multiple sclerosis and other autoimmune diseases. (C) 2014 Elsevier Ltd. All rights reserved.

Sphingolipids constitute bioactive molecules with functional implications in homeostasis and pathogenesis of various diseases. However, the role of sphingolipids as possible disease biomarkers in chronic liver disease remains largely unexplored. In the present study we used mass spectrometry and spectrofluorometry methods in order to quantify various sphingolipid metabolites and also assess the activity of an important corresponding regulating enzyme in the serum of 72 healthy volunteers as compared to 69 patients with non-alcoholic fatty liver disease and 69 patients with chronic hepatitis C virus infection. Our results reveal a significant upregulation of acid sphingomyelinase in the serum of patients with chronic liver disease as compared to healthy individuals (p0.001). Especially in chronic hepatitis C infection acid sphingomyelinase activity correlated significantly with markers of hepatic injury (r=0.312, p=0.009) and showed a high discriminative power. Accumulation of various (dihydro-) ceramide species was identified in the serum of patients with non-alcoholic fatty liver disease (p0.001) and correlated significantly to cholesterol (r=0.448, p0.001) but showed a significant accumulation in patients with normal cholesterol values as well (p0.001). Sphingosine, a further bioactive metabolite, was also upregulated in chronic liver disease (p0.001). However, no significant correlation to markers of hepatic injury was identified.Chronic hepatitis C virus infection and non-alcoholic fatty liver disease induce a significant upregulation of serum acid sphingomyelinase which appears as a novel biomarker in chronic hepatopathies. Further studies are required to elucidate the potential of the sphingolipid signaling pathway as putative therapeutic target in chronic liver disease.

Sphingosine-1-phosphate (S1P) is a pleiotropic lipid mediator that acts either on G protein-coupled S1P receptors on the cell surface or via intracellular target sites. In addition to the well established effects of S1P in angiogenesis, carcinogenesis and immunity, evidence is now continuously accumulating which demonstrates that S1P is an important regulator of fibrosis. The contribution of S1P to fibrosis is of a Janus-faced nature as S1P exhibits both pro- and anti-fibrotic effects depending on its site of action. Extracellular S1P promotes fibrotic processes in a S1P receptor-dependent manner, whereas intracellular S1P has an opposite effect and dampens a fibrotic reaction by yet unidentified mechanisms. Fibrosis is a result of chronic irritation by various factors and is defined by an excess production of extracellular matrix leading to tissue scarring and organ dysfunction. In this review, we highlight the general effects of extracellular and intracellular S1P on the multistep cascade of pathological fibrogenesis including tissue injury, inflammation and the action of pro-fibrotic cytokines that stimulate ECM production and deposition. In a second part we summarize the current knowledge about the involvement of S1P signaling in the development of organ fibrosis of the lung, kidney, liver, heart and skin. Altogether, it is becoming clear that targeting the sphingosine kinase-1/S1P signaling pathway offers therapeutic potential in the treatment of various fibrotic processes. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Neutral ceramidase (NCDase) and sphingosine kinases (SphKs) are key enzymes regulating cellular sphingosine-1-phosphate (S1P) levels. In this study we found that stress factor-induced apoptosis of rat renal mesangial cells was significantly reduced by dexamethasone treatment. Concomitantly, dexamethasone increased cellular S1P levels, suggesting an activation of sphingolipid-metabolizing enzymes. The cell-protective effect of glucocorticoids was reversed by a SphK inhibitor, was completely absent in SphK1-deficient cells, and was associated with upregulated mRNA and protein expression of NCDase and SphK1. Additionally, in vivo experiments in mice showed that dexamethasone also upregulated SphK1 mRNA and activity, and NCDase protein expression in the kidney. Fragments (2285, 1724, and 1126 bp) of the rat NCDase promoter linked to a luciferase reporter were transfected into rat kidney fibroblasts and mesangial cells. There was enhanced NCDase promoter activity upon glucocorticoids treatment that was abolished by the glucocorticoid receptor antagonist RU-486. Single and double mutations of the two putative glucocorticoid response element sites within the promoter reduced the dexamethasone effect, suggesting that both glucocorticoid response elements are functionally active and required for induction. Our study shows that glucocorticoids exert a protective effect on stress-induced mesangial cell apoptosis in vitro and in vivo by upregulating NCDase and SphK1 expression and activity, resulting in enhanced levels of the protective lipid second messenger S1P.

Sphingosine kinases (SK) catalyze the production of sphingosine-1-phosphate which in turn regulates cell responses such as proliferation and migration. Here, we show that exposure of the human endothelial cell line EA.hy 926 to hypoxia stimulates a increased SK-1, but not SK-2, mRNA, protein expression, and activity. This effect was due to stimulated SK-1 promoter activity which contains two putative hypoxia-inducible factor-responsive-elements (HRE). By deletion of one of the two HREs, hypoxia-induced promoter activation was abrogated. Furthermore, hypoxia upregulated the expression of HIF-1alpha and HIF-2alpha, and both contributed to SK-1 gene transcription as shown by selective depletion of HIF-1alpha or HIF-2alpha by siRNA. The hypoxia-stimulated SK-1 upregulation was functionally coupled to increased migration since the selective depletion of SK-1, but not of SK-2, by siRNAs abolished the migratory response. In summary, these data show that hypoxia upregulates SK-1 activity and results in an accelerated migratory capacity of endothelial cells. SK-1 may thus serve as an attractive therapeutic target to treat diseases associated with increased endothelial migration and angiogenesis such as cancer growth and progression.