Hepatitis C Virus Infection Upregulates Plasma Phosphosphingolipids and Endocannabinoids and Downregulates Lysophosphoinositols.

A mass spectrometry-based lipidomic investigation of 30 patients with chronic hepatitis C virus (HCV) infection and 30 age- and sex-matched healthy blood donor controls was undertaken. The clustering and complete separation of these two groups was found by both unsupervised and supervised multivariate data analyses. Three patients who had spontaneously cleared the virus and three who were successfully treated with direct-acting antiviral drugs remained within the HCV-positive metabotype, suggesting that the metabolic effects of HCV may be longer-lived. We identified 21 metabolites that were upregulated in plasma and 34 that were downregulated (p < 1 × 10-16 to 0.0002). Eleven members of the endocannabinoidome were elevated, including anandamide and eight fatty acid amides (FAAs). These likely activated the cannabinoid receptor GPR55, which is a pivotal host factor for HCV replication. FAAH1, which catabolizes FAAs, reduced mRNA expression. Four phosphosphingolipids, d16:1, d18:1, d19:1 sphingosine 1-phosphate, and d18:0 sphinganine 1-phosphate, were increased, together with the mRNA expression for their synthetic enzyme SPHK1. Among the most profoundly downregulated plasma lipids were several lysophosphatidylinositols (LPIs) from 3- to 3000-fold. LPIs are required for the synthesis of phosphatidylinositol 4-phosphate (PI4P) pools that are required for HCV replication, and LPIs can also activate the GPR55 receptor. Our plasma lipidomic findings shed new light on the pathobiology of HCV infection and show that a subset of bioactive lipids that may contribute to liver pathology is altered by HCV infection.

Sphk1 and Sphk2 Differentially Regulate Erythropoietin Synthesis in Mouse Renal Interstitial Fibroblast-like Cells.

Erythropoietin (Epo) is a crucial hormone regulating red blood cell number and consequently the hematocrit. Epo is mainly produced in the kidney by interstitial fibroblast-like cells. Previously, we have shown that in cultures of the immortalized mouse renal fibroblast-like cell line FAIK F3-5, sphingosine 1-phosphate (S1P), by activating S1P1 and S1P3 receptors, can stabilize hypoxia-inducible factor (HIF)-2α and upregulate Epo mRNA and protein synthesis. In this study, we have addressed the role of intracellular iS1P derived from sphingosine kinases (Sphk) 1 and 2 on Epo synthesis in F3-5 cells and in mouse primary cultures of renal fibroblasts. We show that stable knockdown of Sphk2 in F3-5 cells increases HIF-2α protein and Epo mRNA and protein levels, while Sphk1 knockdown leads to a reduction of hypoxia-stimulated HIF-2α and Epo protein. A similar effect was obtained using primary cultures of renal fibroblasts isolated from wildtype mice, Sphk1-/-, or Sphk2-/- mice. Furthermore, selective Sphk2 inhibitors mimicked the effect of genetic Sphk2 depletion and also upregulated HIF-2α and Epo protein levels. The combined blockade of Sphk1 and Sphk2, using Sphk2-/- renal fibroblasts treated with the Sphk1 inhibitor PF543, resulted in reduced HIF-2α and Epo compared to the untreated Sphk2-/- cells. Exogenous sphingosine (Sph) enhanced HIF-2α and Epo, and this was abolished by the combined treatment with the selective S1P1 and S1P3 antagonists NIBR-0213 and TY52156, suggesting that Sph was taken up by cells and converted to iS1P and exported to then act in an autocrine manner through S1P1 and S1P3. The upregulation of HIF-2α and Epo synthesis by Sphk2 knockdown was confirmed in the human hepatoma cell line Hep3B, which is well-established to upregulate Epo production under hypoxia. In summary, these data show that sphingolipids have diverse effects on Epo synthesis. While accumulation of intracellular Sph reduces Epo synthesis, iS1P will be exported to act through S1P1+3 to enhance Epo synthesis. Furthermore, these data suggest that selective inhibition of Sphk2 is an attractive new option to enhance Epo synthesis and thereby to reduce anemia development in chronic kidney disease.

Sphingosine 1-Phosphate Receptor 5 (S1P5) Knockout Ameliorates Adenine-Induced Nephropathy.

S1P and its receptors have been reported to play important roles in the development of renal fibrosis. Although S1P5 has barely been investigated so far, there are indications that it can influence inflammatory and fibrotic processes. Here, we report the role of S1P5 in renal inflammation and fibrosis. Male S1P5 knockout mice and wild-type mice on a C57BL/6J background were fed with an adenine-rich diet for 7 days or 14 days to induce tubulointerstitial fibrosis. The kidneys of untreated mice served as respective controls. Kidney damage, fibrosis, and inflammation in kidney tissues were analyzed by real-time PCR, Western blot, and histological staining. Renal function was assessed by plasma creatinine ELISA. The S1P5 knockout mice had better renal function and showed less kidney damage, less proinflammatory cytokine release, and less fibrosis after 7 days and 14 days of an adenine-rich diet compared to wild-type mice. S1P5 knockout ameliorates tubular damage and tubulointerstitial fibrosis in a model of adenine-induced nephropathy in mice. Thus, targeting S1P5 might be a promising goal for the pharmacological treatment of kidney diseases.

Loss of sphingosine kinase 2 enhances Wilm's tumor suppressor gene 1 and nephrin expression in podocytes and protects from streptozotocin-induced podocytopathy and albuminuria in mice.

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 ß (TGFß) 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.

Novel compounds with dual S1P receptor agonist and histamine H3 receptor antagonist activities act protective in a mouse model of multiple sclerosis.

The sphingosine 1-phosphate (S1P) receptor 1 (S1P1) has emerged as a therapeutic target for the treatment of multiple sclerosis (MS). Fingolimod (FTY720) is the first functional antagonist of S1P1 that has been approved for oral treatment of MS. Previously, we have developed novel butterfly derivatives of FTY720 that acted similar to FTY720 in reducing disease symptoms in a mouse model of experimental autoimmune encephalomyelitis (EAE). In this study, we have synthesized a piperidine derivative of the oxazolo-oxazole compounds, denoted ST-1505, and its ring-opened analogue ST-1478, and characterised their in-vitro and in-vivo functions. Notably, the 3-piperidinopropyloxy moiety resembles a structural motif of pitolisant, a drug with histamine H3R antagonistic/inverse agonist activity approved for the treatment of narcolepsy. Both novel compounds exerted H3R affinities, and in addition, ST-1505 was characterised as a dual S1P1+3 agonist, whereas ST-1478 was a dual S1P1+5 agonist. Both multitargeting compounds were also active in mice and reduced the lymphocyte numbers as well as diminished disease symptoms in the mouse model of MS. The effect of ST-1478 was dependent on SK-2 activity suggesting that it is a prodrug like FTY720, but with a more selective S1P receptor activation profile, whereas ST-1505 is a fully active drug even in the absence of SK-2. In summary, these data suggest that the well soluble piperidine derivatives ST-1505 and ST-1478 hold promise as novel drugs for the treatment of MS and other autoimmune or inflammatory diseases, and by their H3R antagonist potency, they might additionally improve cognitive impairment during disease.

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

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.

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.

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 TGFß/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.

Design, Synthesis, and Structure-Activity Relationship Studies of Dual Inhibitors of Soluble Epoxide Hydrolase and 5-Lipoxygenase.

Inhibition of multiple enzymes of the arachidonic acid cascade leads to synergistic anti-inflammatory effects. Merging of 5-lipoxygenase (5-LOX) and soluble epoxide hydrolase (sEH) pharmacophores led to the discovery of a dual 5-LOX/sEH inhibitor, which was subsequently optimized in terms of potency toward both targets and metabolic stability. The optimized lead structure displayed cellular activity in human polymorphonuclear leukocytes, oral bioavailability, and target engagement in vivo and demonstrated profound anti-inflammatory and anti-fibrotic efficiency in a kidney injury model caused by unilateral ureteral obstruction in mice. These results pave the way for investigating the therapeutic potential of dual 5-LOX/sEH inhibitors in other inflammation- and fibrosis-related disease models.

Sorafenib Treatment and Modulation of the Sphingolipid Pathway Affect Proliferation and Viability of Hepatocellular Carcinoma In Vitro.

Hepatocellular carcinoma (HCC) shows a remarkable heterogeneity and is recognized as a chemoresistant tumor with dismal prognosis. In previous studies, we observed significant alterations in the serum sphingolipids of patients with HCC. This study aimed to investigate the in vitro effects of sorafenib, which is the most widely used systemic HCC medication, on the sphingolipid pathway as well as the effects of inhibiting the sphingolipid pathway in HCC. Huh7.5 and HepG2 cells were stimulated with sorafenib, and inhibitors of the sphingolipid pathway and cell proliferation, viability, and concentrations of bioactive metabolites were assessed. We observed a significant downregulation of cell proliferation and viability and a simultaneous upregulation of dihydroceramides upon sorafenib stimulation. Interestingly, fumonisin B1 (FB1) and the general sphingosine kinase inhibitor SKI II were able to inhibit cell proliferation more prominently in HepG2 and Huh7.5 cells, whereas there were no consistent effects on the formation of dihydroceramides, thus implying an involvement of distinct metabolic pathways. In conclusion, our study demonstrates a significant downregulation of HCC proliferation upon sorafenib, FB1, and SKI II treatment, whereas it seems they exert antiproliferative effects independently from sphingolipids. Certainly, further data would be required to elucidate the potential of FB1 and SKI II as putative novel therapeutic targets in HCC.

Ceramide Kinase Is Upregulated in Metastatic Breast Cancer Cells and Contributes to Migration and Invasion by Activation of PI 3-Kinase and Akt.

Ceramide kinase (CerK) is a lipid kinase that converts the proapoptotic ceramide to ceramide 1-phosphate, which has been proposed to have pro-malignant properties and regulate cell responses such as proliferation, migration, and inflammation. We used the parental human breast cancer cell line MDA-MB-231 and two single cell progenies derived from lung and bone metastasis upon injection of the parental cells into immuno-deficient mice. The lung and the bone metastatic cell lines showed a marked upregulation of CerK mRNA and activity when compared to the parental cell line. The metastatic cells also had increased migratory and invasive activity, which was dose-dependently reduced by the selective CerK inhibitor NVP-231. A similar reduction of migration was seen when CerK was stably downregulated with small hairpin RNA (shRNA). Conversely, overexpression of CerK in parental MDA-MB-231 cells enhanced migration, and this effect was also observed in the non-metastatic cell line MCF7 upon CerK overexpression. On the molecular level, CerK overexpression increased the activation of protein kinase Akt. The increased migration of CerK overexpressing cells was mitigated by the CerK inhibitor NVP-231, by inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway and the Rho kinase, but not by inhibition of the classical extracellular signal-regulated kinase (ERK) pathway. Altogether, our data demonstrate for the first time that CerK promotes migration and invasion of metastatic breast cancer cells and that targeting of CerK has potential to counteract metastasis in breast cancer.

Downregulation of S1P Lyase Improves Barrier Function in Human Cerebral Microvascular Endothelial Cells Following an Inflammatory Challenge.

Sphingosine 1-phosphate (S1P) is a key bioactive lipid that regulates a myriad of physiological and pathophysiological processes, including endothelial barrier function, vascular tone, vascular inflammation, and angiogenesis. Various S1P receptor subtypes have been suggested to be involved in the regulation of these processes, whereas the contribution of intracellular S1P (iS1P) through intracellular targets is little explored. In this study, we used the human cerebral microvascular endothelial cell line HCMEC/D3 to stably downregulate the S1P lyase (SPL-kd) and evaluate the consequences on endothelial barrier function and on the molecular factors that regulate barrier tightness under normal and inflammatory conditions. The results show that in SPL-kd cells, transendothelial electrical resistance, as a measure of barrier integrity, was regulated in a dual manner. SPL-kd cells had a delayed barrier build up, a shorter interval of a stable barrier, and, thereafter, a continuous breakdown. Contrariwise, a protection was seen from the rapid proinflammatory cytokine-mediated barrier breakdown. On the molecular level, SPL-kd caused an increased basal protein expression of the adherens junction molecules PECAM-1, VE-cadherin, and ß-catenin, increased activity of the signaling kinases protein kinase C, AMP-dependent kinase, and p38-MAPK, but reduced protein expression of the transcription factor c-Jun. However, the only factors that were significantly reduced in TNFα/SPL-kd compared to TNFα/control cells, which could explain the observed protection, were VCAM-1, IL-6, MCP-1, and c-Jun. Furthermore, lipid profiling revealed that dihydro-S1P and S1P were strongly enhanced in TNFα-treated SPL-kd cells. In summary, our data suggest that SPL inhibition is a valid approach to dampenan inflammatory response and augmente barrier integrity during an inflammatory challenge.

Renal Mesangial Cells Isolated from Sphingosine Kinase 2 Transgenic Mice Show Reduced Proliferation and are More Sensitive to Stress-Induced Apoptosis.

BACKGROUND/AIMS: Sphingosine 1-phosphate (S1P) is considered as a key molecule regulating various cell functions including cell growth and death. It is produced by two sphingosine kinases (SK) denoted as SK-1 and SK-2. Whereas SK-1 has been extensively studied and has been appointed a role in promoting cell growth, the function of SK-2 is controversial, and both pro-proliferative and pro-apoptotic functions have been suggested. In this study we investigated whether renal mesangial cells isolated from transgenic mice overexpressing the human Sphk2 gene (hSK2-tg) showed an altered cell response towards growth-inducing and apoptotic stimuli. METHODS: hSK2-tg mice were generated by using a Quick KnockinR strategy. Renal mesangial cells were isolated by a differential sieving method and further cultivated in vitro. Lipids were quantified by mass spectrometry. Protein expression was determined by Western blot analysis, cell proliferation was determined by 3H-thymidine incorporation, and apoptosis was determined by a DNA fragmentation ELISA. RESULTS: We show here that kidneys and mesangial cells from hSK2-tg mice express the hSK2 as well as the endogenous mouse mSK2. hSK2 and mSK2 predominantly resided in the cytosol of quiescent transgenic cells. However, S1P accumulated strongly in the nucleus and only minimally in the cytosol of transgenic cells. Functionally, hSK2-tg cells proliferated less than control cells under normal growth conditions and were also more sensitive towards stress-induced apoptosis. On the molecular level, this was reflected by reduced ERK and Akt/PKB activation, and upon staurosporine treatment, by a sensitized mitochondrial pathway as manifested by reduced anti-apoptotic Bcl-XL expression and increased cleavage of caspase-9, downstream caspase-3 and PARP-1. CONCLUSION: Altogether, these data demonstrate that SK-2 exerts an antiproliferative and apoptosis-sensitizing effect in renal mesangial cells which suggests that selective inhibitors of SK-2 may promote proliferation and reduce apoptosis and this may have impact on the outcome of proliferation-associated diseases such as mesangioproliferative glomerulonephritis.

Sphingosine Kinase-2 Deficiency Ameliorates Kidney Fibrosis by Up-Regulating Smad7 in a Mouse Model of Unilateral Ureteral Obstruction.

Kidney fibrosis is a hallmark of chronic kidney disease and leads to extracellular matrix accumulation, organ scarring, and loss of kidney function. In this study, we investigated the role of sphingosine kinase-2 (SPHK2) on the progression of tubular fibrosis by using a mouse unilateral ureteral obstruction (UUO) model. We found that SPHK2 protein and activity are up-regulated in fibrotic renal tissue. Functionally, Sphk2-deficient (Sphk2-/-) mice showed an attenuated fibrotic response to UUO compared with wild-type mice, as demonstrated by reduced collagen abundance and decreased expression of fibronectin-1, collagen I, α-smooth muscle actin, connective tissue growth factor (CTGF), and plasminogen activator inhibitor (PAI-1). More important, these changes were associated with increased expression of the antifibrotic protein Smad7 and higher levels of sphingosine in Sphk2-/- UUO kidneys. Mechanistically, sphingosine ameliorates transforming growth factor-ß-induced collagen accumulation, CTGF, and PAI-1 expression, but enhances Smad7 protein expression in primary kidney fibroblasts. In a complementary approach, in human Sphk2-overexpressing mice, UUO resulted in exacerbated signs of fibrosis with increased collagen accumulation, higher expression levels of fibronectin-1, collagen I, α-smooth muscle actin, CTGF, and PAI-1, but decreased Smad7 expression. SPHK2 plays an important role in kidney fibrogenesis by modulating transforming growth factor-ß signaling. Thus, SPHK2 might be an attractive new target for the treatment of fibrosis in chronic kidney disease.

Biglycan- and Sphingosine Kinase-1 Signaling Crosstalk Regulates the Synthesis of Macrophage Chemoattractants.

In its soluble form, the extracellular matrix proteoglycan biglycan triggers the synthesis of the macrophage chemoattractants, chemokine (C-C motif) ligand CCL2 and CCL5 through selective utilization of Toll-like receptors (TLRs) and their adaptor molecules. However, the respective downstream signaling events resulting in biglycan-induced CCL2 and CCL5 production have not yet been defined. Here, we show that biglycan stimulates the production and activation of sphingosine kinase 1 (SphK1) in a TLR4- and Toll/interleukin (IL)-1R domain-containing adaptor inducing interferon (IFN)-ß (TRIF)-dependent manner in murine primary macrophages. We provide genetic and pharmacological proof that SphK1 is a crucial downstream mediator of biglycan-triggered CCL2 and CCL5 mRNA and protein expression. This is selectively driven by biglycan/SphK1-dependent phosphorylation of the nuclear factor NF-κB p65 subunit, extracellular signal-regulated kinase (Erk)1/2 and p38 mitogen-activated protein kinases. Importantly, in vivo overexpression of soluble biglycan causes Sphk1-dependent enhancement of renal CCL2 and CCL5 and macrophage recruitment into the kidney. Our findings describe the crosstalk between biglycan- and SphK1-driven extracellular matrix- and lipid-signaling. Thus, SphK1 may represent a new target for therapeutic intervention in biglycan-evoked inflammatory conditions.

Upregulation of the S1P3 receptor in metastatic breast cancer cells increases migration and invasion by induction of PGE2 and EP2/EP4 activation.

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.

Sphingosine-1-Phosphate Receptor-2 Antagonists: Therapeutic Potential and Potential Risks.

The sphingosine-1-phosphate (S1P) signaling system with its specific G-protein-coupled S1P receptors, the enzymes of S1P metabolism and the S1P transporters, offers a multitude of promising targets for drug development. Until today, drug development in this area has nearly exclusively focused on (functional) antagonists at the S1P1 receptor, which cause a unique phenotype of immunomodulation. Accordingly, the first-in class S1P1 receptor modulator, fingolimod, has been approved for the treatment of relapsing-remitting multiple sclerosis, and novel S1P1 receptor (functional) antagonists are being developed for autoimmune and inflammatory diseases such as psoriasis, inflammatory bowel disease, lupus erythematodes, or polymyositis. Besides the S1P1 receptor, also S1P2 and S1P3 are widely expressed and regulate many diverse functions throughout the body. The S1P2 receptor, in particular, often exerts cellular functions which are opposed to the functions of the S1P1 receptor. As a consequence, antagonists at the S1P2 receptor have the potential to be useful in a contrasting context and different areas of indication compared to S1P1 antagonists. The present review will focus on the therapeutic potential of S1P2 receptor antagonists and discuss their opportunities as well as their potential risks. Open questions and areas which require further investigations will be emphasized in particular.

Sphingosine kinase 2 deficient mice exhibit reduced experimental autoimmune encephalomyelitis: Resistance to FTY720 but not ST-968 treatments.

The immunomodulatory drug FTY720 is presently approved for the treatment of relapsing-remitting multiple sclerosis. It is a prodrug that requires activation by sphingosine kinase 2 (SK-2) to induce T cell homing to secondary lymphoid tissue. In this study, we have investigated the role of SK-2 in experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. We show that SK-2 deficiency reduced clinical symptoms of EAE. Furthermore, in SK-2-deficient mice, the protective effect of FTY720 on EAE was abolished, while the non-prodrug FTY720-derivative ST-968 was still fully active. Protection was paralleled by reduced numbers of T-lymphocytes in blood and a reduced blood-brain-barrier leakage. This correlated with reduced mRNA expression of ICAM-1, VCAM-1, but enhanced expression of PECAM-1. A similar regulation of permeability and of PECAM-1 was seen in primary cultures of isolated mouse brain vascular endothelial cells and in a human immortalized cell line upon SK-2 knockdown. In summary, these data demonstrated that deletion of SK-2 exerts a protective effect on the pathogenesis of EAE in C57BL/6 mice and that SK-2 is essential for the protective effect of FTY720 but not of ST-968. Thus, ST-968 is a promising novel immunomodulatory compound that may be a valuable alternative to FTY720 under conditions where SK-2 activity is limited.

FTY720 and two novel butterfly derivatives exert a general anti-inflammatory potential by reducing immune cell adhesion to endothelial cells through activation of S1P(3) and phosphoinositide 3-kinase.

Sphingosine-1-phosphate (S1P) is a key lipid regulator of a variety of cellular responses including cell proliferation and survival, cell migration, and inflammatory reactions. Here, we investigated the effect of S1P receptor activation on immune cell adhesion to endothelial cells under inflammatory conditions. We show that S1P reduces both tumor necrosis factor (TNF)-α- and lipopolysaccharide (LPS)-stimulated adhesion of Jurkat and U937 cells to an endothelial monolayer. The reducing effect of S1P was reversed by the S1P1+3 antagonist VPC23019 but not by the S1P1 antagonist W146. Additionally, knockdown of S1P3, but not S1P1, by short hairpin RNA (shRNA) abolished the reducing effect of S1P, suggesting the involvement of S1P3. A suppression of immune cell adhesion was also seen with the immunomodulatory drug FTY720 and two novel butterfly derivatives ST-968 and ST-1071. On the molecular level, S1P and all FTY720 derivatives reduced the mRNA expression of LPS- and TNF-α-induced adhesion molecules including ICAM-1, VCAM-1, E-selectin, and CD44 which was reversed by the PI3K inhibitor LY294002, but not by the MEK inhibitor U0126.In summary, our data demonstrate a novel molecular mechanism by which S1P, FTY720, and two novel butterfly derivatives acted anti-inflammatory that is by suppressing gene transcription of various endothelial adhesion molecules and thereby preventing adhesion of immune cells to endothelial cells and subsequent extravasation.

Sphingosine kinase 2 deficiency increases proliferation and migration of renal mouse mesangial cells and fibroblasts.

Both of the sphingosine kinase (SK) subtypes SK-1 and SK-2 catalyze the production of the bioactive lipid molecule sphingosine 1-phosphate (S1P). However, the subtype-specific cellular functions are largely unknown. In this study, we investigated the cellular function of SK-2 in primary mouse renal mesangial cells (mMC) and embryonic fibroblasts (MEF) from wild-type C57BL/6 or SK-2 knockout (SK2ko) mice. We found that SK2ko cells displayed a significantly higher proliferative and migratory activity when compared to wild-type cells, with concomitant increased cellular activities of the classical extracellular signal regulated kinase (ERK) and PI3K/Akt cascades, and of the small G protein RhoA. Furthermore, we detected an upregulation of SK-1 protein and S1P3 receptor mRNA expression in SK-2ko cells. The MEK inhibitor U0126 and the S1P1/3 receptor antagonist VPC23019 blocked the increased migration of SK-2ko cells. Additionally, S1P3ko mesangial cells showed a reduced proliferative behavior and reduced migration rate upon S1P stimulation, suggesting a crucial involvement of the S1P3 receptor. In summary, our data demonstrate that SK-2 exerts suppressive effects on cell growth and migration in renal mesangial cells and fibroblasts, and that therapeutic targeting of SKs for treating proliferative diseases requires subtype-selective inhibitors.

Transforming growth factor ß2 (TGF-ß2)-induced connective tissue growth factor (CTGF) expression requires sphingosine 1-phosphate receptor 5 (S1P5) in human mesangial cells.

Transforming growth factor ß2 (TGF-ß2) is well known to stimulate the expression of pro-fibrotic connective tissue growth factor (CTGF) in several cell types including human mesangial cells. The present study demonstrates that TGF-ß2 enhances sphingosine 1-phosphate receptor 5 (S1P5) mRNA and protein expression in a time and concentration dependent manner. Pharmacological and siRNA approaches reveal that this upregulation is mediated via activation of classical TGF-ß downstream effectors, Smad and mitogen-activated protein kinases. Most notably, inhibition of Gi with pertussis toxin and downregulation of S1P5 by siRNA block TGF-ß2-stimulated upregulation of CTGF, demonstrating that Gi coupled S1P5 is necessary for TGF-ß2-triggered expression of CTGF in human mesangial cells. Overall, these findings indicate that TGF-ß2 dependent upregulation of S1P5 is required for the induction of pro-fibrotic CTGF by TGF-ß. Targeting S1P5 might be an attractive novel approach to treat renal fibrotic diseases.