Expression Of Sphingosine Kinase Research Articles

Abstract LB-369: Sphingosine-1-phosphate (S1P) as a novel target in renal cancer (RCC)

Abstract Although treatment of patients (pts) with metastatic RCC with VEGFR2 inhibitors induces disease regression, these responses tend to be short-lived. Mouse models of VEGFR TKI treatment of RCC were used to study the mechanisms of resistance and identify novel targets. Drug-induced growth arrest was associated with enhanced expression of sphingosine kinase (sphk) whose only product is the bioactive signaling lipid, S1P. To explore the role of this pathway in mice bearing 786-O RCC were treated with a neutralizing antibody against S1P. Vehicle treated tumors increased by 2mm after 5.8 ± 4.1 days (n=6) and treatment with 10mg/kg or 50mg/kg anti-S1P led to growth after 15.3 ± 3.5 days (n=7) and 20.5 ± 3.9 days (n=6) respectively (P<0.01 for PBS vs. 10mg/kg or 50mg/kg), demonstrating that anti-S1P exhibits single agent activity in treatment naïve tumors. To assess the activity of S1P ab in the setting of sunitinib resistance, mice whose tumors developed resistance to sunitinib, were switched to either an anti-isotype negative control antibody, or anti-S1P. Treatment with anti-S1P produced relative tumor stabilization: 23.0 ± 16.5 days (n=10) vs. isotype control 6.9 ± 2.1 days for switch to isotype (n=7, P=0.01). In an A498 RCC tumor xenograft model, vehicle and isotype treated tumors increased by 2mm after 5.0 ± 1.17 days (n=7) compared to15.3 ± 5.8 days (n=6) for mice treated with 50mg/kg anti-S1P (P<0.01) and time to reach sacrifice size of 20mm was 30.7 ± 3.8 days (n=7) in vehicle and isotype treated tumors and 45.0 ± 8.6 days (n=6) with 50mg/kg anti-S1P (P=0.01). S1P levels in pts with RCC were found to be significantly higher than control pts (2.7 ± 0.032uM (n=23) vs 0.588 ± 0.057 M (n=20, P<0.0001). Thus, the S1P pathway is a novel target for the treatment of both treatment naïve and VEFGR TKI refractory RCC and anti-S1P therapy is an attractive new option for clinical investigation in RCC patients. The humanized anti-S1P mAb, ASONEPTM, has recently completed Phase I clinical trials in cancer and planning for Phase II efficacy trials is underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-369.

Abstract 5262: Resistance to fenretinide in neuroblastoma is associated with increased expression of sphingosine kinase and sphingosine-1-phosphate receptors and targeting sphingosine kinase reverses resistance

Abstract The synthetic retinoid fenretinide, N-(4-hydroxyphenyl) retinamide (4-HPR), is selectively cytotoxic to cancer cells via increases in dihydroceramide levels and generation of reactive oxygen species and has clinical activity against recurrent neuroblastoma (J Clin Oncol 27: 15s abstr 10009, 2009). Metabolism of ceramides into various sphingolipid species, including sphingosine, and phosphorylation of dihydrosphingosine and sphingosine by sphingosine kinase 1 (SPHK1) are anti-apoptotic and thought to counter cytotoxicity mediated by ceramides. Multistep selection for resistance to 4-HPR was performed with a 4-HPR sensitive neuroblastoma cell line, SMS-KCNR (IC99=4.6 uM), resulting in the 4-HPR resistant line, KCNR-FR (IC99=19.9 uM). We generated a TaqMan low-density array (TLDA) that measures expression of 42 genes involved in ceramide synthesis and metabolism by quantitative RT-PCR. Genes overexpressed in KCNR-FR relative to SMS-KCNR included ceramide desaturase 2 (14.1-fold), sphingosine-1-phosphate receptors S1PR2, 3, and 4 (2.4, 1.6, and 1.3-fold, respectively), and SPHK1 (5.2-fold). These data point toward 4-HPR resistance being mediated by sphingosine-1-phosphate or dihydrosphingosine-1-phosphate, both of which are generated via phosphorylation of sphingosine by SPHK1. SPHK1-specific RNAi knockdown decreased SPHK1 mRNA expression (36% ± 0.007 of scrambled RNAi, p<0.05) and significantly increased apoptosis (35.8 ± 1.7%; p<0.05) in KCNR-FR treated with 4-HPR relative to scrambled RNAi controls (8.8 ± 6.5%; p<0.05). Safingol (L-threo-dihydrosphingosine), a putative sphingosine kinase inhibitor in phase I trials, partially reversed KCNR-FR resistance to 4-HPR (Combination Index = 0.56). Thus, selection for resistance to 4-HPR in neuroblastoma was associated with increased expression of multiple components of the anti-apoptotic sphingosine kinase signaling pathway, and such resistance can be partially overcome by targeting sphingosine kinase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5262.

886 SPHINGOSINE KINASE 1 KNOCK DOWN IMPROVES ERECTILE FUNCTION IN STREPTOZOTOCIN- INDUCED DIABETIC RATS AND DECREASES RHO-KINASE ACTIVITY IN VITRO

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research II1 Apr 2010886 SPHINGOSINE KINASE 1 KNOCK DOWN IMPROVES ERECTILE FUNCTION IN STREPTOZOTOCIN- INDUCED DIABETIC RATS AND DECREASES RHO-KINASE ACTIVITY IN VITRO Guillermo Villegas, Moses Tar, Arnold Melman, and Michael DiSanto Guillermo VillegasGuillermo Villegas More articles by this author , Moses TarMoses Tar More articles by this author , Arnold MelmanArnold Melman More articles by this author , and Michael DiSantoMichael DiSanto More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1642AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that is mainly formed by phosphorylation of sphingosine by sphingosine kinase 1 (SPHK1) and can be reverted to sphingosine by sphingosine-1-phosphate phosphatase 1 (SPP1). Our recent studies show that S1P plays a mayor role in the Ca+2 sensitization pathway and contractility of corpus cavernosum smooth muscle (CCSM). Furthermore we demonstrated that SPHK1 expression is elevated in Type 1 diabetic patients with impotence while SPHK1 overexpression in vivo induces erectile dysfunction (ED) in normal rats and upregulates the expression of Rho-kinase (ROCK). Our goals were a) to asses if knocking down expression of SPHK1 in CCSM of diabetic (DM) rats improves erectile function and therefore confirm the determinant role of S1P in the etiology of DM-induced ED b) To examine if SPHK1 expression modulates smooth muscle calcium sensitization by increasing or decreasing ROCK activity and c) to determine whether ROCK is modulated directly by SPHK1 or by S1P. METHODS 1) SPHK1 mRNA was silenced to prevent the synthesis of S1P by using small hairpin RNA (sh) cloned into a shRNA expression vector (pSiren/SPHK1) 2) Human SPP1 and SPHK1 were cloned into the pVax vector to overexpress these proteins 3) For in vivo studies, 2 month strepotozotocin induced diabetic rats (STZ) with ED were injected in the CC with 100 μg pSiren/SPHK1 or pSiren/Luciferase (MOC) and erectile function assessed via cavernous nerve stimulation-induced increase in intracavernous pressure (ICP) one week post-injection 4) For the in vitro studies rat and human primary CCSM cells were transfected with either pSiren/SPHK1, pVax/SPP1 or pVax/SPHK1. In addition S1P receptors were blocked with suramin or N-ethylmaleimide. Cells were harvested at 48 hours, lysed for protein and RNA extraction and analyzed by real time PCR and Western blot. RESULTS a) pSiren/SPHK1 injected STZ rats showed an improved erectile function especially at lower stimulations compared to MOC b) Transfection of CCSM cells with either SPHK1 shRNA or SPP1 significant decreased ROCK activity and c) Blocking of the S1P receptors also downregulated ROCK activity. CONCLUSIONS We showed that in STZ rats, treatment of CC with SPHK1 shRNA improves erectile function confirming the direct role S1P plays in DM-induced ED. Our results further suggest that S1P mediates Ca+2 sensitization of CCSM cells by modulating ROCK activity and that the mechanism involves S1P and not SPHK1. Bronx, NY© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e346-e347 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Guillermo Villegas More articles by this author Moses Tar More articles by this author Arnold Melman More articles by this author Michael DiSanto More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Efficacy of a novel sphingosine kinase inhibitor in experimental Crohn’s disease

Activation of sphingosine kinase (SK) is a key response to many inflammatory processes. The present studies test the hypothesis that an orally available SK inhibitor, ABC294640, would be effective in rodent models of Crohn's disease. Trinitrobenzene sulfonic acid (TNBS) was administered rectally to mice and rats. Rats were treated with ABC294640 orally alone or in combination with olsalazine and disease progression was monitored. For both rodent species, treatment with ABC294640 attenuated disease progression. Colon samples from the ABC294640-treated animals had improved histology and cytokine parameters when compared with vehicle-treated animals. The expression of SK was similarly increased in TNBS-treated animals and in human colon tissue specimens from inflammatory bowel disease patients relative to normal, control patients. Sphingosine kinase may be a critical mediator of colonic damage during intestinal inflammation, and pharmacologic inhibitors of this enzyme may prove useful in the treatment of Crohn's disease.

Altered expression of sphingosine kinase 1 and sphingosine-1-phosphate receptor 1 in mouse hippocampus after kainic acid treatment

Kainic acid (KA) induces hippocampal cell death and astrocyte proliferation. There are reports that sphingosine kinase (SPHK)1 and sphingosine-1- phosphate (S1P) receptor 1 (S1P 1) signaling axis controls astrocyte proliferation. Here we examined the temporal changes of SPHK1/S1P 1 in mouse hippocampus during KA-induced hippocampal cell death. Mice were killed at 2, 6, 24, or 48 h after KA (30 mg/kg) injection. There was an increase in Fluoro-Jade B-positive cells in the hippocampus of KA-treated mice with temporal changes of glial fibrillary acidic protein (GFAP) expression. The lowest level of SPHK1 protein expression was found 2 h after KA treatment. Six hours after KA treatment, the expression of SPHK1 and S1P 1 proteins steadily increased in the hippocampus. In immunohistochemical analysis, SPHK1 and S1P 1 are more immunoreactive in astrocytes within the hippocampus of KA-treated mice than in hippocampus of control mice. These results indicate that SPHK1/S1P 1 signaling axis may play an important role in astrocytes proliferation during KA-induced excitotoxicity.

HSP-70 mitigates LPS/SKI-induced cell damage by increasing sphingosine kinase 1 (SK1)

Heat shock proteins (HSPs) are potent protectors of cellular integrity against environmental stresses, including toxic microbial products. To investigate the mechanism of HSP-70 cell protection against bacterial lipopolysaccharide (LPS), we established a stable HSP-70 gene-transfected RAW 264.7 murine macrophage model of LPS-induced cell death. Bacterial LPS increases the activity of sphingosine kinase 1 (SK1), which catalyzes formation of sphingosine-1-phosphate (S1P). S1P functions as a critical signal for initiation and maintenance of diverse aspects of immune cell activation and function. When mouse macrophages were incubated with Escherichia coli LPS (1 μg/ml) and sphingosine kinase inhibitor (SKI, 5 μM), 90% of cells died. Neither LPS nor SKI alone at these doses damaged the cells. The LPS/SKI-induced cell death was partially reversed by overexpression of HSP-70 in gene-transfected macrophages. The specificity of HSP-70 in this reversal was demonstrated by transfection of HSP-70-specific siRNA. Down-regulation of HSP-70 expression after transfection of siRNA specific for HSP-70 was associated with increased LPS/SKI-induced cell damage. Overexpression of human or murine HSP-70 (HSPA1A and Hspa1a, respectively) increased both cellular SK1 mRNA and protein levels. Cellular heat shock also increased SK1 protein. These studies confirm the importance of SK1 as a protective moiety in LPS-induced cell injury and demonstrate that HSP-70-mediated protection from cells treated with LPS/SKI is accompanied by upregulating expression of SK1. HSP-70-mediated increases in SK1 and consequent increased levels of S1P may also play a role in protection of cells from other processes that lead to programmed cell death.

The Sphingosine 1-Phosphate (S1P) Signaling Pathway Is Regulated During Pregnancy in Sheep1

Because sphingosine 1-phosphate (S1P) is a potent stimulator of angiogenesis, we hypothesized that the S1P pathway is activated to stimulate endometrial/placental angiogenesis during pregnancy. We initially localized S1P signaling pathway members in the gravid and nongravid uterine horns of unilaterally pregnant ewes. Sphingosine kinase-1 expression was greater in gravid compared to nongravid horns. In situ hybridization revealed elevated expression of sphingosine 1-phosphate phosphatase (SGPP1) in gravid interplacentomal endometrial stroma on Days 20 and 40 compared to the nongravid uterine horn, but expression increased in endometrium of the nongravid uterine horn between Days 40 and 120. SGPP1 expression increased in placentomes late in gestation. Sphingosine 1-phosphate lyase mRNA was modestly expressed at Day 20 and then decreased. In contrast, sphingosine 1-phosphate receptor 1 (S1PR1) mRNA increased in endometrium and caruncular stroma of the gravid uterine horn. Treatment with FTY720 and VPC23019, S1P receptor antagonists, blocked human and ovine endothelial cell invasion using an in vitro model of sprouting angiogenesis. Knockdown of S1PR1 with siRNA reduced invasion responses as well. We previously reported that delta-like 4 (DLL4) and A disintegrin and metalloproteinase with thrombospondin-like repeats 1 (ADAMTS1) participate in endothelial cell invasion stimulated by S1P and growth factors in vitro, and thus investigated whether their expression correlated with areas undergoing angiogenesis in vivo. DLL4 expression was similar to S1PR1, while ADAMTS1 mRNA was expressed by endometria of both nongravid and gravid horns, as well as conceptus and placentomes. These results establish that S1P signaling pathway members and S1P- and growth factor-regulated genes are prominent in uterine and placental tissue and in some cases are correlated with areas undergoing angiogenesis. Thus, S1P signaling may be crucial for proper fetal-placental development.

An Active Form of Sphingosine Kinase-1 Is Released in the Extracellular Medium as Component of Membrane Vesicles Shed by Two Human Tumor Cell Lines

Expression of sphingosine kinase-1 (SphK-1) correlates with a poor survival rate of tumor patients. This effect is probably due to the ability of SphK-1 to be released into the extracellular medium where it catalyzes the biosynthesis of sphingosine-1-phosphate (S1P), a signaling molecule endowed with profound proangiogenic effects. SphK-1 is a leaderless protein which is secreted by an unconventional mechanism. In this paper, we will show that in human hepatocarcinoma Sk-Hep1 cells, extracellular signaling is followed by targeting the enzyme to the cell surface and parallels targeting of FGF-2 to the budding vesicles. We will also show that SphK-1 is present in a catalitycally active form in vesicles shed by SK-Hep1 and human breast carcinoma 8701-BC cells. The enzyme substrate sphingosine is present in shed vesicles where it is produced by neutral ceramidase. Shed vesicles are therefore a site for S1P production in the extracellular medium and conceivably also within host cell following vesicle endocytosis.

Increased ceramide synthase 2 and 6 mRNA levels in breast cancer tissues and correlation with sphingosine kinase expression

Intervention in the ceramide metabolic pathway is emerging as a novel means to regulate cancer and to modify the activity of chemotherapeutic drugs. We now study mRNA expression levels of the six ceramide synthase (CerS) genes in breast cancer tissue. CerS2 and CerS6 mRNA was significantly elevated in breast cancer tissue compared to paired normal tissue, with approximately half of the individuals showing elevated CerS2 and CerS6 mRNA. A significant correlation was found between CerS2 and CerS6 expression, and between CerS4 and CerS2/CerS6 expression. Moreover, patients that expressed higher CerS2 or 4 mRNA levels tended to show no changes in sphingosine kinase 1 levels, and likewise patients that expressed no change in CerS2 or CerS4 mRNA levels tended to express higher levels of sphingosine kinase 1. Together these results suggest an important role for the CerS genes in breast cancer etiology or diagnosis.

Does Sphingosine Kinase 1 (SPHK1) Play a Role in Endometriosis?

Sphingolipids are important constituents of cell membranes, which play key roles as regulatory molecules for various cellular functions. Sphingosine-1-phosphate produced by sphingosine kinase 1 (SPHK1) promotes cell proliferation, regulates angiogenesis, and influences invasion as well as the attachment of cells. Since these processes are believed to be involved in the development of endometriosis, we analyzed the expression of SPHK1 in human eutopic and ectopic endometrium using immunohistochemistry and microarray analysis. Epithelial cells of both eutopic and ectopic endometrium showed highly variable immunostaining with polyclonal antibody directed against SPHK1. However, strong expression of SPHK1 was largely restricted to the epithelial endometrial cells of peritoneal endometriotic lesions (n = 8/23, 34.8 %). Only n = 1/15 (6.7 %) of the adenomyosis samples and n = 3/41 (7.3 %) of the eutopic endometrium samples displayed strong antigen expression (p = 0.008, χ 2 test). No association between SPHK1 and Ki-67 expression was detectable. Still further research is needed in order to clarify the role of sphingolipids in the development of endometriosis, and particularly in invasive growth.

Transforming growth factor-β2 upregulates sphingosine kinase-1 activity, which in turn attenuates the fibrotic response to TGF-β2 by impeding CTGF expression

Transforming growth factor-beta2 (TGF-beta2) stimulates the expression of pro-fibrotic connective tissue growth factor (CTGF) during the course of renal disease. Because sphingosine kinase-1 (SK-1) activity is also upregulated by TGF-beta, we studied its effect on CTGF expression and on the development of renal fibrosis. When TGF-beta2 was added to an immortalized human podocyte cell line we found that it activated the promoter of SK-1, resulting in upregulation of its mRNA and protein expression. Further, depletion of SK-1 by small interfering RNA or its pharmacological inhibition led to accelerated CTGF expression in the podocytes. Over-expression of SK-1 reduced CTGF induction, an effect mediated by intracellular sphingosine-1-phosphate. In vivo, SK-1 expression was also increased in the podocytes of kidney sections of patients with diabetic nephropathy when compared to normal sections of kidney obtained from patients with renal cancer. Similarly, in a mouse model of streptozotocin-induced diabetic nephropathy, SK-1 and CTGF were upregulated in podocytes. In SK-1 deficient mice, exacerbation of disease was detected by increased albuminuria and CTGF expression when compared to wild-type mice. Thus, SK-1 activity has a protective role in the fibrotic process and its deletion or inhibition aggravates fibrotic disease.

Expression of sphingosine kinase 2 in synovial fibroblasts of rheumatoid arthritis contributing to apoptosis by a sphingosine analogue, FTY720

Gene expression profiles in synovial tissues from rheumatoid arthritis (RA) patients have yielded useful information on the pathogenetic process of the synovitis. In one group of them, sphingosine kinase 2 (SPHK2), a nuclear protein regulating cell proliferation, seemed to be highly expressed, undergoing a different pathogenetic process of synovitis. In the present study it was clarified that SPHK2 was expressed in the synovial fibroblasts of the synovial tissues obtained from the knee joints of the RA patients. In the cultured synovial fibroblasts from these patients, SPHK2 was more highly expressed than that in the human macrophage cell line, THP-1 and human dermal fibroblasts. SPHK2 was expressed in and around the nucleus and transferred to the cytoplasm and cell surface by the administration of epidermal growth factor, associated with the increased expression of sphingosine-1-phosphate. A sphingosine analogue, FTY720, which is activated by phosphorylation specifically by SPHK2, mediated apoptotic signaling of the cultured synovial fibroblasts. These findings suggest that SPHK2 may regulate the autonomous proliferation of synovial fibroblasts as one of the predisposing genes to RA and could be a target for a novel therapeutic strategy for RA.

Sphingosine Kinase 1 Is Associated with Gastric Cancer Progression and Poor Survival of Patients

The present study was to investigate the clinical significance of sphingosine kinase 1 (SPHK1), an oncoenzyme, in the development and progression of gastric cancer. mRNA and protein levels of SPHK1 expression in normal gastric epithelial cells, gastric cancer cell lines, and paired gastric cancer lesions and the adjacent noncancerous tissues were examined using reverse transcription-PCR and Western blotting. Immunohistochemistry was employed to analyze SPHK1 expression in 175 clinicopathologically characterized gastric cancer cases. Statistical analyses were applied to derive prognostic and diagnostic associations. Levels of SPHK1 mRNA and protein were higher in gastric cancer cell lines than in normal gastric epithelial cells. SPHK1 protein level was up-regulated in gastric cancer lesions compared with that in the paired adjacent noncancerous tissues. Gastric cancer tissues from 115 of 175 (65.7%) patients revealed high level of SPHK1 protein expression in contrast to the undetectable or marginally detectable expression of SPHK1 in the adjacent noncancerous gastric tissues. Significantly different expression levels of SPHK1 were found in patients at different clinical stages (P=0.003), T classification (P=0.035), and M classification (P=0.020). Patients with higher SPHK1 expression had shorter overall survival time, whereas those with lower SPHK1 expression survived longer. Further multivariate analysis suggested that SPHK1 up-regulation was an independent prognostic indicator for the disease. SPHK1 protein could be a useful marker for the prognosis of gastric cancer. Further study on the potential use of SPHK1 as a therapeutic target is also warranted.

Prognostic and predictive value of enzymes of the sphingolipid metabolism in breast cancer.

Abstract Abstract #6014 Backround:
 Sphingolipids do not only have structural roles for the cell membrane, but also act as effector molecules and second messengers in signal transduction with links to cancer initiation, progression and treatment response. Knowledge of sphingolipid's impact on breast cancer and its prognosis is limited to in vitro data. Clinical data is rare but needed.
 Material and Methods: We analyzed gene expression of 43 proteins from the sphingolipid pathways in different subtypes of breast cancer using microarray data of 1269 tumor samples (test set n=171; validation sets n=1098). Kaplan Meier analysis of disease free survival was performed to examine the prognostic value of different markers. In addition immunohistochemistry was done to determine whether the genes of the sphingolipid rheostat are expressed in cancer or stroma cells of the tumor.
 Results: Significant relationships of several genes of the sphingolipid metabolism with clinical parameters were obtained. Sphingosine kinase 1 (SPHK1), ceramide galactosyltransferase (UGT8), and Ganglioside GD3-Synthase (ST8SIA1) displayed higher expression among ER negative tumors. In contrast, glucosylceramidesynthase (GCS), dihydroceramidesynthases (LASS4, LASS 6) and acid ceramidase (ASAH1) were higher expressed in ER positive samples. Survival analysis revealed a significant worse outcome of patients with high SPHK1 expression. LASS 6 and GCS gene expression had no prognostic impact when the estrogen receptor status was taken into account in multivariate analyses. Immunohistochemistry identified the carcinoma cells as the major source of SPHK1 expression in the tumor.
 Conclusions: These studies of a large cohort of clinical breast cancer samples reveal that sphingolipids do play a role in breast cancer. Enzymes of the sphingolipid metabolism have impact on the prognosis of breast cancer patients and targeting the sphingolipid rheostat can open new treatment options. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6014.

The effects of markedly raised intracellular sphingosine kinase-1 activity in endothelial cells

The enzyme sphingosine kinase-1 (SK1) promotes the formation of sphingosine-1-phosphate (S1P), which is an important survival factor for endothelial cells (EC). Modest increases in intracellular SK1 activity in the EC are known to confer a survival advantage upon the cells. Here, we investigated the effects of more dramatic increases in intracellular SK1 in the EC. We found that these cells show reduced cell survival under conditions of stress, enhanced caspase-3 activity, cell cycle inhibition, and cell-cell junction disruption. We propose that alterations in the phosphorylation state of the enzyme may explain the differential effects on the phenotype with modest versus high levels of enforced expression of SK1. Our results suggest that SK1 activity is subject to control in the EC, and that this control may be lost in conditions involving vascular regression.

Dynamic regulation of sphingosine-1-phosphate homeostasis during development of mouse metanephric kidney

Branching morphogenesis of the metanephric kidney is critically dependent on the delicate orchestration of diverse cellular processes including proliferation, apoptosis, migration, and differentiation. Sphingosine-1-phosphate (S1P) is a potent lipid mediator influencing many of these cellular events. We report increased expression and activity of both sphingosine kinases and S1P phosphatases during development of the mouse metanephric kidney from induction at embryonic day 11.5 to maturity. Sphingosine kinase activity exceeded S1P phosphatase activity in embryonic kidneys, resulting in a net accumulation of S1P, while kinase and phosphatase activities were similar in adult tissue, resulting in reduced S1P content. Sphingosine kinase expression was greater in the metanephric mesenchyme than in the ureteric bud, while the S1P phosphatase SPP2 was expressed at greater levels in the ureteric bud. Treatment of cultured embryonic kidneys with sphingosine kinase inhibitors resulted in a dose-dependent reduction of ureteric bud tip numbers and increased apoptosis. Exogenous S1P rescued kidneys from apoptosis induced by kinase inhibitors. Ureteric bud tip number was unaffected by exogenous S1P in kidneys treated with N,N-dimethylsphingosine, although tip number increased in those treated with d,l-threo-dihydrosphingosine. S1P1 and S1P2 were the predominant S1P receptors expressed in the embryonic kidney. S1P1 expression increased during renal development while expression of S1P2 decreased, and both receptors were expressed predominantly in the metanephric mesenchyme. These results demonstrate dynamic regulation of S1P homeostasis during renal morphogenesis and suggest that differential expression of S1P metabolic enzymes and receptors provides a novel mechanism contributing to the regulation of kidney development.

Deactivation of Sphingosine Kinase 1 by Protein Phosphatase 2A

Sphingosine kinase 1 (SK1) is an important regulator of cellular signaling that has been implicated in a broad range of cellular processes. Cell exposure to a wide array of growth factors, cytokines, and other cell agonists can result in a rapid and transient increase in SK activity via an activating phosphorylation. We have previously identified extracellular signal-regulated kinases 1 and 2 (ERK1/2) as the kinases responsible for the phosphorylation of human SK1 at Ser(225), but the corresponding phosphatase targeting this phosphorylation has remained undefined. Here, we provide data to support a role for protein phosphatase 2A (PP2A) in the deactivation of SK1 through dephosphorylation of phospho-Ser(225). The catalytic subunit of PP2A (PP2Ac) was found to interact with SK1 using both GST-pulldown and coimmunoprecipitation analyses. Coexpression of PP2Ac with SK1 resulted in reduced Ser(225) phosphorylation of SK1 in human embryonic kidney (HEK293) cells. In vitro phosphatase assays showed that PP2Ac dephosphorylated both recombinant SK1 and a phosphopeptide based on the phospho-Ser(225) region of SK1. Finally, both basal and tumor necrosis factor-alpha-stimulated cellular SK1 activity were regulated by molecular manipulation of PP2Ac activity. Thus, PP2A appears to function as an endogenous regulator of SK1 phosphorylation.

Clinical Significance of Sphingosine Kinase-1 Expression in Human Astrocytomas Progression and Overall Patient Survival

To characterize the expression of sphingosine kinase-1 (SPHK1) in human astrocytomas and to investigate the association between SPHK1 expression and progression of astrocytomas. The expression of SPHK1 in normal human astrocytes, astrocytoma cell lines, and four pairs of matched astrocytoma tissues and their adjacent normal brain tissues were detected by quantitative reverse transcription-PCR and Western blot. In addition, SPHK1 protein expression was examined in 243 cases of histologically characterized astrocytomas by immunohistochemistry. Statistical analyses were applied to test for prognostic and diagnostic associations. SPHK1 in astrocytoma cell lines was elevated at both mRNA and protein levels, and the SPHK1 mRNA and protein were significantly up-regulated by up to 6.8- and 40-fold, respectively, in primary astrocytomas compared with those in the adjacent noncancerous brain tissues. Immunohistochemical analysis showed that 100 of 243 (41.2%) paraffin-embedded archival astrocytoma biopsies exhibited high expression of SPHK1. Statistical analysis suggested that the up-regulation of SPHK1 was significantly correlated with the histologic grade of astrocytoma (P=0.000) and that patients with high SPHK1 level exhibited shorter survival time (P<0.001). Multivariate analysis revealed that SPHK1 up-regulation might be an independent prognostic indicator for the survival of patients with astrocytoma. SPHK1 might represent a novel and useful prognostic marker for astrocytoma and play a role during the development and progression of the disease.

Specific and overlapping sphingosine-1-phosphate receptor functions in human synoviocytes: impact of TNF-α

Sphingosine-1-phosphate (S1P), via interaction with its G protein-coupled receptors, regulates various physiological and pathological responses. The present study investigated the role of S1P/S1P receptor signaling in several functional responses of human fibroblast-like synoviocytes (FLSs) that may contribute to the pathogenesis of rheumatoid arthritis (RA). We report that FLSs express the S1P(1), S1P(2), and S1P(3) receptors. Moreover, exogenously applied S1P induces FLS 1) migration, 2) secretion of inflammatory cytokines/chemokines, and 3) protection from apoptosis. Using specific S1P receptor agonists/antagonists, we determined that S1P stimulates FLS migration through S1P(1) and S1P(3), induces cytokine/chemokine secretion through S1P(2) and S1P(3), and protects from cell apoptosis via S1P(1). The S1P-mediated cell motility and cytokine/chemokine secretion seem to be regulated by the p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, and Rho kinase signal transduction pathways. Interestingly, treatment of FLSs with tumor necrosis factor-alpha increases S1P(3) expression and correlates with the enhancement of S1P-induced cytokine/chemokine production. Our data suggest that S1P(1), S1P(2), and S1P(3) play essential roles in the pathogenesis of RA by modulating FLS migration, cytokine/chemokine production, and cell survival. Moreover, the cytokine-rich environment of the inflamed synovium may synergize with S1P signaling to exacerbate the clinical manifestations of this autoimmune disease.

Involvement of sphingosine kinase in plant cell signalling

In mammalian cells sphingosine-1-phosphate (S1P) is a well-established messenger molecule that participates in a wide range of signalling pathways. The objective of the work reported here was to investigate the extent to which phosphorylated long-chain sphingoid bases, such as sphingosine-1-phosphate and phytosphingosine-1-phosphate (phytoS1P) are used in plant cell signalling. To do this, we manipulated Arabidopsis genes capable of metabolizing these messenger molecules. We show that Sphingosine kinase1 (SPHK1) encodes an enzyme that phosphorylates sphingosine, phytosphingosine and other sphingoid long-chain bases. The stomata of SPHK1-KD Arabidopsis plants were less sensitive, whereas the stomata of SPHK1-OE plants were more sensitive, than wild type to ABA. The rate of germination of SPHK1-KD was enhanced, whereas the converse was true for SPHK1-OE seed. Reducing expression of either the putative Arabidopsis S1P phosphatase (SPPASE) or the DPL1 gene, which encodes an enzyme with S1P lyase activity, individually, had no effect on guard-cell ABA signalling; however, stomatal responses to ABA in SPPASEDPL1 RNAi plants were compromised. Reducing the expression of DPL1 had no effect on germination; however, germination of SPPASE RNAi seeds was more sensitive to applied ABA. We also found evidence that expression of SPHK1 and SPPASE were coordinately regulated, and discuss how this might contribute to robustness in guard-cell signalling. In summary, our data establish SPHK1 as a component in two separate plant signalling systems, opening the possibility that phosphorylated long-chain sphingoid bases such as S1P and phytoS1P are ubiquitous messengers in plants.