Targeting the sphingosine kinase/sphingosine 1-phosphate pathway to treat chronic inflammatory kidney diseases

Chronic kidney diseases including glomerulonephritis are often accompanied by acute or chronic inflammation that leads to an increase in extracellular matrix (ECM) production and subsequent glomerulosclerosis. Glomerulonephritis is one of the leading causes for end-stage renal failure with high morbidity and mortality, and there are still only a limited number of drugs for treatment available. In this MiniReview, we discuss the possibility of targeting sphingolipids, specifically the sphingosine kinase 1 (SphK1) and sphingosine 1-phosphate (S1P) pathway, as new therapeutic strategy for the treatment of glomerulonephritis, as this pathway was demonstrated to be dysregulated under disease conditions. Sphingosine 1-phosphate is a multifunctional signalling molecule, which was shown to influence several hallmarks of glomerulonephritis including mesangial cell proliferation, renal inflammation and fibrosis. Most importantly, the site of action of S1P determines the final effect on disease progression. Concerning renal fibrosis, extracellular S1P acts pro-fibrotic via activation of cell surface S1P receptors, whereas intracellular S1P was shown to attenuate the fibrotic response. Interference with S1P signalling by treatment with FTY720, an S1P receptor modulator, resulted in beneficial effects in various animal models of chronic kidney diseases. Also, sonepcizumab, a monoclonal anti-S1P antibody that neutralizes extracellular S1P, and a S1P-degrading recombinant S1P lyase are promising new strategies for the treatment of glomerulonephritis. In summary, especially due to the bifunctionality of S1P, the SphK1/S1P pathway provides multiple target sites for the treatment of chronic kidney diseases.

Sphingosine 1-Phosphate Produced by Sphingosine Kinase 2 Intrinsically Controls Platelet Aggregation In Vitro and In Vivo

RATIONALE Platelets are known to play a crucial role in hemostasis. Sphingosine kinases (Sphk) 1 and 2 catalyze the conversion of sphingosine to the bioactive metabolite sphingosine 1-phosphate (S1P). Although platelets are able to secrete S1P on activation, little is known about a potential intrinsic effect of S1P on platelet function. OBJECTIVE To investigate the role of Sphk1- and Sphk2-derived S1P in the regulation of platelet function. METHODS AND RESULTS We found a 100-fold reduction in intracellular S1P levels in platelets derived from Sphk2(-/-) mutants compared with Sphk1(-/-) or wild-type mice, as analyzed by mass spectrometry. Sphk2(-/-) platelets also failed to secrete S1P on stimulation. Blood from Sphk2-deficient mice showed decreased aggregation after protease-activated receptor 4-peptide and adenosine diphosphate stimulation in vitro, as assessed by whole blood impedance aggregometry. We revealed that S1P controls platelet aggregation via the sphingosine 1-phosphate receptor 1 through modulation of protease-activated receptor 4-peptide and adenosine diphosphate-induced platelet activation. Finally, we show by intravital microscopy that defective platelet aggregation in Sphk2-deficient mice translates into reduced arterial thrombus stability in vivo. CONCLUSIONS We demonstrate that Sphk2 is the major Sphk isoform responsible for the generation of S1P in platelets and plays a pivotal intrinsic role in the control of platelet activation. Correspondingly, Sphk2-deficient mice are protected from arterial thrombosis after vascular injury, but have normal bleeding times. Targeting this pathway could therefore present a new therapeutic strategy to prevent thrombosis.

Downregulation of sphingosine 1-phosphate (S1P) receptor 1 by dexamethasone inhibits S1P-induced mesangial cell migration

Sphingosine 1-phosphate (S1P) is generated by sphingosine kinase (SK)-1 and -2 and acts mainly as an extracellular ligand at five specific receptors, denoted S1P1-5. After activation, S1P receptors regulate important processes in the progression of renal diseases, such as mesangial cell migration and survival. Previously, we showed that dexamethasone enhances SK-1 activity and S1P formation, which protected mesangial cells from stress-induced apoptosis. Here we demonstrate that dexamethasone treatment lowered S1P1 mRNA and protein expression levels in rat mesangial cells. This effect was abolished in the presence of the glucocorticoid receptor antagonist RU-486. In addition, in vivo studies showed that dexamethasone downregulated S1P1 expression in glomeruli isolated from mice treated with dexamethasone (10 mg/kg body weight). Functionally, we identified S1P1 as a key player mediating S1P-induced mesangial cell migration. We show that dexamethasone treatment significantly lowered S1P-induced migration of mesangial cells, which was again reversed in the presence of RU-486. In summary, we suggest that dexamethasone inhibits S1P-induced mesangial cell migration via downregulation of S1P1. Overall, these results demonstrate that dexamethasone has functional important effects on sphingolipid metabolism and action in renal mesangial cells.

Transforming growth factor β2 (TGF-β2)-induced connective tissue growth factor (CTGF) expression requires sphingosine 1-phosphate receptor 5 (S1P5) in human mesangial cells

Transforming growth factor β2 (TGF-β2) 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-β2 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-β downstream effectors, Smad and mitogen-activated protein kinases. Most notably, inhibition of Gi with pertussis toxin and downregulation of S1P5 by siRNA block TGF-β2-stimulated upregulation of CTGF, demonstrating that Gi coupled S1P5 is necessary for TGF-β2-triggered expression of CTGF in human mesangial cells. Overall, these findings indicate that TGF-β2 dependent upregulation of S1P5 is required for the induction of pro-fibrotic CTGF by TGF-β. Targeting S1P5 might be an attractive novel approach to treat renal fibrotic diseases.

Ligand-induced Trafficking of the Sphingosine-1-phosphate Receptor EDG-1

The endothelial-derived G-protein–coupled receptor EDG-1 is a high-affinity receptor for the bioactive lipid mediator sphingosine-1-phosphate (SPP). In the present study, we constructed the EDG-1–green fluorescent protein (GFP) chimera to examine the dynamics and subcellular localization of SPP–EDG-1 interaction. SPP binds to EDG-1–GFP and transduces intracellular signals in a manner indistinguishable from that seen with the wild-type receptor. Human embryonic kidney 293 cells stably transfected with the EDG-1–GFP cDNA expressed the receptor primarily on the plasma membrane. Exogenous SPP treatment, in a dose-dependent manner, induced receptor translocation to perinuclear vesicles with a τ1/2 of ∼15 min. The EDG-1–GFP–containing vesicles are distinct from mitochondria but colocalize in part with endocytic vesicles and lysosomes. Neither the low-affinity agonist lysophosphatidic acid nor other sphingolipids, ceramide, ceramide-1-phosphate, or sphingosylphosphorylcholine, influenced receptor trafficking. Receptor internalization was completely inhibited by truncation of the C terminus. After SPP washout, EDG-1–GFP recycles back to the plasma membrane with a τ1/2 of ∼30 min. We conclude that the high-affinity ligand SPP specifically induces the reversible trafficking of EDG-1 via the endosomal pathway and that the C-terminal intracellular domain of the receptor is critical for this process.

Sphingosine kinase 1 is critically involved in nitric oxide-mediated human endothelial cell migration and tube formation

Sphingosine kinases (SKs) convert sphingosine to sphingosine 1-phosphate (S1P), which is a bioactive lipid that regulates a variety of cellular processes including proliferation, differentiation and migration.

A novel mode of action of the putative sphingosine kinase inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKI II): induction of lysosomal sphingosine kinase 1 degradation

Sphingosine kinase 1 (SK1) is a key enzyme in the generation of sphingosine 1-phosphate (S1P) which critically regulates a variety of important cell responses such as proliferation and migration. Therefore, inhibition of SK-1 has been suggested to be an attractive approach to treat tumor growth and metastasis formation.

The sphingosine kinase 1 and S1P1 axis specifically counteracts LPS-induced IL-12p70 production in immune cells of the spleen

Sphingosine-1-phosphate (S1P) has been implicated in angiogenesis, inflammation, cancerogenesis, neurological excitability and immune regulation and is synthesized by two different sphingosine kinases (SphK). It was suggested that mice lacking the gene for SphK1 exhibit no obvious phenotype, because SphK2 compensates for its absence. However, recent investigations revealed that under challenge SphK1 contributed to pro-inflammatory processes favoring Th2 and Th17 rather than Th1-type reactions. To investigate the immune modulatory role of SphK1 as opposed to SphK2 specifically for the Th1 propagating IL-12p70 we compared WT and SphK1(-/-) splenocytes and Flt3-ligand differentiated BMCs of WT and SphK1(-/-), representing dendritic cells as major producers of IL-12p70, incubated with LPS. We determined the impact on IL-12p70 in comparison to other inflammatory cytokines, and on DC and macrophage surface marker expression, SphK mRNA, protein expression and enzymatic activity in splenocytes. Our data demonstrated that SphK1 deficiency enhanced LPS-induced IL-12p70 production although SphK2 was present. To further characterize SphK1-dependent IL-12p70 regulation we exogenously applied S1P, SEW2871 and the new potent S1P1 agonist CYM5442. Both S1P and S1P1-specific analogs fully compensated the increase of IL-12p70 production in SphK1-deficient splenocytes. The use of pertussis toxin, to block G(i)-coupled signaling downstream of S1P1, again increased IL-12p70 and neglected the compensation achieved by addition of S1P and S1P1 agonists pointing on the importance of this specific S1P-receptor. Given that, in parallel to a prominent IL-12p35 increase following LPS stimulation, LPS also enhanced SphK expression and total SphK activity, we concluded that SphK1-derived S1P acting via S1P1 is a major mechanism of this negative IL-12p70 feedback loop, which did not affect other cytokines. Moreover, our data showed that SphK2 activity failed to compensate for SphK1 deficiency. These findings clearly point to a divergent and cytokine-specific impact of immune cell SphK1 and SphK2 in chronic inflammation and cancer.

Loss of sphingosine kinase-1 in carcinoma cells increases formation of reactive oxygen species and sensitivity to doxorubicin-induced DNA damage

Sphingosine kinases (SK) catalyse the formation of sphingosine 1-phosphate, which is a key lipid mediator regulating cell responses such as proliferation, survival and migration. Here we have investigated the effect of targeted inhibition of SK-1 on cell damage and elucidated the mechanisms involved.

Targeting sphingosine kinase 1 in carcinoma cells decreases proliferation and survival by compromising PKC activity and cytokinesis

Sphingosine kinases (SK) catalyze the phosphorylation of proapoptotic sphingosine to the prosurvival factor sphingosine 1-phosphate (S1P), thereby promoting oncogenic processes. Breast (MDA-MB-231), lung (NCI-H358), and colon (HCT 116) carcinoma cells were transduced with shRNA to downregulate SK-1 expression or treated with a pharmacologic SK-1 inhibitor. The effects of SK-1 targeting were investigated by measuring the level of intracellular sphingosine, the activity of protein kinase C (PKC) and cell cycle regulators, and the mitotic index. Functional assays included measurement of cell proliferation, colony formation, apoptosis, and cell cycle analysis. Downregulation of SK-1 or its pharmacologic inhibition increased intracellular sphingosine and decreased PKC activity as shown by reduced phosphorylation of PKC substrates. In MDA-MB-231 cells this effect was most pronounced and reduced cell proliferation and colony formation, which could be mimicked using exogenous sphingosine or the PKC inhibitor RO 31-8220. SK-1 downregulation in MDA-MB-231 cells increased the number of cells with 4N and 8N DNA content, and similar effects were observed upon treatment with sphingosine or inhibitors of SK-1 or PKC. Examination of cell cycle regulators unveiled decreased cdc2 activity and expression of Chk1, which may compromise spindle checkpoint function and cytokinesis. Indeed, SK-1 kd cells entered mitosis but failed to divide, and in the presence of taxol also failed to sustain mitotic arrest, resulting in further increased endoreduplication and apoptosis. Our findings delineate an intriguing link between SK-1, PKC and components of the cell cycle machinery, which underlines the significance of SK-1 as a target for cancer therapy.

Thiazolidinedione-dependent activation of sphingosine kinase 1 causes an anti-fibrotic effect in renal mesangial cells

PPARγ agonists [thiazolidinediones (TZDs)] are known to exert anti-fibrotic effects in the kidney. In addition, we previously demonstrated that sphingosine kinase 1 (SK-1) and intracellular sphingosine-1-phosphate (S1P), by reducing the expression of connective tissue growth factor (CTGF), have a protective role in the fibrotic process.

Histamine increases sphingosine kinase-1 expression and activity in the human arterial endothelial cell line EA.hy 926 by a PKC-alpha-dependent mechanism

Sphingosine 1-phosphate (S1P) is a potent mitogenic signal generated from sphingosine by the action of sphingosine kinases (SKs). In this study, we show that in the human arterial endothelial cell line EA.hy 926 histamine induces a time-dependent upregulation of the SK-1 mRNA and protein expression which is followed by increased SK-1 activity. A similar upregulation of SK-1 is also observed with the direct protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, SK-2 activity is not affected by neither histamine nor TPA. The increased SK-1 protein expression is due to stimulated de novo synthesis since cycloheximide inhibited the delayed SK-1 protein upregulation. Moreover, the increased SK-1 mRNA expression results from an increased promoter activation by histamine and TPA. In mechanistic terms, the transcriptional upregulation of SK-1 is dependent on PKC and the extracellular signal-regulated protein kinase (ERK) cascade since staurosporine and the MEK inhibitor U0126 abolish the TPA-induced SK-1 induction. Furthermore, the histamine effect is abolished by the H1-receptor antagonist diphenhydramine, but not by the H2-receptor antagonist cimetidine. Parallel to the induction of SK-1, histamine and TPA stimulate an increased migration of endothelial cells, which is prevented by depletion of the SK-1 by small interfering RNA (siRNA). To appoint this specific cell response to a specific PKC isoenzyme, siRNA of PKC-alpha, -delta, and -epsilon were used to selectively downregulate the respective isoforms. Interestingly, only depletion of PKC-alpha leads to a complete loss of TPA- and histamine-triggered SK-1 induction and cell migration. In summary, these data show that PKC-alpha activation in endothelial cells by histamine-activated H1-receptors, or by direct PKC activators leads to a sustained upregulation of the SK-1 protein expression and activity which, in turn, is critically involved in the mechanism of endothelial cell migration.

Prolactin upregulates sphingosine kinase-1 expression and activity in the human breast cancer cell line MCF7 and triggers enhanced proliferation and migration

Sphingosine kinases (SK) catalyze the formation of sphingosine-1-phosphate (S1P) which plays a crucial role in cell growth and survival. Here, we show that prolactin (PRL) biphasically activates the SK-1, but not the SK-2 subtype, in the breast adenocarcinoma cell-line MCF7. A first peak occurs after minutes of stimulation and is followed by a second delayed activation after hours of stimulation. A similar biphasic effect on SK-1 activity is seen for 17beta-estradiol (E(2)). The delayed activation of SK-1 derives from an upregulated mRNA and protein expression and is due to increased SK-1 promoter activity and mechanistically involves STAT5 activation as well as protein kinase C and the classical mitogen-activated protein kinases. Furthermore, glucocorticoids also block both hormone-induced SK-1 expression and activity. Functionally, long-term stimulation of MCF7 cells with PRL or E(2) is well known to trigger increased cell proliferation and migration. Both hormone-induced cell responses critically involve SK-1 activation since the depletion of SK-1, but not SK-2, by siRNA transfection abolishes the hormone-induced cell proliferation and migration. In summary, our data show that PRL and E(2) cause a pronounced delayed SK-1 activation which is due to increased gene transcription, and critically determines the capability of cells to grow and move. Thus, the SK-1 may represent a novel attractive target for anti-tumor therapy.