Sphingosine Kinase 2 Inhibition and Blood Sphingosine 1-Phosphate Levels

Abstract

Sphingosine 1-phosphate (S1P) levels are significantly higher in blood and lymph than in tissues. This S1P concentration difference is necessary for proper lymphocyte egress from secondary lymphoid tissue and to maintain endothelial barrier integrity. Studies with mice lacking either sphingosine kinase (SphK) type 1 and 2 indicate that these enzymes are the sole biosynthetic source of S1P, but they play different roles in setting S1P blood levels. We have developed a set of drug-like SphK inhibitors, with differing selectivity for the two isoforms of this enzyme. Although all SphK inhibitors tested decrease S1P when applied to cultured U937 cells, only those inhibitors with a bias for SphK2 drove a substantial increase in blood S1P in mice and this rise was detectable within minutes of administration of the inhibitor. Blood S1P also increased in response to SphK2 inhibitors in rats. Mass-labeled S1P was cleared more slowly after intravenous injection into SphK2 inhibitortreated mice or mice lacking a functional SphK2 gene; thus, the increased accumulation of S1P in the blood appears to result from the decreased clearance of S1P from the blood. Therefore, SphK2 appears to have a function independent of generating S1P in cells. Our results suggest that differential SphK inhibition with a drug might afford a method to manipulate blood S1P levels in either direction while lowering tissue S1P levels.

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

“…Interestingly, application of these latter pyrrolidyl guanidine compounds to mice caused an increase of plasma S1P levels and this fits well to the situation of SK-2 knockout mice which also show enhanced plasma S1P levels [34]. The increased plasma S1P was explained with a dysregulated removal of plasma S1P that is mediated by SK-2 although mechanistically still unclear [33]. These novel series of SK-2 inhibitors will certainly need to be tested in tumor models to support the results obtained with the first generation compound ABC294640.…”

“…Very recently, more potent SK-2 inhibitors were synthesized such as K145 [32], and pyrrolidyl guanidine compounds SLC5111312 and SLM6081442 [33]. Interestingly, application of these latter pyrrolidyl guanidine compounds to mice caused an increase of plasma S1P levels and this fits well to the situation of SK-2 knockout mice which also show enhanced plasma S1P levels [34].…”

Recent advances in the development of sphingosine kinase inhibitors

“…It is worth noting that knockdown of SK1 and SK2 has in some cases been seen to result in compensatory increases in expression of the other isoform [39,54,55] adding some further complexity to the system. Furthermore, SK1 has been shown to be up-regulated in SK2 −/− mice [56] with increased S1P in circulation of these mice [49,[57][58][59] leading to increased severity of chronic intestinal inflammation and colitis-associated cancer [56]. This may mean that targeting SK2 alone could be detrimental in some inflammatory disorders and some cancers, suggesting that in some disease settings it may be beneficial to target both SK isoforms concurrently to off-set any compensation by the other isoform.…”

“…Despite these advances, the hunt for selective SK1 and SK2 inhibitors that recapitulate genetic knockdown studies has proven controversial. The findings that a number of potent SK1 inhibitors fail to induce cancer cell death despite effectively reducing cellular S1P levels [45][46][47], as well as discrepancies with SK2-selective inhibitors either increasing [48,49] or decreasing [50,51] circulating S1P levels in mice have necessitated re-evaluation of how rigorously inhibitors need to be assessed for on-and off-target effects, particularly within the sphingolipid pathway. Indeed, important off-targets have been recently reported for a number of commonly-used SK inhibitors [52,53], meaning re-evaluation of previously published work is required in light of this.…”

Signaling Microdomains within the Myoendothelial Junction

“…For S1Ps, one half of the sphingolipid extract was centrifuged at maximum speed for 10min, and the supernatant was dried down and resuspended in methanol. Both ceramides and S1Ps were analyzed by liquid chromatography-mass spectrometry following previously established methods 172 . Values were normalized to an internal standard and then to protein concentration.…”