4- or 8.2-fold in CXCL4-stimulated cells, while in the same samples SphK2 (SPHK2), which is barely detectable in monocytes and macrophages, is down-regulated by 89 or 34%, respectively. S1P-degrading enzyme sphingosine-1-phosphate phosphohydrolase 2 (SGPP2) mRNA expression is rapidly up-regulated by 190-fold within 4 h of stimulation with CXCL4 and decreases thereafter (19-fold of unstimulated control), and sphingosine-1-phosphate lyase 1 (SGPL1) expression increases 1.6- Tanespimycin purchase or 1.3-fold in the presence of CXCL4 (Fig. 1, lower panels). These data clearly show that CXCL4 regulates expression of genes involved in S1P metabolism

in human monocytes. Next, we were interested in whether SphK1 is directly activated in CXCL4-stimulated monocytes. Activation of SphK1 was tested by its membrane translocation as well as by its ability to phosphorylate exogenous sphingosine in the presence of Triton X-100 14. Monocytes were stimulated for up to 30 min in the presence of 4 μM CXCL4. Subsequently, cytosol and membrane fractions were isolated and membrane fractions were tested for SphK1 by western blot analysis. As shown in Fig. 2, stimulation with CXCL4 provoked a rapid biphasic increase in membrane-bound SphK1 as well as SphK1 enzyme activity reaching

a first maximum after 30 s of stimulation. After 2 min amounts of membrane-bound SphK1 and SphK1 enzyme activity decreased again, while a second peak occurred after 10–30 min of stimulation. In summary, CXCL4 stimulates activation and membrane translocation of SphK1 in human monocytes. However, CXCL4-induced activation of Dabrafenib SphK1 is not accompanied by the release of S1P into the extracellular medium. This was evident from experiments where monocytes (1×106 cells/mL) were activated with CXCL4 (4 μM) for 30 min, 4 and 18 h and release was determined by competitive ELISA. Under these experimental conditions, S1P concentrations in supernatants of CXCL4 stimulated monocytes never reached levels of detection limit of the ELISA (about 30 nM; data not shown). To test whether SphK signaling is involved in CXCL4-induced monocyte

functions, the cells were preincubated in the presence or absence of increasing concentrations of SKI 17. Subsequently, the cells were stimulated with 4 μM CXCL4 and production of ROS was recorded for 60 min. Preincubation of the cells with SKI resulted in a significant ADAM7 and dose-dependent reduction of CXCL4-mediated respiratory burst by 73% at 1 μM SKI to 98% at 27 μM SKI (Fig. 3A). These data provided first evidence that activation of SphK is involved in the generation of ROS in CXCL4-treated monocytes. To investigate whether the same pathway is involved in the control of CXCL4-mediated protection from spontaneous apoptosis in monocytes, the cells were pretreated with inhibitors as indicated in Fig. 3A and subsequently cultured for 72 h in the presence or absence of 4 μM CXCL4. To assess the proportion of apoptotic cells, the cultured monocytes were labeled with annexin V.