Moreover, in 2003 Allan et al. described that inhibition of caspase-9 is mediated through phosphorylation by Erk 36. Our observation that inhibition of either Erk 3 or SphK (Fig. 3) each results in strongly reduced cytokine release from CXCL4-stimulated monocytes, extent corresponding findings for TNF-activated monocytes
and C5a-treated macrophages 15, 16. Having in mind that in CXCL4-treated monocytes cytokine release as well as rescue from apoptosis are regulated by SphK1 as well as Erk the question arise whether these molecules might regulate each other. By investigating this possibility C59 wnt research buy in more detail, we could clearly demonstrate that Erk phosphorylation is totally blocked in SKI-treated monocytes (Fig. 5), indicating that Erk is located downstream of SphK. Finally, the finding that high dosages of S1P reduce apoptosis rates in monocytes might be related to its ability to induce Erk phosphorylation (Fig. 6D). The activation of Erk by SphK or S1P has been reported by others too. Monick et al. as well as Chandru and Boggaram demonstrated that in lung epithelial cells treatment with S1P leads to the phosphorylation of Erk 37, 38. According to our results, S1P mediates
only a partial but significant reduction of apoptosis (compared with unstimulated cells; Fig. 6A), which might be the result of a shorter duration of Erk activation as compared with the more prolonged Erk phosphorylation induced by CXCL4 (Fig. 6D). Taken Selleck RAD001 together, we identified SphK1 as an essential signaling element in CXCL4-induced monocyte activation. By several lines of evidence
we could ASK1 demonstrate that CXCL4-mediated ROS formation, as well as cytokine/chemokine expression, and rescue from apoptosis depends on SphK1 activity. Furthermore, our data indicate that the protective effect of CXCL4 on monocyte survival involves sequential activation of SphK and Erk resulting in an inhibition of caspase activation. Further studies will address the question by which mechanisms CXCL4 regulates SphK activity and whether SphK/S1P is involved in CXCL4-induced monocyte differentiation. Human natural CXCL4 was purified in our laboratory from release supernatants of thrombin-stimulated platelets, as described previously 39. Antibodies directed against Erk1 p44 (serum K-23; cross-reactive to Erk2 p42), and phospho-Erk (clone E-4) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany), while anti-SphK1 antiserum was purchased from ABGENT (Bioggio-Lugano, Switzerland). Alexa680-conjugated goat anti-mouse IgG was obtained from MoBiTec (Göttingen, Germany), and IRDye800-conjugated goat anti-rabbit IgG was from Biotrend (Köln, Germany). Inhibitors directed against SphK (SKI; 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole 17, or DMS), MEK/Erk (PD098059; 2′-amino-3′-methoxyflavone), Gi proteins (PTX), and D-erythro-S1P were purchased from Calbiochem (Schwalbach, Germany).