cytoskeletons. Therefore, the effects of cytochalasin D, an inhibitor selleck of actin polymerization, and colchicine, an inhibitor of microtubule polymerization, were Inhibitors,Modulators,Libraries examined on MSU internalization by OBs. Cytochalasin D pretreatment abrogated the formation of vacuoles as sociated with MSU phagocytosis. In contrast, colchicine did not inhibit the appearance of vacuoles containing MSU. Mechanisms underlying phagocytosis also impli cate several intracellular signaling pathways that lead to cytoskeleton reorganization and ingestion of particles. From that point of view, pharmacological inhibitors can help decipher signaling pathways associated with MSU phagocytosis by OBs. The phosphoinositide 3 kinases that control cytoskeleton dynamics, signal trans duction, and membrane trafficking were targeted by two pan PI3K inhibitors, wortmannin and LY294002.
Both inhibitors reduced by 50% the vacuole formation process, suggesting a role of PI3K in the internalization of MSU by OBs. Protein kinase C can also be in volved in the transduction of phagocytic signals. The inhibitor of pan PKC isoforms GF109203X and the inhibitor of classic type Inhibitors,Modulators,Libraries PKC isoforms G6976 Phosphorylation levels after 1 hour of MSU stimulation were higher than those recorded at 5 and 20 minutes. Thus, a 1 hour MSU stimulation of OBs was associated with a phosphorylation increase of p38 by 86% and ERK 1 2 by 94%, whereas the phosphorylation of Src kinases tended to be inhibited, Yes, Hck, Fyn or unchanged, Lck. Additionally, phosphorylation of the serine threonine protein kinases TOR and p70S6K was decreased by the presence of MSU.
Pharmacologic modulation of phagocytosis Considering Inhibitors,Modulators,Libraries these results on signaling pathways suggest ing that MSU modulated the phosphorylation status of various kinases, the investigation was pursued to deter mine the role in OBs of those kinases that are known to be implicated Inhibitors,Modulators,Libraries in phagocytosis, a dynamic mechanism of endocytizing particles. The engulfment of large particles is governed by the microfilament and microtubule were found to reduce by approximately 60% and 70% MSU vacuole formation, respectively, thereby support ing an involvement of PKC in this process. The extracel lular kinase inhibitor PD98069 reduced by 44% the MSU induced formation of vacuoles, confirm ing an implication of these MAPK in the process of vacuole formation by OBs.
As Syk tyrosine kinases have been shown Inhibitors,Modulators,Libraries to control phagocytosis, the Syk in hibitor piceatannol was tested on MSU activated OBs. Piceatannol reduced the MSU induced formation of vac uoles by 58%, indicating an involvement of Syk kinases in this process. Surprisingly, the inhibition of Src kinases by PP2 failed to modulate the MSU induced formation of vacuoles, whereas PP2 completely inhibited Src ki nases in MSU activated neutrophils. Conversely, OB preincubated with the p38 MAPK in hibitor SB203580 research only exhibited a twofold increase of MSU induced vacuole formation.