Moreover, the addition of MSU to OBs transfected with green fluorescent protein tagged LC3 showed sellectchem a rapid increase of labeled vac uoles in their cytosol, as well as MSU coated with GFP Inhibitors,Modulators,Libraries tagged LC3. These results indicate that MSU in human OBs induced endogenous LC3 conversion and stimulated the process of autophagy while they were pro gressively engulfed in OBs. After our pharmacologic study that indi cated activation of signaling pathways involved in both Inhibitors,Modulators,Libraries autophagy and phagocytosis, and because giant vacuoles containing MSU appeared comparatively late versus the rapid generation of autophagosomes, was the primum movens to destroy these solid particles autophagy or phagocytosis Dynasore, a dynamin inhibitor, was used to abrogate the phagocytic pathways by blocking vesicle formation.
Interestingly, pretreatment of OBs with dynasore totally abolished the MSU induced cleavage of LC3 I into LC3 II, suggesting that phagocytosis Inhibitors,Modulators,Libraries precedes autophagy and that MSU activated autophagy directly depends on crystal phagocyt osis by OBs. MSU stimulates NLRP3 in OBs MSU microcrystals ingested by macrophages have been shown to stimulate the production of IL 1B through the NLRP3 inflammasome. Because NLRP3 is expressed by OBs, we examined next whether MSU in OBs is capable of activating the NLRP3 inflammasome. As a first step, we investigated whether IL 1B was produced by OBs in the presence of 0. 5 mg MSU 106 cells for 24 and 48 hours of culture. No extracellular IL 1B or intracellular pro IL 1B, even in the presence of 1 mM ATP, which activates NLRP 3, was detected in MSU stimulated OBs.
However, OBs ex posed to MSU increased their expression of NLRP3 protein, which Inhibitors,Modulators,Libraries peaked at 12 hours of MSU stimulation and decreased after 24 hours, as evaluated with densitom etry. Conversely, NF ��B is activated by solid particles ingested by OBs and by MSU in monocytic cells. Its activation was assessed through the kinetic phosphor ylation of its inhibitor I��B in OBs in the presence of MSU. No modification of I��B phosphorylation was detected in OBs activated by MSU, whereas TNF addition to OBs was typically Inhibitors,Modulators,Libraries associated with changes of I��B phosphorylation. Overall, these results indicate that OBs respond to MSU by a primary non conventional phagocytosis followed by a secondary autophagy, by activating NLRP3 protein without con comitant IL 1B generation, and by no signal through the NF ��B pathway.
MSU stimulated autophagy is regulated by NLRP3 Under certain conditions like bacterial infection of macrophages, another inflammasome, the selleck catalog NLRC4 Ipaf inflammasome, has been reported to downregulate autophagy independent of IL 1B production. In addition, members of the NLR protein family, like NOD1 and NOD2, are intracellular sensors that in duce autophagy independent of NF ��B.