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OBJECTIVE To identify the role of GSK3 isoform inhibition on inflammasome activation.METHODS The NLRP3 inflammasome was activated by typical LPS/ATP and host-derived metabolites in primary mouse macrophages.The pharmacological inhibition of GSK3 isoforms on inflammasome activation was assayed by quantifying IL-1βin the supernatant,and activated caspase-1in cell lysates using highly selective inhibitors.Further molecular mechanisms were investigated by protein pulldown assay,confocal imaging using forced gene expression system and endogenous protein tagged mouse macrophages.RESULTS Pharmacological inhibition of GSK3-β,but not GSK3-αisoform suppressed NLRP3 inflammasome activation in response to ATP,urate crystal and the microbial alkaloid toxin staurosporine.GSK3-βinhibition did not inhibit melanoma 2(AIM2)inflammasome activation in response to double-stranded DNA(dsDNA)and did not affect non-canonical caspase-11 inflammasome activation.GSK3-βinhibition suppressed high glucose mediated NLRP3 inflammasome activation.Mechanistically,GSK3-βinhibition blocked NLRP3 inflammasome by preventing pro IL-1βtranscription,reducing caspase-1 activation and ASC speck formation.GSK3-βinhibition blocked NLRP3 inflammasome activation without affecting the level of reactive oxygen species(ROS)which is a crucial component in initialing inflammasome activation.Further studies revealed that GSK3-βdirectly binds to ASC by both co-forced expression and endogenous protein level.Interestingly,we found ASC can be glycosylated in response to inflammasome activation,and GSK3-βinhibition reduced ASC glycosylation.Consistently,the O-Glc NAc transferase(OGT)deficient mouse macrophages showed the significant reduction of mature IL-1βsecretion in response to NLRP3 inflammasome activation.CONCLUSION Our results demonstrate a critical role of metabolism-sensing GSK3-βpathway in mediating NLRP3 inflammasome activation,thus defining a new therapeutic target for sterile inflammation.
OBJECTIVE To identify the role of GSK3 isoform inhibition on inflammasome activation. METHODS The NLRP3 inflammasome was activated by typical LPS / ATP and host-derived metabolites in primary mouse macrophages. The pharmacological inhibition of GSK3 isoforms on inflammasome activation was assayed by quantifying IL-1βin the supernatant, and activated caspase-1 in cell lysates using highly selective inhibitors. Further molecular mechanisms were investigated by protein pulldown assay, confocal imaging using forced gene expression system and endogenous protein tagged mouse macrophages. -alpha isoform suppressed NLRP3 inflammasome activation in response to ATP, urate crystal and the microbial alkaloid toxin staurosporine. GSK3-ßinhibition did not inhibit melanoma 2 (AIM2) inflammasome activation in response to double-stranded DNA (dsDNA) and did not affect non-canonical caspase-11 inflammasome activation. GSK3-β inhibitor inhibits high glucose medi ated NLRP3 inflammasome activation. Mechanistically, GSK3-ßinhibition blocked NLRP3 inflammasome by preventing pro-IL-1β transcription, reducing caspase-1 activation and ASC speck formation. GSK3-ßinhibition blocked NLRP3 inflammasome activation without affecting the level of reactive oxygen species (ROS) which is a crucial component in initializing inflammasome activation. Facurther studies revealed that GSK3- [beta] directly binds to ASC by both co-forced expression and endogenous protein level. Rapid found, we found ASC can be glycosylated in response to inflammasome activation, and GSK3- [beta] inhibition reduced ASC glycosylation .Consistently, the O-Glc NAc transferase (OGT) deficient mouse macrophages showed the significant reduction of mature IL-1β secretion in response to NLRP3 inflammasome activation. CONCLUSION Our results demonstrate a critical role of metabolism-sensing GSK3-βpathway in mediating NLRP3 inflammasome activation , thus defining a new therapeutic target for sterile inflammation.