LEF1 Promotes Inflammatory Responses In Mouse Condylar Chondrocytes Via Activation Of Macrophage NLRP3 Inflammasomes

Background and objective:Temporomandibular joint osteoarthritis(TMJOA)is a common disease of maxillofacial region that causes joint pain and affects oral and maxillary function,often accompanied by degenerative changes of condylar cartilage.Currently,most clinical treatments for TMJOA can be categorized as symptomatic treatments,with related molecular mechanism rarely explored.The inflammatory cytokines such as interleukin-1 beta(IL-1β)produced by macrophages have a great destructive effect on articular cartilage.The release of IL-1β from macrophages is regulated by the NLRP3 inflammasomes,whose activation is closely related to TMJOA.Lymphoid enhancer binding factor 1(LEF1)is a key transcription factor in the Wnt/β-catenin signaling pathway,which can bind β-catenin to promote the transcription of downstream target genes.The Wnt/β-catenin pathway showed changes in expression in the mouse model of osteoarthritis,and β-catenin participated in the regulation of NLRP3 inflammasomes to promote the activation of NLRP3 inflammasomes by binding to NLRP3.Current studies have shown that abnormal LEF1 expression is involved in the regulation of the pathological process of osteoarthritis,but the exact mechanism has not been fully elucidated.The purpose of this study was to explore the effects of IL-1β on mandibular condylar cartilage cells(MCCs),and the regulation of LEF1 on the activation process of NLRP3 inflammasomes in macrophages and the release of IL-1β hoping to provide a new entry point for the treatment of TMJOA.Methods:(1)The effect of IL-1β on synthesis and degradation of MCCs extracellular matrixMCCs were extracted and identified by Alcian Blue staining and COL-Ⅱimmunofluorescence staining.With MCCs treated with IL-1β for 24 h,the expression changes of COL-Ⅱ,MMP-3 and MMP-9 in MCCs were detected by qRT-PCR and Western blot,and the changes of aggrecan in extracellular matrix of MCCs were observed by Alcian Blue staining.(2)The expression of LEF1 with the activation of NLRP3 inflammasomesDatabase was used to find LEF1 expression in synovium of patients with or without TMJOA;THP-1 and BMDM extracted from mice were stimulated with LPS and ATP,qRT-PCR and western blot were used to explore the level of LEF1 expression.(3)The interaction between LEF1 and NLRP3 proteinThe interaction between LEF1 and NLRP3 was demonstrated by silver stain experiment and co-immunoprecipitation,and the interacting domain of LEF1 or NLRP3 was further explored by transfecting LEF1 and NLRP3 related plasmid in HEK293T cells.(4)The effect of LEF1 knockdown on inflammasomes activation and IL-1β releasesiRNA was used to knockdown LEF1 expression in THP-1,and after LPS+ATP were used to activate macrophages,qRT-PCR and western blot were used to detect NLRP3 inflammasomes activation related indicators,and IL-1β expression in cell supernatant was detected by ELISA.Results:(1)IL-1β promoted the degradation of extracellular matrix of primary MCCs and inhibited its synthesisCell morphology,Alcian Blue staining and COL-Ⅱ immunofluorescence showed that MCCs was successfully extracted and cultured.After IL-1β stimulation,MCCs expressed more MMP-3,MMP-9 and less COL-Ⅱ at mRNA and protein levels compared with the control group;Alcian Blue staining showed that aggrecan in extracellular matrix of MCCs decreased after IL-1β stimulation.(2)LEF1 expression increased in NLRP3 inflammasomes activated macrophagesLimited by sample size,the data obtained from GEO database could not prove that LEF1 expression was significantly increased in synovial inflammation;qRT-PCR and western blot results showed that the expression of LEF1 in THP-1 cells and mouse macrophages was significantly increased by LPS+ATP activation of inflammasomes.(3)There is interaction between LEF1 and NLRP3 proteinCo-immunoprecipitation results showed the interaction of LEF1 and NLRP3 protein in THP-1 and HEK293T cells,and the combining domains are HMG domain of LEF1 and NACHT domain of NLRP3.(4)Knockdown of LEF1 inhibited activation of NLRP3 inflammasomes and IL-1βrelease in macrophagesqRT-PCR and western blot showed that in the control group and LPS+ATP group,knockdown of LEF1 expression in THP-1 resulted in decreased cleavage of caspase-1,while mRNA and protein levels of NLRP3 and ASC and mRNA level of caspase-1 had no significant changes.The level of IL-1β protein in the supernatant of the cells was detected by ELISA,which showed that there was less IL-1β release in the LEF1 knockdown group,and the decrease of IL-1β was statistically different after activation with LPS and ATP.Conclusion:(1)IL-1β could disturb the balance of MCCs extracellular matrix synthesis and degradation,resulting in decreased synthesis and increased degradation.(2)With activation of NLRP3 inflammasomes in human THP-1 cells and mouse primary macrophages,LEF1 expression level was significantly up-regulated.(3)LEF1 could bind to NLRP3 protein.(4)Knockdown of LEF1 could lower the activation level of NLRP3 inflammasomes and reduce the release of IL-1β.In conclusion,LEF1 might promote the activation of NLRP3 inflammasomes in macrophages and increase IL-1β secretion by binding NLRP3 protein,thus promoting the degradation of extracellular matrix of condylar cartilage and inhibiting its synthesis.This study revealed that LEF1 might be a key target in the treatment of NLRP3 inflammasomes related diseases such as TMJOA,providing a new direction for improving cartilage damage in TMJOA.