The Role And Mechanism Of NCOA4 In Ferroptosis Of Chondrocytes And The Progression Of Osteoarthritis

Aims: To investigate whether NCOA4 expression is correlated with OA development;To investigate the role of NCOA4 in chondrocyte ferroptosis and OA pathogenesis;To investigate the role of NCOA4-mediated ferritinophagy in chondrocyte ferroptosis and regulatory mechanism of NCOA4 expression;To explore whether inhibiton of JNK-JUNNCOA4 axis could protect against OA.Methods: The articular cartilage of patients with OA after knee arthroplasty and those from traumatic amputees were collected to detect the expression level of NCOA4.The expression of NCOA4 in articular cartilage was also observed in aging mice and posttraumatic OA model of mice.IL-1β was used to estanblish an OA-related inflammatory environment for chondrocytes and the expression of NCOA4 was examined in inflammatory chondrocytes.NCOA4 was knocked down or overexpressed in chondrocytes to explore the role of NCOA4 in chondrocyte ferroptosis and extracellular matrix(ECM)metabolism.The destabilization of the medial meniscus(DMM)surgery in knee joint of mouse was used to establish the post-traumatic OA model of mouse.Adenoassociated virus 9-NCOA4(AAV9-NCOA4)was intra-articularly injected to over-express NCOA4 in articular cartilage,and the effect of NCOA4 on the progression of DMM OA model was investigated.The inreraction between NCOA4 and FTH1 in chondrocytes was examined using co-immunoprecipitation(Co-IP).The impacts of NCOA4 knockdown on the expression of FTH1 and autophagic flux were investigated.The agonists or inhibitors for autophagy were used to investigate the occurance of ferritinophagy in chondrocytes.Additionally,the rescue experiment by inhibiting FTH1 was conducted to verify the role of NCOA4-mediated ferritinophagy during chondrocyte ferroptosis.The transcription factors of Ncoa4 and the binding sites were predicted through multiple databases.The binding between JUN and Ncoa4 promoter was verified by chromatin immunoprecipitation(Ch IP)-PCR and Ch IP-q PCR.Furthermore,the expression changes of NCOA4,FTH1,and ferroptosis-related protein were examined after knocking down JUN or inhibiting JNK.The rescue experiment by overexpressing NCOA4 was conducted to verify the role of NCOA4 in the effects caused by inhibiting JNK/JUN axis.The effects of the JNK-JUN signal selective inhibitor,SP600125,on the expression of NCOA4 in articular cartilage and DMM-OA model were observed.At the same time,intra-articular injection of AAV9-NCOA4 was performed to investigate whether overexpression of NCOA4 in articular cartilage could affect the protevtive effect of SP600125.Results: We found that NCOA4 was highly expressed in articular cartilage of patients with OA,cartilage of aged mice,cartilage of DMM model of mice,and IL-1β-induced chondrocytes.NCOA4 knockdown inhibited IL-1β-induced or FAC-induced chondrocyte ferroptosis and ECM degradation.Contrarily,overexpression of NCOA4 promoted IL-1β-caused effects in chondrocytes.In addition,intra-articular injection of AAV9-NCOA4 aggravated DMM-induced cartilage degeneration and osteophyte formation.Co-IP experiment showed that NCOA4 could interact with ferritin.In addition,IL-1β could induce ferritinophagy in chondrocytes.The results showed that knockdown of NCOA4 could inhibit degradation of ferritin and autophagic flux.Inhibiting FTH1 could partially reverse the effect of NCOA4 knockdown in chondrocytes.The JUN was predicted as a potential transcription factor of Ncoa4 through mutiple databases.Ch IP-PCR and Ch IPq PCR results showed that JUN could directly bind to the promoter of Ncoa4 and initial the transcription of Ncoa4.We futher found that knocking down JUN or inhibiting JNK could inhibit the expression of NCOA4 and upregulate protein level of FTH1,eventually attenuating IL-1β-induced chondrocyte ferroptosis and ECM degradation.However,overexpression of NCOA4 could partially reverse the effects caused by knocking down JUN or inhibiting JNK in chondrocytes.Inhibiting JNK-JUN axis with SP600125 could reduce the expression of NCOA4 in cartilage of DMM-induced mice and further attenuated DMM-induced cartilage degeneration with a lower Osteoarthritis Research Society International(OARSI)score and osteophyte formation.Intra-articular injection of AAV9-NCOA4 could weaken the role of SP600125 for cartilage degeneration and osteophyte formation.Conclusions: NCOA4 expression is positively correlated with OA development;NCOA4contributes to IL-1β-induced or FAC-induced chondrocyte ferroptosis and OA pathogenesis;NCOA4-mediated ferritinophagy is involved in ferroptotic process of chondrocyte and NCOA4 is upregulated in a JNK-JUN signal-dependent manner;Inhibiton of JNK-JUN-NCOA4 axis with SP600125 could protect against DMM-induced OA,suggesting that inhibition of this axis could be a potential target for OA treatment.