JNK-mediated BNIP3 Hyperphosphorylation On The Regulation Mechanism Of Mitophagy/Apoptosis In Rheumatoid Arthritis Fibroblast-like Synoviocytes Under Hypoxia And The Intervention Of Geniposide

One of the important substrates of mitochondrial oxidative phosphorylation in cellular metabolism is oxygen,which is crucial in the regulation of cell survival.Studies have demonstrated that moderate hypoxia induces hyperproliferation of cells and promotes their survival,while extreme hypoxia induces cell death.The study of cell metabolism changes under different oxygen concentrations is helpful for the in-depth understanding of the body’s adaptive regulation mechanism.Hypoxia is one of the important features of synovial microenvironment in rheumatoid arthritis（RA）and plays an important role in synovial hyperplasia.In terms of cell survival,Fibroblast like synovial cells（FLSs）are relatively affected by hypoxia.In contrast,Fibroblast-like cells from patients with rheumatoid arthritis（RA-FLSs）are particularly resistant to hypoxia-induced cell death.In the study of the mechanism of hypoxia-regulated mitophagy,it was found that,as a mitochondrial outer membrane protein,Bcl-2 and adenovirus E1B 19k Da-interacting protein 3（BNIP3）mediates the recognition of mitochondria by the autophagic isolation membrane by binding to the Autophagy membrane protein microtubule associated protein 1A/1B light chain 3（LC3）.In addition,BNIP3 is regulated by hypoxia and is also one of the key target genes of hypoxia-inducible factor-1α（HIF-1α）.Studies revealed the molecular mechanism by which BNIP3 is transcriptionally up-regulated under hypoxia to activate mitophagy.In addition to the influence of BNIP3 on the regulation of mitophagy at the transcriptional level,little is known about the mechanism of its post-translational modification regulating mitophagy.RA-FLSs showed abnormal proliferation and insufficient apoptosis in the hypoxic microenvironment of synovium.Studies have shown that mitophagy was involved in the adaptive response of cells under hypoxia.Previous studies have shown that geniposide（GE）has anti-inflammatory effects,but the effect of GE on mitophagy of RA-FLSs under hypoxia is unclear.1 OBJECTIVESThe purposes of this study were to explore the role of BNIP3 in the regulation of mitophagy in the adaptive response of OA-FLSs/RA-FLSs to hypoxia,and to reveal that hypoxia regulated BNIP3 through phospho-proteasome degradation pathway and ubiquitination modification,and the specific regulation of post-translational modification on mitophagy and its upstream regulation mechanism,and clarify the intervention effect of GE on mitophagy of RA-FLSs.2 METHODSHE staining,immunohistochemistry and Western blot were used to detect the changes of autophagy and apoptosis in joint synovium of AA rats,CCK-8,flow cytometry and Brd U were used to detect cell proliferation,AV/PI,TUNEL and Western blot were used to detect cell apoptosis,electron microscope,laser confocal microscope and Western blot were used to observe the function of mitochondrial autophagy,and ROS and JC-1 were used to detect the state of mitochondria status,and Western blot and RT-q PCR were used to detect BNIP3 and HIF-1α.Silencing of BNIP3 was achieved by si RNA technology.RA-FLSs were treated with cycloheximide（CHX）and TNF-αto observe whether BNIP3 is the product of caspase cleavage,and RA-FLSs were treated with MG-132（1μM）and CHX（5μg/m L）to detect the expression of BNIP3 The amount changes to confirm whether it can be modified by ubiquitination and its degradation rate under hypoxia.In vitro Lambda protein phosphatase experiments verified whether the phosphorylation modification resulted in the transition of the BNIP3 band.RA-FLSs were treated with phosphatase inhibitor（Okadaic Acid,OA）to observe the changes of BNIP3 expression under 21%and 2%O2 to confirm whether phosphorylation modification is involved in the regulation of BNIP3.We further confirmed that S66/T72 is the main site of BNIP3phosphorylation modification by constructing S66/T72A to block phosphorylation and S66D or S66E point mutations that mimic phosphorylation.The expression changes of BNIP3 were observed by JNK inhibitor and activator,and JNK kinase was further knocked down by si RNA to observe its effect on the phosphorylation level of BNIP3and the occurrence of mitophagy.The interaction between p-JNK and BNIP3 was detected by immunocoprecipitation.3 RESULTS3.1 Changes in the amount of mitophagy under hypoxia and the regulatory effect of BNIP3 mediated mitophagy on cell survivalWith the prolongation of modeling time,apoptosis in the synovial membrane of AA rats was gradually inhibited,and autophagy was continuously increased,and this process was accompanied by aggravation of hypoxia.After treatment with 3-MA（15mg/kg）to inhibit autophagy,the apoptosis of synovial cells in AA rats was increased,the degree of hypoxia of joint synovium was improved,and synovial hyperplasia was relieved.After 24 h of treatment with 2%O₂,the survival ability of RA-FLSs increased significantly.After ROS probe molecular staining,the fluorescence intensity and mitochondrial membrane potential did not change significantly.The apoptosis rate of RA-FLSs was low,but the mitophagy in RA-FLSs increased significantly.Treatment with 0.5%O₂for 24 h significantly inhibited the survival of RA-FLSs,significantly increased the fluorescence intensity of ROS in RA-FLSs,induced the decrease of mitochondrial membrane potential in a large number of RA-FLSs,increased the apoptosis rate,and significantly inhibited mitophagy.Under21%O_2,after 3-MA treatment inhibited the occurrence of mitophagy,the accumulation of ROS and mitochondrial membrane potential in RA-FLSs did not change significantly,and the apoptosis rate did not change significantly.After 3-MA treatment at 2%and 0.5%O₂,the accumulation of ROS in RA-FLSs increased significantly,the mitochondrial membrane potential decreased,and a large number of apoptosis of RA-FLSs were induced.After hypoxia,the survival rate of OA-FLSs decreased,while the proliferation activity of RA-FLSs further increased.Hypoxia induced an increase in apoptosis and inhibition of mitophagy in OA-FLSs,but not in RA-FLSs.Hypoxia led to a more lasting adaptive response.RA-FLSs displayed a more significant increase in the expression of genes transcriptionally regulated by HIF-1α.Interestingly,they showed higher BNIP3 expression than OA-FLSs,and showed stronger mitophagy and proliferation activities.BNIP3 si RNA experiment confirmed the potential role of BNIP3 in the survival of RA-FLSs.Inhibition of BNIP3 resulted in the decrease of cell proliferation,mitophagy and the increase of apoptosis.3.2 Regulation of BNIP3 expression by ubiquitin-protease degradation and phosphorylation under hypoxia and its mediated changes in mitophagyAfter CHX（5μg/m L）treatment,the expression level of BNIP3 in RA-FLSs was significantly decreased,and its half-life was about 6 h.MG-132（1μM）can promote the accumulation of BNIP3 under normoxia or hypoxia,suggesting that BNIP3 can be rapidly degraded by the ubiquitin-proteasome pathway.After treatment withλ-PP,multiple bands of BNIP3 were moved down to 21.5 k Da under different oxygen concentrations,and OA（0-100 n M）promoted the movement of BNIP3 bands from21.5 k Da to 30 k Da in a dose-dependent manner.It was confirmed that phosphorylation modification was the reason for the emergence of multiple bands of BNIP3.Through OA combined with CHX（5μg/m L）and MG-132（1μM）treatment,it was found that phosphorylation can significantly slow down the degradation rate of BNIP3,and the maintenance of its stability is closely related to phosphorylation modification.By constructing S66/T72A to block BNIP3 phosphorylation and S66D or S66E point mutations that mimic phosphorylation,it was found that phosphorylation of S66 can enhance the protein stability of BNIP3 and enhance BNIP3-mediated mitophagy.Knockdown of JNK not only induces a decrease in the phosphorylation level of BNIP3,but also induces a significant decrease in the expression of BNIP3 protein.JNK participates in the regulation of its ubiquitination modification and proteasome degradation pathway by phosphorylating the S66 site of BNIP3.3.3 Interventional effect of GE on mitophagy and its mechanismGE（60 mg/kg）reduced abnormal synovial hyperplasia in AA rats,reduced the expressions of LC3-II/LC3-I,Beclin-1,Bcl-XL,HIF-1α,BNIP3,and increased the expression of Bax.GE（100μM）inhibited the abnormal proliferation of RA-FLSs under hypoxia,down-regulated mitophagy,leads to ROS accumulation and reduction of mitochondrial membrane potential,induced apoptosis,and down-regulated JNK-BNIP3 pathway.4 CONCLUSIONS1 Under 2%O₂,RA-FLSs induced an increase in mitophagy to inhibit apoptosis and induce cell proliferation.Under 0.5%O₂,although the amount of mitophagy in RA-FLSs increased,the amount of apoptosis increased and the cell survival rate decreased.However,RA-FLSs maintained intracellular redox balance through mitophagy to promote cell survival under hypoxia.The mitophagy of OA-FLSs was too little to maintain the redox balance of mitochondria,resulting in apoptosis.The difference of mitophagy between OA-FLSs and RA-FLSs under hypoxia is mediated by the expression of BNIP3.2 Under hypoxia,JNK participates in the regulation of ubiquitination proteasome degradation and phosphorylation modification pathway of BNIP3 in RA-FLSs,and finally increases the occurrence of mitophagy to play a new mechanism of promoting cell survival.That is,hypoxia activates s66 site of BNIP3 phosphorylated by JNK,then enhances the stability of BNIP3,increases the occurrence of mitophagy and induces cell survival.3 In short,GE down-regulates the overactive JNK-BNIP3 signaling pathway until the occurrence of mitophagy,which leads to the accumulation of ROS and the decrease of mitochondrial membrane potential to induce increased apoptosis of RA-FLSs,and inhibits the excessive gain of RA-FLSs under hypoxia to play a role in the treatment of RA,this process may be related to the inhibition of BNIP3phosphorylation modification after down-regulation of JNK expression,and the induction of BNIP3 to undergo ubiquitin-proteasome degradation.