The Role Of YAP1-Mediated Autophagy In Osteoarthritis Development

Objective: Chondrocytes play an important role in osteoarthritis(OA)progression,which is the most common degenerative joint disease.However,the studies of OA underlying pathological mechanisms are rare.Thus,the purpose of this study was to study the role and molecular mechanism of Yes-associated protein 1(YAP1)in regulating chondrocytes autophagy in OA,and to elucidate the relationship between the OA occurrence and chondrocytes.Methods: In this study,we investigated the role of YAP1-regulated autophagy in chondrocyte proliferation,apoptosis,and differentiation by using real time PCR,Western blot,CCK-8 assay,cell cycle and apoptosis analysis by flow cytometry,immunohistochemistry,immunofluorescence,co-immunoprecipitation and luciferase reporter gene assays.Results: To study the role of YAP1 in OA development,we constructed a mice OA model,and used articular cartilage tissues for Real time PCR,Western blot and immunohistochemistry(IHC)assays.Our results showed that YAP1 was highly expressed in articular cartilage tissues of OA mice,suggesting that YAP1 may be involved in the OA development,and inhibit YAP1 expression may control the OA development.To further observe and detect the effects of YAP1 on cell proliferation,cycle,apoptosis and differentiation in chondrocytes,we employed ATDC5 cells as experimental subjects,and then overexpressed or knocked down Yap1 by adenovirus to detect cell proliferation,cell cycle and apoptosis.Our results showed that Yap1 overexpression inhibited the cell proliferation and cycle,and promoted cell apoptosis.On the other hand,knocked down Yap1 could promote the cell proliferation and cycle,and inhibit cell apoptosis.Real time PCR and Western blot were also used to explore the effect of YAP1 on the differentiation of ATDC5 cells.It was found that Yap1 overexpression could significantly inhibit the chondrocyte differentiation-related genes Runx2,Ocn and Col-1 mRNA and proteins expression while knocking down Yap1 could increase these mRNA and proteins expression.These results indicated that YAP1,as an important OA promoter,could promote the OA development byregulating cell proliferation and apoptosis,and also relate to the chondrocyte differentiation-related genes Runx2,Ocn and Col-1 mRNA and proteins expression.To further investigate the effect of chondrocytes autophagy on the OA development,we used Western blot and Real time PCR to examine the effect of YAP1 expression on ATDC5 cells autophagy.Our results showed that overexpression Yap1 could inhibit Beclin1 and LC-3BII expression in ATDC5 cells,and knocked down Yap1 could promote LC-3BII expression in ATDC5 cells.We also found that YAP1 and Beclin1 co-localize in ATDC5 cells,and their interaction was enhanced in OA tissues by immunofluorescence and co-immunoprecipitation assays.After bioinformatics analysis of the YAP1 and Beclin1 structures,we found that YAP1 contained multiple WW domains,which were involved in the interaction of YAP1 and Beclin1.It was further found that the WW domain was associated with the inhibition of Beclin1 ubiquitination activity.These results indicated that YAP1 could inhibit the ubiquitination and degradation of Beclin1 by interacting with Beclin1,thereby inhibiting Beclin1-mediated autophagy,and finally promoting chondrocyte apoptosis and inhibiting chondrocyte proliferation and differentiation.To investigate the clinical role of YAP1 and the key role of YAP1 in the OA treatment,we examined the effects of seven OA treatment drugs(Paracetamol,Diacerein,99Tc-MDP,DSG,O3,Vitamin C and Rapamycin)on YAP1 expression.Western blot analysis showed that all of the seven drugs could inhibit the expression of YAP1 protein and promote the expression of autophagy-related proteins Beclin1 and LC-3BII.To further investigate the molecular mechanism of drug inhibition on YAP1 protein expression,ATDC5 cells were incubated with ActD for 1 hour,and treated with drugs for 10 hours.Real time PCR revealed that Paracetamol,Diacerein and 99Tc-MDP could inhibit Yap1 mRNA expression by acting on promoters,while DSG,O3,Vitamin C,and Rapamycin acting on 3'UTR.The results of luciferase reporter gene assay system showed that Paracetamol,Diacerein and 99Tc-MDP inhibited the activation of Yap1 promoter,while DSG,O3,Vitamin C and Rapamycin promoted the degradation of Yap1 3'UTR.To study the mechanism of Paracetamol,Diacerein and 99Tc-MDP on Yap1 promoter,we used bioinformatics analysis to query multiple transcription factor databases,and constructed overexpression and knockdown transcription factors plasmids and luciferase reporter gene of Yap1 promoter.By detecting the fluorescence intensity of the reporter geneof Yap1 promoter,we found that knockdown of the transcription factors AP2? and SP1 could inhibit the Yap1 promoter luciferase and the Yap1 mRNA expression.Meanwhile,overexpression of AP2? and SP1 could activate Yap1 promoter and promote Yap1 mRNA expression.Furthermore,ChIP assay showed that the transcription factors AP2? and SP1 could bind Yap1 promoter(-300,+20 bp).These results indicated that the transcription factors AP2? and SP1 could significantly regulate the Yap1 promoter activity.To further investigate the regulation of AP2? and SP1 on chondrocyte proliferation,apoptosis and differentiation,we detected the effects of AP2? and SP1 knockdown or overexpression on proliferation,apoptosis and differentiation genes expression in ATDC5 cells by using CCK-8,flow cytometry and Real time PCR.Our results showed that knockdown of AP2? and SP1 could promote cell proliferation,inhibit cell apoptosis,and up-regulate the cell differentiation genes expression.Whereas AP2? and SP1 overexpression could inhibit the cell proliferation,promote cell apoptosis and down-regulate cell differentiation genes expression.These results revealed that AP2? and SP1 play an important role in regulation of chondrocyte proliferation,apoptosis and differentiation.In addition,in order to study the mechanism of drugs DSG,O3,Vitamin C and Rapamycin on Yap1 3'UTR,we searched databases such as Targetscan,miRNAMAP and microRNA.org,and found that miRNAs such as miR-103,miR-107,miR-124 a,miR-129-3p,miR-299,miR-5624-5p,miR-33-3p and miR-6918-5p may bind to the Yap1 3'UTR.The overexpressed or knockdown-treated ATDC5 cells were detected by using fluorescein reporter gene system,Real time PCR,CCK-8 and Annexin-V/PI.The miRNAs miR-5624-5p,miR-33-3p and miR-6918-5p were found to significantly reduce the luciferase activity of Yap1 3'UTR,while their inhibitors were found to promote the luciferase activity of Yap13'UTR,significantly inhibit the proliferation,promote apoptosis and inhibit differentiation gene expression in ATDC5 cells.These results revealed the regulation of miR-5624-5p,miR-33-3p and miR-6918-5p on Yap1 mRNA expression in OA.Conclusion:(1)YAP1 is an important OA promoter.Inhibiting YAP1 expression can control the OA development.(2)YAP1 promotes the OA development and affects the chondrocytes differentiation by regulating the cell proliferation and apoptosis;and can inhibit Beclin1-mediated autophagy,promote chondrocytes apoptosis,inhibit chondrocyte proliferation and differentiation by interacting with Beclin1.(3)Seven OA treatment drugscan inhibit the YAP1 protein expression.Paracetamol,Diacerein and 99Tc-MDP act on Yap1 promoter,while DSG,O3,Vitamin C and Rapamycin acting on Yap1 3'UTR.(4)The transcription factors AP2? and SP1 and three miRNAs(miR-5624-5p,miR-33-3p and miR-6918-5p)play important role in Yap1 mRNA expression,and also will be important for drug therapy target of osteoarthritis.