RANKL Mediates Cartilage Degeneration Via Activation Of NF-?B-ADAMTS5 Signaling Pathway

Objective:To identify the direct role of receptor activator of nuclear factor kappa-B ligand(RANKL)on cartilage degeneration and identify a novel therapeutic way for joint cartilage degeneration.Methods:The effects of RANKL on ATDC5 chondrocytes and bovine cartilage were investigated in vitro and ex vivo.The expressions of chondrocyte related genes and proteins were examined.In addition,the role of RANKL on chondrocyte degeneration was further investigated in vivo using a mice osteoarthritis(OA)model.In part 1:The expression of RANK and RANKL in ATDC5 cells were examined using RT-PCR.We analyzed the expression of chondrogenic differentiation markers using RT-qPCR.RANK expression was then analyzed by western blotting.Degeneration of cartilage was analyzed by H&E,safranin-O and alician blue staining and Markin score.In part 2:The mRNA expression of Col2a1,MMP9,MMP13 and ADAMTS5 was detected using RT-qPCR.The protein expression of MMP13 and ADAMTS5 were detected using Western blot.The protein expression of total IKB a and phospho IKB a were detected using western blot.Immunofluorescence was carried out to examine p65 nuclear translocation.The inhibitory effects of I?B-? inhibitors(BAY 11-7082)on RANKL-induced ADAMTS5 was detected by western blot.RANK small interfering RNA(siRNA)was used to silence RANK in ATDC5 cells.In part 3:The mouse tibia was dissected for micro-CT analysis.RT-qPCR was performed to analyze the expression of TRAP,Col2a1,and Col10a of femurs.A standard,3-step,avidin-biotin complex immnohistochemical staining protocol was used.The data were analyzed by paired t test using SPSS 17.0 software package.Results:1)The expressions of RANKL and RANK were detected in chondrocytes.The role of RANKL on cartilage was then investigated.We noticed RANKL induced chondrocytedegeneration,evidenced by reduced type ? collagen expression.Further ex vivo experiments demonstrated RANKL induced bovine cartilage degeneration evidenced by decreased SO and AB staining in RANKL treatment group as well as decreased cell number.2)Therefore,we investigated why RANKL can induce cartilage degeneration.We found RANKL can activate I?B? phosphorylation and subsequently i?B? degradation in ATDC5 cells.Consequently,p65 nuclear translation was noticed and finally activated the expression of ADAMTS5,which is detrimental to cartilage extracellular matrix.Importantly,inhibition of NF-?B signaling pathway by BAY 11-7082 or RANK silencing both rescued RANKL induced ADAMTS5 upregulation.3)Finally,the role of RANKL on cartilage degeneration was further investigated in a mice OA model.We did confirm the destructive effect of RANKL on cartilage,as Col ?downregulation and ADAMTS5 upregulation.Conclusions:Our study explored the direct role of RANKL on cartilage degeneration via NF-?B-ADAMTS5 signaling pathway.The inhibition of this pathway may provide a new strategy for TM J-OA treatment.