MicroRNA-145 Modulates TNF-?-driven Cartilage Matrix Degradation And The Underlying Mechanism

Objectives:Osteoarthritis(OA)is the most common degenerative joint disorder,characterized by cartilage destruction,subchondral bone remodeling,and synovial inflammation.So far,there is no effective treatment for OA until the end stage of disease necessitating joint replacement.The initiation of OA have been mainly attributed to an imbalance between the anabolism and catabolism of the articular cartilage.Pro-inflammatory cytokines(such as IL-1?,TNF-?,and IL-6)are critical mediators of cartilage dyshomeostasis.During OA pathogenesis,IL-1? is mainly associated with cartilage destruction,whereas TNF-? is implicated in driving of the inflammatory cascades.It is known that inflammation presents cascade amplification,therefore,targeting TNF-? signaling may be a powerful strategy to cure OA.However,the negative regulation of TNF-?-mediated signaling remains undefined.Micro RNAs(miRNAs)are small non-coding RNAs of 19–24 nucleotides in length and exert their regulatory functions by m RNA degradation or translational inhibition.Recent studies have identified several OA-related miRNAs,including miR-140,miR-27 b,miR-125 b,miR-146 a,and miR-30 a.However,merely targeting a single molecule among matrix-degrading enzymes(MMPs and ADAMTS)may limit their efficiency in protecting cartilage from degradation.Inversely,due to the cascade amplification,miRNAs implicated in TNF-?-mediated signaling may have much broader effects on the production of matrix-degrading enzymes and therefore targeting them should be more effective in curing OA.This study aims to screen the miRNA involved in regulating the downstream signaling pathway of TNF-? and to investigate its role in TNF-?-mediated signaling pathways and cartilage matrix degradation and the underlying mechanism.Content and Methods:Primary cultured rat chondrocytes were treated with TNF-? before analyzing miRNA expression profiles via micro-array.Expression of miR-145 in OA and normal cartilage was measured by quantitative real-time PCR(q RT-PCR).Chromatin immunoprecipitation assay(Ch IP)was used to examine the binding of p65 to the promoter region of miR-145.MiR-145 mimics or inhibitor were transfected into chondrocytes,and expression levels of matrix-degrading enzymes(MMP-3,MMP-13,and Adamts-5)induced by TNF-? were detected by q RT-PCR and Western blot.MiR-145 binding with the putative site in the MKK4 3?UTR was validated by luciferase reporter assay.The effect of miR-145 on TNF-?-induced NF-?B and MAPK activation were analyzed by Western blot.The binding of c-Jun and ATF-2 to the promoter regions of candidate genes was examined by Ch IP.Chondrocytes were infected with lentiviruses carrying MKK4 sh RNA(Len-sh MKK4)or MKK4 gene(Len-MKK4),and the effect on TNF-?-induced expression of matrix-degrading enzymes was examined by q RT-PCR and Western blot.Proteins in human and rat OA cartilage were identified via immunostaining.Experimental OA in rat was surgically induced by destabilisation of the medial meniscus(DMM),and therapeutic experiment was performed via intra-articular injection of miR-145 agomir(agomir-145),Len-sh MKK4,JNK-specific inhibitors(SP600125),p38-specific inhibitors(SB203508),or TNF-? secretion inhibitor(CC-5013).Cartilage destruction was evaluated by histopathological analysis with safranin O staining.Results:MiR-145 was sensitive to TNF-?,characterized by a rapid reduction upon TNF-? stimulation in chondrocytes and a negative correlation with TNF-? secretion during OA progression.In turn,miR-145 overexpression broadly restrained the production of TNF-?-induced matrix-degrading enzymes(MMP-3,MMP-13,Adamts-5).Mechanism studies unveiled that miR-145 inhibited TNF-?-mediated JNK and p38 activation by suppressing MKK4 phosphorylation,and subsequently reduced the nuclear accumulation of p-c-Jun and p-ATF2,eventually resulting in the alteration of catabolic genes transcription.Moreover,p-ATF2 interacted with the promoter of Mmp-13,whereas p-c-Jun bound to promoters of Mmp-3 and Adamts-5.MKK4 depletion resulted in decreased matrix-degrading enzymes production,JNK and p38 inactivation,and an inhibition of cartilage degradation in vivo.On the contrary,inhibition of miR-145,as well as enforced expression of MKK4,enhanced TNF-?-mediated signaling and worsened cartilage degradation.Intra-articular(IA)injection of agomir-145,Len-sh MKK4,SP600125,SB203508,or CC-5013 to rat with surgery-induced OA alleviated degeneration of articular cartilage.Conclusion:Altogether,we elucidate a novel regulatory mechanism by discovering that miR-145 is a crucial negative regulator of TNF-?-mediated signaling activation and cartilage matrix degradation mechanically through the MKK4-JNK/p38-c-Jun/ATF2 axis during OA pathogenesis,and demonstrate the potential utility of miR-145 and MKK4 as therapy targets for OA.