The Mechanisms Study Of MTORC1 And Ihh/PTHrP In Degenerative Cartilage Of Temporomandibular Joint Osteoarthritis Induced By Abnormal Occlusion

Temporomandibular disorder（TMD）is a wide spread disease with several aetiologies which are increasingly being identified in both medical and dental practices.Temporomandibular joint osteoarthritis（TMJ OA）is a chronic degenerative joint disorder that destroys articular cartilage with a high socioeconomic cost in TMD.Biomechanical factors play important roles in the pathogenesis of OA.Temporomandibular joint（TMJ）,which relates biomechanically to dental occlusion,is a site frequently insulted by OA.OA is a common pathology which relates to progressive cartilage degradation.The cartilage thickness and matrix are lost through the abnormal biomechanical forces and apoptosis,autophagy and terminal differentiation of chondrocyte occurs in OA.Under severe endoplasmic reticulum stress（ERS）conditions,apoptosis is initiated which is a form of programmed cell death that represents the degradative turnover of cells within organisms.The apoptosis induced by emerging chronic or unresolved perturbations in ERS is termed as ERS pathway-apoptosis（ERS-apoptosis）.Stress pathways often sequentially elicit autophagy and apoptosis within the same cell.Autophagy inhibits the extension and blocks the induction of apoptosis by removing excessive cellular components,while apoptosis-associated caspase activation turns on the autophagy-lysosome process.MTORC1 is a sensitive target of rapamycin which is a negative regulator of autophagy and functions as an upstream suppresser.However,the modulating function of MTORC1 in apoptosis and autophagy of TMJ OA remains unknown.Therefore,it is interesting to determine whether MTORC1 plays a vital role between autophagic program and ERS-apoptosis process in the biomechanically induced OA progression.One of another main characters of OA is accelerated cellular terminal differentiation.Cells located in articular cartilage are differentiated from early to late stage.The deep zone chondrocytes are hypertrophic,from which stage the cells undergo terminal differentiation,while the superficial zone cells are proliferative.Many signaling pathways are involved in modulating chondrocyte differentiation and hypertrophy in the growth plate among which Indian hedgehog（Ihh）,and parathyroid hormone-related peptide（PTHrP）feedback loop play a key role.However,little is known about the chondrocyte biological activity and the molecular mechanisms of the biomechanically accelerated chondrocyte terminal differentiation in osteoarthritic degenerative TMJ cartilage.Recently,we have an in vivo abnormal dental occlusion model termed unilateral anterior cross（UAC）which could lead to OA-like lesions in condylar cartilage.UAC induced the apoptosis,subchondral bone resorption and upregulation of terminal differentiation accompanied with upregulation of related factors and a progressive degradation of cartilage matrix and inhibited proliferation.We also reported that fluid flow shear stress（FFSS）which simulated the abnormal biomachenical stimulation induced cartilage degradation and chondrocyte death in vitro.These in vitro and in vivo models are useful tools to facilitate the investigation of molecular mechanisms through which abnormal biomechanical forces induce chondrocyte death,autophagy and terminal differentiation and the onset of TMJ OA.Using these models,we firstly detected whether ERS-apoptosis,autophagy and terminal differentiation existed in TMJ OA and the related signaling pathways,on behalf of revealing the key role of ERS-apoptosis,autophagy and terminal differentiation on cartilage degradation.Then,we detected the location of MTORC1 in condylar chondrocyte and its regulation mechanisms under UAC/FFSS causes cartilage degeneration by modulating the autophagy and ERS-apoptosis programs in TMJ chondrocytes.At last,a study was conducted to clarify the relationship of Ihh and PTHrP signaling in modulating the enhanced chondrocyte terminal differentiation in dental stimulated TMJ osteoarthritic cartilage.A gain-and loss-of-function strategy which contained intervene experiment,tissue specific inducible genetic knockout and injection of inhibitor locally was used in UAC/FFSS model to reveal the mechanisms of MTORC1,Ihh and PTHrP signaling in the apoptosis,autophagy and terminal differentiation of TMJ OA chondrocyte.The main results:1.UAC stimulation could induce degenerative response of TMJ condylar cartilage by the loss of chondrocyte,reduced cartilage thickness and downregulation of proteoglycan.We found that both FFSS and UAC actively induced UPR in the deep zone chondrocytes,as demonstrated by dramatic increases in expression of HSPA5,EIF2AK3,ERN1 and ATF6.Extensive loading stimulation caused calcium overload and accumulation of ERS.Interestingly,UAC activated pro-death p-EIF2AK3-mediated ERS-apoptosis programs by upregulation of CASP12 and DDIT3.Furthermore,pro-survival p-ERN1-mediated autophagic flux in chondrocytes by the upregulation of BECN1 and LC3B-II were detected at early stage（<8 week）.After 8 weeks UAC stimulation,the inhibition of autophagy was revealed by the inhibition of autophagosome-lysosome fusion.Further,we found that both FFSS and UAC stimulated the deep zone chondrocytes to undergo terminal differentiation together with decreasing the chondrocyte matrix gene levels and increasing cellular differentiation markers,such as Alp、Col10、Mmp13、Opn and Runx2.However,cells in the superficial zone were robust.2.With the treatment of GSK2606414,rapamycin,et al.,data from UAC demonstrated that MTORC1 was inhibited at early stage（<8 week）,and the negative regulated autophagy was activated.At late stage（>8 week）,the upregulation of MTORC1activated the p-EIF2AK3 pathway to induce the ERS-apoptosis.At the same time,this upregulation of p-EIF2AK3 could prevent the fusion of autophagosome-lysosome（detected by the colocalization of LAMP2 and LC3B-II）.Next,we found that MTORC1,a downstream of p-ERN-1 through AMPK signaling,suppressed autophagy but promoted p-EIF2AK3 mediated ERS-apoptosis.Inhibition of MTORC1 by TMJ local injection of rapamycin in rats or inducible ablation of Mtor selectively in chondrocytes in mice promoted chondrocyte autophagy and suppressed apoptosis,and reduced TMJ cartilage loss induced by UAC.In contrast,MTORC1 activation by TMJ local administration of MHY1485 or genetic deletion of Tsc1,an upstream MTORC1 suppressor,resulted in opposite effects.3.We demonstrated that PTHrP was expressed at the superficial zone chondrocytes where as its unique receptor,PTH1R,was located at the deep zone chondrocytes.The chondrocyte terminal differentiation process was mediated by the enhanced Ihh signaling（which was also located at deep zone chondrocyte）and reducing the expression of PTH1R in the UAC/FFSS induced degenerative TMJ cartilage.The UAC-promoted chondrocytes terminal differentiation and OA-like lesions were rescued when Smo was knockout,but were enhanced when Pth1r was knockout or downregulation.These rescue and enhancing effects were verified by administration of the Ihh inhibitor or PTH1R activator in FFSS treatment.Importantly,the relieving effect of Smo knockout was attenuated when Pth1r was additionally double knockout.Conclusions:1.Collectively,mechanical stimuli by UAC/FFSS can induce the accumulation of Ca²⁺in ER and the eccurrence of chondrocyte ERS-apoptosis and autophagy in the degraded cartilage of TMJ OA.Furthermore,the chondrocyte terminal differentiation and matrix degradation focus on the deep zone condylar cartilage.2.Our results establish that autophagy and ERS-apoptosis are both involved in the progression of biomechanically induced TMJ OA lesions.At early OA stage,both the p-ERN1 and p-EIF2AK3 were activated and MTORC1 was inhibited in TMJ chondrocytes.At late stage,MTORC1-p-EIF2AK3-mediated ERS-apoptosis were predominant,while p-ERN1 and autophagic flux were inhibited.We further revealed that MTORC1 plays a role in switching the p-ERN1-mediated autophagic flux to the p-EIF2AK3-mediated ERS-apoptosis program in chondrocytes in TMJ OA progression.Thus,inhibition of MTORC1 provides a novel therapeutic strategy for prevention and treatment of OA.3.Our findings demonstrate that both Ihh and PTHrP signaling have modulatory effects on chondrocyte terminal differentiation by UAC stimulation of the TMJ.Inhibiting one of them will impact on the other;however,suppression of Ihh signaling is dependent on PTHrP signaling.This result provides novel insights into new therapeutic strategies for TMJ OA by protecting chondrocytes through promotion of the feedback loop between PTHrP and Ihh signaling.