MTORC1 coordinates the autophagy and apoptosis signaling in articular chondrocytes in osteoarthritic temporomandibular joint

A switch from autophagy to apoptosis is implicated in chondrocytes during the osteoarthritis (OA) progression with currently unknown mechanism(s). In this study we utilized a flow fluid shear stress (FFSS) model in cultured chondrocytes and a unilateral anterior crossbite (UAC) animal model. We found that both FFSS and UAC actively induced endoplasmic reticulum stress (ERS) in the temporomandibular joints (TMJ) chondrocytes, as demonstrated by dramatic increases in expression of HSPA5, p-EIF2AK3, p-ERN1 and ATF6. Interestingly, both FFSS and UAC activated not only pro-death p-EIF2AK3-mediated ERS-apoptosis programs but also pro-survival p-ERN1-mediated autophagic flux in chondrocytes. Data from FFSS demonstrated that MTORC1, a downstream of p-ERN1, suppressed autophagy but promoted p-EIF2AK3 mediated ERS-apoptosis. Data from UAC model demonstrated that at early 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. Inhibition of MTORC1 by TMJ local injection of rapamycin in rats or inducible ablation of MTORC1 expression 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 <i>Tsc1</i>, an upstream MTORC1 suppressor, resulted in opposite effects. Collectively, our results establish that aberrant mechanical loading causes cartilage degeneration by activating, at least in part, the MTORC1 signaling which modulates the autophagy and apoptosis programs in TMJ chondrocytes. Thus, inhibition of MTORC1 provides a novel therapeutic strategy for prevention and treatment of OA. <b>Abbreviations</b> : ACTB: actin beta; ATF6: activating transcription factor 6; BECN1: beclin 1; BFL: bafilomycin A₁; CASP12: caspase 12; CASP3: caspase 3; DAPI: 4ʹ,6-diamidino-2-phenylindole; DDIT3: DNA-damage inducible transcript 3; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERS: endoplasmic reticulum stress; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; FFSS: flow fluid shear stress; HSPA5/GRP78/BiP: heat shock protein 5; LAMP2: lysosome-associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin complex 1; OA: osteoarthritis; PRKAA1/2/AMPK1/2: protein kinase, AMP-activated, alpha 1/2 catalytic subunit; RPS6: ribosomal protein S6; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TG: thapsigargin; TMJ: temporomandibular joints; TSC1/2: tuberous sclerosis complex 1/2; UAC: unilateral anterior crossbite; UPR: unfolded protein response; XBP1: x-box binding protein 1.

骨关节炎（osteoarthritis, OA）进展过程中，软骨细胞存在从自噬向凋亡的表型转换，但其潜在分子机制目前仍未阐明。本研究分别构建了体外培养软骨细胞的流体剪切应力（flow fluid shear stress, FFSS）模型，以及单侧前牙反牙合（unilateral anterior crossbite, UAC）动物模型。
研究结果显示，流体剪切应力与单侧前牙反牙合均可显著激活颞下颌关节（temporomandibular joints, TMJ）软骨细胞的内质网应激（endoplasmic reticulum stress, ERS），具体表现为HSPA5、p-EIF2AK3、p-ERN1及ATF6的表达水平显著升高。有趣的是，二者不仅激活了促死亡的p-EIF2AK3介导的内质网应激-凋亡通路，同时也激活了促生存的p-ERN1介导的自噬流。
基于流体剪切应力模型的实验数据表明，作为p-ERN1下游靶点的MTORC1（mechanistic target of rapamycin complex 1, MTORC1）可抑制自噬，并促进p-EIF2AK3介导的内质网应激-凋亡通路。而单侧前牙反牙合动物模型的数据显示，造模早期颞下颌关节软骨细胞同时激活p-ERN1与p-EIF2AK3通路，且MTORC1活性受到抑制；造模晚期则以MTORC1-p-EIF2AK3介导的内质网应激-凋亡通路为主导，p-ERN1通路与自噬流均受到抑制。
研究者通过大鼠颞下颌关节局部注射雷帕霉素（rapamycin, Rapa）抑制MTORC1，或在小鼠软骨细胞中特异性诱导敲除MTORC1表达，均可促进软骨细胞自噬、抑制凋亡，并减轻单侧前牙反牙合诱导的颞下颌关节软骨退变。与之相反，通过颞下颌关节局部给予MHY1485激活MTORC1，或遗传敲除MTORC1上游负调控因子TSC1（tuberous sclerosis complex 1），则会产生完全相反的生物学效应。
综上，本研究结果证实，异常机械负荷可通过激活MTORC1信号通路（至少部分通过调控颞下颌关节软骨细胞的自噬与凋亡程序）引发软骨退变。因此，抑制MTORC1可为骨关节炎的预防与治疗提供全新的治疗策略。
<b>缩略语表</b>：ACTB：肌动蛋白β（actin beta）；ATF6：激活转录因子6（activating transcription factor 6）；BECN1：Beclin 1（beclin 1）；BFL：巴弗洛霉素A₁（bafilomycin A₁）；CASP12：半胱氨酸天冬氨酸蛋白酶12（caspase 12）；CASP3：半胱氨酸天冬氨酸蛋白酶3（caspase 3）；DAPI：4',6-二脒基-2-苯基吲哚（4ʹ,6-diamidino-2-phenylindole）；DDIT3：DNA损伤诱导转录因子3（DNA-damage inducible transcript 3）；EIF2AK3/PERK：真核翻译起始因子2α激酶3（eukaryotic translation initiation factor 2 alpha kinase 3, EIF2AK3/PERK）；ER：内质网（endoplasmic reticulum）；ERS：内质网应激（endoplasmic reticulum stress）；ERN1/IRE1：内质网到细胞核信号转导1（endoplasmic reticulum to nucleus signaling 1, ERN1/IRE1）；FFSS：流体剪切应力（flow fluid shear stress）；HSPA5/GRP78/BiP：热休克蛋白5（heat shock protein 5, HSPA5/GRP78/BiP）；LAMP2：溶酶体相关膜蛋白2（lysosome-associated membrane protein 2）；MAP1LC3B/LC3B：微管相关蛋白1轻链3β（microtubule associated protein 1 light chain 3 beta, MAP1LC3B/LC3B）；MTOR：雷帕霉素靶蛋白激酶（mechanistic target of rapamycin kinase）；MTORC1：哺乳动物雷帕霉素靶蛋白复合物1（mechanistic target of rapamycin complex 1, MTORC1）；OA：骨关节炎（osteoarthritis）；PRKAA1/2/AMPK1/2：AMP活化蛋白激酶α1/2催化亚基（protein kinase, AMP-activated, alpha 1/2 catalytic subunit, PRKAA1/2/AMPK1/2）；RPS6：核糖体蛋白S6（ribosomal protein S6）；Rapa：雷帕霉素（rapamycin）；SQSTM1/p62：隔离体1（sequestosome 1, SQSTM1/p62）；TEM：透射电子显微镜（transmission electron microscopy）；TG：毒胡萝卜素（thapsigargin）；TMJ：颞下颌关节（temporomandibular joints, TMJ）；TSC1/2：结节性硬化复合物1/2（tuberous sclerosis complex 1/2, TSC1/2）；UAC：单侧前牙反牙合（unilateral anterior crossbite, UAC）；UPR：未折叠蛋白反应（unfolded protein response）；XBP1：X盒结合蛋白1（x-box binding protein 1）。