Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H⁺-ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A₁ or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p < 0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p < 0.001). Importantly, the same effect was observed in ATG5 (autophagy-related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity.

雷帕霉素机制性靶点（丝氨酸/苏氨酸激酶）复合物1（MTORC1）是一类位于合成代谢与分解代谢过程关键节点的蛋白信号复合物，其活性依赖于广泛的环境信号。目前学界普遍认为，溶酶体通过产生胞内氨基酸池来促进MTORC1的激活。氨基酸可通过刺激MTORC1转位至溶酶体膜来激活该复合物，此时MTORC1会与结合于溶酶体膜的液泡型H⁺-ATPase（v-ATPase）质子泵形成超级复合物。该转位过程与MTORC1的激活均依赖功能正常的溶酶体。本研究发现，与这一公认观点相悖，在骨骺软骨细胞中，使用v-ATPase抑制剂巴弗洛霉素A₁或康卡霉素A抑制溶酶体活性，反而会强效激活MTORC1信号通路。MTORC1的活性可通过其两个经典下游靶点——核糖体蛋白S6（RPS6）与真核翻译起始因子4E结合蛋白1（EIF4EBP1）的磷酸化形式进行可视化检测。经48小时抑制剂处理后可观测到RPS6的最大磷酸化水平，其增幅高达12倍（p < 0.018）。该MTORC1激活现象在骨骼器官培养体系中进一步得到验证，且可强效促进骨骼纵向生长（p < 0.001）。值得注意的是，在自噬相关5（ATG5）缺陷的骨骼组织中也观测到了相同效应，这表明溶酶体对MTORC1的抑制作用不依赖于巨自噬途径。综上，本研究数据表明，在骨骺软骨细胞中，溶酶体以不依赖巨自噬的方式抑制MTORC1活性，且该抑制作用可能依赖于v-ATPase的功能。