Silencing COX-2 blocks PDK1/TRAF4-induced AKT activation to inhibit fibrogenesis during skeletal muscle atrophy.
Silencing COX-2 blocks PDK1/TRAF4-induced AKT activation to inhibit fibrogenesis during skeletal muscle atrophy.
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沉默 COX-2 可阻断 PDK1/TRAF4 诱导的 AKT 激活，从而抑制骨骼肌萎缩期间的纤维形成
DOI：
10.1016/j.redox.2020.101774
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发表时间：
2021-01
期刊：
影响因子：
11.4
通讯作者：
Cao X
中科院分区：
文献类型：
--
作者：
Chen H;Qian Z;Zhang S;Tang J;Fang L;Jiang F;Ge D;Chang J;Cao J;Yang L;Cao X
骨骼肌萎缩的高患病率可导致虚弱和疲劳，并对健康和生活质量造成巨大负担。在骨骼肌退变过程中，过量的成纤维细胞和细胞外基质（ECM）积累，取代和损害驻留的肌纤维，并导致肌肉质量的损失。环氧合酶-2（考克斯-2）是前列腺素合成的限速酶，在炎症、氧化应激等病理生理过程中起着积极的调节作用。在我们的研究中，我们发现人受试者和小鼠模型的受损肌肉与未受损区域相比过表达考克斯-2，并且在TGF-β刺激后成纤维细胞中考克斯-2也上调。观察选择性考克斯-2抑制剂塞来昔布对纤维化的影响。塞来昔布通过抑制成纤维细胞分化、增殖和迁移以及灭活TGF-β依赖性信号通路、非经典TGF-β通路和抑制活性氧（ROS）和氧化应激的产生来介导抗纤维化作用。塞来昔布对考克斯-2的体内药理学抑制可减少组织纤维化，增加骨骼肌纤维保存，表现为ECM形成和肌成纤维细胞蓄积减少，p-ERK 1/2、p-Smad 2/3、TGF-βR1、VEGF、NOX 2和NOX 4表达降低。表达谱分析进一步发现塞来昔布可抑制PDK 1表达。考克斯-2与PDK 1/AKT信号通路的相互作用尚不清楚，本研究发现考克斯-2可以与PDK 1/AKT信号通路结合形成复合物。通过药物失活或siRNA敲低成纤维细胞中的考克斯-2抑制TGF-β处理诱导的PDK 1表达和AKT磷酸化。此外，si-COX-2通过阻断AKT与E3泛素连接酶TRAF 4的相互作用，抑制TGF-β诱导的K63泛素化。综上所述，我们发现阻断考克斯-2可以抑制损伤性肌萎缩后的纤维化，并通过抑制上游PDK 1的表达和阻止TRAF 4向AKT的募集来抑制AKT信号通路，提示考克斯-2/PDK 1/AKT信号通路有望成为未来治疗肌萎缩的靶点。考克斯-2在体内人和小鼠的损伤肌肉中以及在体外用TGF-β处理的成纤维细胞中上调。药理学抑制考克斯-2可抑制TGF-β依赖性途径、非经典TGF-β途径和氧化应激。塞来昔布减弱ECM形成和组织纤维化以改善骨骼肌萎缩。考克斯-2与PDK 1和AKT相互作用。沉默考克斯-2通过抑制PDK 1表达和抑制AKT与TRAF 4之间的相互作用而使AKT失活。
Skeletal muscle atrophy with high prevalence can induce weakness and fatigability and place huge burden on both health and quality of life. During skeletal muscle degeneration, excessive fibroblasts and extracellular matrix (ECM) accumulated to replace and impair the resident muscle fiber and led to loss of muscle mass. Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in synthesis of prostaglandin, has been identified as a positive regulator in pathophysiological process like inflammation and oxidative stress. In our study, we found injured muscles of human subjects and mouse model overexpressed COX-2 compared to the non-damaged region and COX-2 was also upregulated in fibroblasts following TGF-β stimulation. Then we detected the effect of selective COX-2 inhibitor celecoxib on fibrogenesis. Celecoxib mediated anti-fibrotic effect by inhibiting fibroblast differentiation, proliferation and migration as well as inactivating TGF-β-dependent signaling pathway, non-canonical TGF-β pathways and suppressing generation of reactive oxygen species (ROS) and oxidative stress. In vivo pharmacological inhibition of COX-2 by celecoxib decreased tissue fibrosis and increased skeletal muscle fiber preservation reflected by less ECM formation and myofibroblast accumulation with decreased p-ERK1/2, p-Smad2/3, TGF-βR1, VEGF, NOX2 and NOX4 expression. Expression profiling further found that celecoxib could suppress PDK1 expression. The interaction between COX-2 and PDK1/AKT signaling remained unclear, here we found that COX-2 could bind to PDK1/AKT to form compound. Knockdown of COX-2 in fibroblasts by pharmacological inactivation or by siRNA restrained PDK1 expression and AKT phosphorylation induced by TGF-β treatment. Besides, si-COX-2 prevented TGF-β-induced K63-ubiquitination of AKT by blocking the interaction between AKT and E3 ubiquitin ligase TRAF4. In summary, we found blocking COX-2 inhibited fibrogenesis after muscle atrophy induced by injury and suppressed AKT signaling pathway by inhibiting upstream PDK1 expression and preventing the recruitment of TRAF4 to AKT, indicating that COX-2/PDK1/AKT signaling pathway promised to be target for treating muscle atrophy in the future. COX-2 is upregulated in injured muscles from humans and mice in vivo and fibroblasts treated with TGF-β in vitro. Pharmacological inhibition of COX-2 inhibited TGF-β-dependent pathway, non-canonical TGF-β pathways and oxidative stress. Celecoxib attenuated ECM formation and tissue fibrosis to ameliorate skeletal muscle atrophy. COX-2 interacted with PDK1 and AKT. Silencing COX-2 inactivated AKT by suppressing PDK1 expression and inhibiting the interaction between AKT and TRAF4.
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