Mitochondrial E3 Ubiquitin Protein Ligase 1 Mediates Cigarette Smoke-Induced Endothelial Cell Death and Dysfunction
Mitochondrial E3 Ubiquitin Protein Ligase 1 Mediates Cigarette Smoke-Induced Endothelial Cell Death and Dysfunction
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DOI：
10.1165/rcmb.2014-0377oc
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发表时间：
2016-02-01
影响因子：
6.4
通讯作者：
Lee, Yun-Song
中科院分区：
文献类型：
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作者：
Kim, Sun-Yong;Kim, Hyo Jeong;Lee, Yun-Song
鉴于Akt在血管内皮细胞（EC）存活和功能中的关键作用，吸烟诱导的Akt减少可能导致吸烟者肺内皮细胞死亡和功能障碍。Akt的负调控机制之一是k48相关的Akt泛素化和随后的蛋白酶体降解。在这里，我们评估了线粒体E3泛素蛋白连接酶1 （MUL1）的参与，最近发现它是一种新的Akt泛素E3连接酶，在吸烟诱导的Akt泛素化及其对肺EC死亡和功能障碍的贡献。在人肺微血管内皮细胞（HLMVECs）中，香烟烟雾提取物（CSE）显著提高了MUL1表达和k48相关的Akt泛素化，而Akt、p-Akt、eNOS和p-eNOS水平降低。MUL1敲除可抑制cse诱导的Akt泛素化/降解和胞质中Akt和p-Akt的减少。此外，MUL1敲除可以减轻eNOS和p-eNOS的减少，并减轻CSE暴露下EC的存活、迁移和管的形成。此外，过表达K284R Akt （mul1泛素化位点的突变体）也会产生类似的效果。暴露于CSE的hlmvec中，在共免疫沉淀和原位接近连接试验中，Akt-MUL1相互作用增加。同样，暴露于香烟烟雾3个月的大鼠肺中邻近结扎试验信号升高，在此期间Mul1水平明显升高。最后，我们发现cse介导的MUL1在HLMVECs中的诱导是由视黄酸受体相关孤儿受体a介导的。综上所述，这些数据表明，吸烟诱导的MUL1升高介导Akt泛素化/降解，可能导致肺EC死亡和功能损伤。
By virtue of the critical roles of Akt in vascular endothelial cell (EC) survival and function, cigarette smoke-induced Akt reduction may contribute to EC death and dysfunction in smokers' lungs. One of the negative Akt regulatory mechanisms is K48-linked Akt ubiquitination and subsequent proteasomal degradation. Here, we assessed the involvement of mitochondrial E3 ubiquitin protein ligase 1 (MUL1), recently revealed as a novel Akt ubiquitin E3 ligase, in cigarette smoke-induced Akt ubiquitination and its contribution to pulmonary EC death and dysfunction. In human lung microvascular ECs (HLMVECs), cigarette smoke extract (CSE) noticeably elevated MUL1 expression and K48-linked Akt ubiquitination, whereas Akt, p-Akt, eNOS, and p-eNOS levels were decreased. MUL1 knockdown suppressed CSE-induced Akt ubiquitination/degradation and cytoplasmic reductions of Akt and p-Akt. Furthermore, MUL1 knockdown attenuated reductions of eNOS and p-eNOS and alleviated EC survival, migration, and tube formation in the presence of CSE exposure. In addition, overexpression of K284R Akt, a mutant for a MUL1-ubiquitination site, produced similar effects. In HLMVECs exposed to CSE, Akt-MUL1 interaction was increased in coimmunoprecipitation and in situ proximity ligation assays. Similarly, the proximity ligation assay signals were elevated in rat lungs exposed to cigarette smoke for 3 months, during which Mul1 levels were noticeably increased. Finally, we found that CSE-mediated MUL1 induction in HLMVECs is mediated by retinoic acid receptor-related orphan receptor a. Taken together, these data suggest that cigarette smoke-induced MUL1 elevation mediates Akt ubiquitination/degradation, potentially leading to pulmonary EC death and functional impairment.