Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) regulates endothelial nitric oxide synthase (eNOS) activity and its localization within the human vein endothelial cells (HUVEC) in culture
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) regulates endothelial nitric oxide synthase (eNOS) activity and its localization within the human vein endothelial cells (HUVEC) in culture
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DOI：
10.1002/jcb.20686
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发表时间：
2006-03-01
影响因子：
4
通讯作者：
Pandolfi, A
中科院分区：
文献类型：
--
作者：
Di Pietro, R;Mariggiò, MA;Pandolfi, A
我们最近证明，肿瘤坏死因子相关凋亡诱导配体（TRAIL）可增加培养的人脐静脉内皮细胞（HUVEC）中内皮一氧化氮合酶（eNOS）磷酸化、NOS活性和一氧化氮（NO）合成，而不诱导细胞凋亡。虽然调节 eNOS 活性的一个重要因素是其在细胞内的定位，但人们对 TRAIL 在调节细胞区室间 eNOS 运输以及参与该机制的细胞骨架中的作用知之甚少。然后，我们在 NOS 活性以及细胞骨架微管结构的特异性抑制剂存在的情况下，通过生化测定和免疫荧光显微镜进行定量和半定量评估。在我们的细胞模型中，TRAIL 处理不仅增加了 NO 水平，而且还引起荧光点从质膜到细胞内部的时间依赖性 NO 迁移。在未受刺激的细胞中，大部分 eNOS 位于细胞膜上。然而，在添加 TRAIL 后 10 分钟内，几乎所有细胞都显示 eNOS 的细胞质定位增加，与未刺激的细胞相比，eNOS 与高尔基体的共定位程度更高。这些效应与跨细胞质应力纤维的形成增加有关，而微管网络没有显着变化。相反，诺考达唑处理诱导的微管破坏和高尔基体散射抑制了 TRAIL 增加的 NOS 活性，表明在培养的 HUVEC 上，TRAIL 通过调节 eNOS 亚细胞分布影响 NO 产生的能力是由细胞骨架和高尔基复合体修饰介导的。
We have recently demonstrated that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increases endothelial nitric oxide synthase (eNOS) phosphorylation, NOS activity, and nitric oxide (NO) synthesis in cultured human umbilical vein endothelial cells (HUVEC), without inducing apoptotic cell death. Although an important factor that regulates eNOS activity is its localization within the cells, little is known about the role of TRAIL in the regulation of eNOS trafficking among cellular compartments and the cytoskeleton involvement in this machinery. Then, we did both quantitative and semi-quantitative evaluations with biochemical assays and immune fluorescence microscopy in the presence of specific inhibitors of NOS activity as well as of cytoskeletal microtubule structures. in our cellular model, TRAIL treatment not only increased NO levels but also caused a time-dependent NO migration of fluorescent spots from the plasma membrane to the inner part of the cells. In unstimulated cells, most of the eNOS was localized at the cell membranes. However, within 10 min following addition of TRAIL, nearly all the cells showed an increased cytoplasm localization of eNOS which appeared co-localized with the Golgi apparatus at a higher extent than in unstimulated cells. These effects were associated to an increased formation of trans-cytoplasm stress fibers with no significant changes of the microtubule network. Conversely, microtubule disruption and Golgi scattering induced with Nocodazole treatment inhibited TRAIL-increased NOS activity, indicating that, on cultured HUVEC, TRAIL ability to affect NO production by regulating eNOS sub-cellular distribution is mediated by cytoskeleton and Golgi complex modifications.