Cathepsin D and H2O2 stimulate degradation of thioredoxin-1 -: Implication for endothelial cell apoptosis
Cathepsin D and H2O2 stimulate degradation of thioredoxin-1 -: Implication for endothelial cell apoptosis
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DOI：
10.1074/jbc.m506985200
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发表时间：
2005-12-30
影响因子：
4.8
通讯作者：
Dimmeler, S
中科院分区：
文献类型：
--
作者：
Haendeler, J;Popp, R;Dimmeler, S
组织蛋白酶D（CatD）是一种溶酶体天冬氨酸蛋白酶，在蛋白质降解和氧化应激、细胞因子和衰老诱导的凋亡过程中起重要作用。所有这些刺激都是内皮细胞凋亡的有效诱导剂。因此，我们研究了CatD在内皮细胞凋亡中的作用，并确定了潜在的机制。用100-500 μ M H2 O2孵育12小时诱导内皮细胞凋亡。为了确定CatD的作用，我们将内皮细胞与CatD抑制剂胃酶抑素A共孵育。胃酶抑素A以及基因敲低CatD消除H2 O2诱导的细胞凋亡。相反，过度表达的CatD野生型，但不是催化失活突变体的CatD（CatDD 295 N）诱导细胞凋亡的基础条件下。为了深入了解潜在的机制，我们研究了CatD对活性氧（ROS）形成的影响。事实上，敲低CatD表达减少H2 O2诱导的ROS形成和细胞凋亡。硫氧还蛋白-1（Thioredoxin-1，Trx）是内皮细胞中最重要的氧化还原调节因子，在细胞凋亡抑制中起着重要作用。因此，我们假设CatD可以改变Trx蛋白水平，从而促进ROS和细胞凋亡的形成。孵育100 μ M H2 O2 6小时降低Trx蛋白水平，而Trx mRNA没有改变。H2 O2诱导的Trx降解被胃酶抑素A和CatD基因敲低所抑制，但不被其他蛋白酶抑制剂所抑制。用重组CatD孵育未刺激的细胞裂解物显著降低体外Trx蛋白水平，这被胃酶抑素A预孵育完全阻断。CatD的过表达减少了细胞中的Trx蛋白。此外，H2 O2孵育导致易位的Trx的溶酶体诱导细胞凋亡之前。总之，CatD通过降解Trx蛋白诱导细胞凋亡，Trx蛋白是内皮细胞中必需的抗细胞凋亡和活性氧清除蛋白。
Cathepsin D (CatD) is a lysosomal aspartic proteinase and plays an important role in the degradation of proteins and in apoptotic processes induced by oxidative stress, cytokines, and aging. All of these stimuli are potent inducers of endothelial cell apoptosis. Therefore, we investigated the role of CatD in endothelial cell apoptosis and determined the underlying mechanisms. Incubation with 100-500 mu M H2O2 for 12 h induced apoptosis in endothelial cells. To determine a role for CatD, we co-incubated endothelial cells with the CatD inhibitor pepstatin A. Pepstatin A as well as genetic knock down of CatD abolished H2O2-induced apoptosis. In contrast, overexpression of CatD wild type but not a catalytically inactive mutant of CatD (CatDD295N) induced apoptosis under basal conditions. To gain insights into the underlying mechanisms, we investigated the effect of CatD on reactive oxygen species (ROS) formation. Indeed, knocking down CatD expression reduced H2O2 induced ROS formation and apoptosis. The major redox regulator in endothelial cells is thioredoxin-1 (Trx), which plays a crucial role in apoptosis inhibition. Thus, we hypothesized that CatD may alter Trx protein levels and thereby promote formation of ROS and apoptosis. Incubation with 100 mu M H2O2 for 6 h decreased Trx protein levels, whereas Trx mRNA was not altered. H2O2-induced Trx degradation was inhibited by pepstatin A and genetic knock down of CatD but not by other protease inhibitors. Incubation of unstimulated cell lysates with recombinant CatD significantly reduced Trx protein levels in vitro, which was completely blocked by pepstatin A pre-incubation. Overexpression of CatD reduced Trx protein in cells. Moreover, H2O2 incubation led to a translocation of Trx to the lysosomes prior to the induction of apoptosis. Taken together, CatD induces apoptosis via degradation of Trx protein, which is an essential anti-apoptotic and reactive oxygen species scavenging protein in endothelial cells.