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Analysis on the mechanisms of NF-κB/Reactive Oxigen Species-mediated survival of hematopoietic cells and its clinical application

NF-κB/活性氧介导造血细胞存活机制分析及其临床应用

基本信息

批准号：
16390278
负责人：
MATSUMURA Itaru
金额：
$ 7.81万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390278/
关键词：
NF-κB apoptosis reactive oxygen species cytokine mitochondrial membrane potential H2O2 OP9システム

项目摘要

To examine the roles for NF-κB family proteins in hematopoiesis, we first expressed dominant negative Rel/NF-κB (IκBSR), which can inhibit the function of all NF-κB family proteins, in a factor-dependent cell line, Ba/F3, in an inducible manner. Although the induced IκBSR neither affected thrombopoietin-dependent nor gp130-mediated growth of Ba/F3 cells, it suppressed interleukin-3- and erythropoietin-dependent growth at low concentrations. In addition, IκBSR enhanced factor-deprived apoptosis, which was associated with the reduction of the mitochondrial membrane potential and accumulation of reactive oxygen species (ROS). Since IκBSR-enhanced apoptosis was cancelled by ROS scavenger enzymes such as MnSOD and thioredoxin X (TRX) almost completely, ROS were supposed be involved in the IκBSR-enhanced apoptosis. When IκBSR was expressed in normal hematopoietic stem/progenitor cells, IκBSR induced apoptosis even in the presence of appropriate cytokines by accumulating ROS, which was also r … More elieved by ROS scavenger enzymes, MCI-186, N-acetyl-cysteine (NAC), and TRX. As for the mechanism of the IκBSR-induced ROS accumulation and apoptosis, semiquantitative reverse transcriptese-PCR analyses showed that the expression of ROS scavenger enzymes (MnSOD, glutathione peroxidase, and TRX) was suppressed by IκBSR. In addition, the expression of anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-XL and A1) was also suppressed. Next, we expressed IκBSR in an inducible fashion at various stages of hematopoiesis using the OP9 system, in which hematopoietic cells are induced to develop from embryonic stem cells. When IκBSR was expressed at the stage of Flk-1^+ cells (putative hemangioblasts), IκBSR inhibited the development of primitive hematopoietic progenitor cells by inducing apoptosis through the ROS accumulation. Furthermore, when IκBSR was expressed affer the development of hematopoietic progenitor cells, it inhibited their terminal differentiation toward erythrocytes, megakaryocytes, and granulocytes by inducing apoptosis through the ROS accumulation. These results indicate that NF-κB is required for preventing apoptosis at multiple steps of hematopoiesis by eliminating ROS. Less

为了研究NF-κB家族蛋白在造血中的作用，我们首先在因子依赖性细胞系Ba/F3中以可诱导的方式表达显性负性Rel/NF-κB（IκBSR），其可以抑制所有NF-κB家族蛋白的功能。虽然诱导的IκBSR既不影响血小板生成素依赖性的Ba/F3细胞的生长，也不影响gp 130介导的Ba/F3细胞的生长，但在低浓度下它抑制白细胞介素3和红细胞生成素依赖性的生长。此外，IκBSR还可促进去因子诱导的细胞凋亡，这与线粒体膜电位降低和活性氧（ROS）积累有关。由于Iκ BSR诱导的细胞凋亡几乎完全被活性氧清除酶MnSOD和硫氧还蛋白X（TRX）所抵消，推测活性氧参与了Iκ BSR诱导的细胞凋亡。当IκBSR在正常造血干/祖细胞中表达时，即使在适当的细胞因子存在下，IκBSR也通过积累ROS诱导细胞凋亡，这也与I κ BSR在正常造血干/祖细胞中的表达有关。 ...更多信息通过ROS清除酶、MCI-186、N-乙酰半胱氨酸（NAC）和TRX实现。至于Iκ BSR诱导活性氧积累和细胞凋亡的机制，半定量逆转录-PCR分析表明，IκBSR抑制活性氧清除酶（MnSOD、谷胱甘肽过氧化物酶和TRX）的表达。此外，抗凋亡Bcl-2家族成员（Bcl-2，Bcl-XL和A1）的表达也受到抑制。接下来，我们使用OP 9系统在造血的各个阶段以诱导方式表达IκBSR，其中造血细胞被诱导从胚胎干细胞发育。当IκBSR在Flk-1^+细胞（假定的成血管细胞）阶段表达时，IκBSR通过ROS积累诱导凋亡来抑制原始造血祖细胞的发育。此外，当IκBSR在造血祖细胞发育后表达时，它通过ROS积累诱导凋亡而抑制其向红细胞、巨核细胞和粒细胞的终末分化。这些结果表明，NF-κB是通过清除ROS在造血的多个步骤中防止细胞凋亡所必需的。少