Calcium Signaling and Cytochrome C Release in Apoptosis

细胞凋亡中的钙信号传导和细胞色素 C 释放

• 批准号: 6613758
• 负责人: Jianjie Ma
• 金额: $ 26.06万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6613758
• 关键词: CHO cells; apoptosis; biological signal transduction; calcium; calcium channel; cell migration; cell proliferation; cytochrome c; cytoplasm; endoplasmic reticulum; epithelium; extracellular; fluorescence; green fluorescent proteins; homeostasis; intracellular; intracellular transport; mitochondria; mitochondrial membrane; neoplastic cell; ovary; prostate; prostate neoplasms; protein transport; tissue /cell culture; transcription factor; transforming growth factors

Apoptosis is a genetically controlled process which plays a major role in the development and maintenance of tissue homeostasis. Endoplasmic reticulum (ER) and mitochondria are two key cellular organelles known to actively participate in apoptosis. While perturbation in cellular Ca homeostasis (i.e. depletion of ER Ca and/or increase in cytosolic Ca) leads to apoptosis, translocation of cytochrome c from mitochondria to cytosol has also been shown to play a critical role in the apoptotic cell death pathway. A family of Bcl-2 related proteins (i.e. Bcl-xL and Bax) serves essential roles in apoptosis, but the functional effects of which on intracellular Ca signaling remain largely unknown. The experiments proposed here will test the hypothesis that ER and mitochondria communicate with each other to release cytochrome c into the cytosol with Ca serving as a messenger in the initiation of apoptosis, and the movements of intracellular Ca and Bax (or its partners) play important roles in determining the fate of the cells to undergo either apoptosis or necrosis. Aim 1 of this proposal will involve developing cultured cell lines(CHO - Chinese hamster ovary cells, and NRP- 154 - epithelial cells derived from prostate tumor tissue) that express both green fluorescent protein-tagged cytochrome c (cyt.c-GFP) and ryanodine receptor which functions as an intracellular Ca release channel. Through controlled release of Ca via activation of ryanodine receptor, and simultaneous measurement of Ca uptake into mitochondria and translocation of cyt.c-GFP from mitochondria, we will establish the spatio- temporal relationship between ER Ca movement and mitochondria cytochrome c translocation, and test the role of intracellular Ca release and extracellular Ca entry in the apoptotic and necrotic cell death pathway. A critical early event in apoptosis is associated with the redistribution of Bax from cytosol to mitochondria and/or ER membranes. Our preliminary data showed that overexpression of Bax can affect ER Ca homeostasis, and perturbation of intracellular Ca can feedback to the translocation process of Bax. Aim 2 of this proposal will probe the relationship between changes in Ca homeostasis and Bax movement in apoptosis, and investigate the cellular and molecular function of Bcl-xL and Bax in the Ca signaling process of apoptosis. The NRP-154 cells are sensitive to TGF-beta-mediated apoptosis, but are resistant to Bax overexpression-mediated apoptosis. This is in contrary to most other known cells where Bax has been described as an ubiquitous stimulator of cell death. Aim 3 of this proposal will search for putative genes that may constitute antagonistic co-factors for Bax-induced apoptosis in the NRP-154 cells, with the hope to further understand the role of Bcl-xL, Bax and Ca in TGF-beta-mediated apoptosis of prostate tissues. Our long term goal of this project is to understand the cellular and molecular mechanism of Ca signaling in apoptosis, and to provide alternative interventions in the therapy of prostate cancer.

细胞凋亡是一个基因控制的过程，在组织稳态的发展和维持中发挥着重要作用。 内质网（ER）和线粒体是已知积极参与细胞凋亡的两个关键细胞器。 虽然细胞 Ca 稳态的扰动（即 ER Ca 的消耗和/或胞质 Ca 的增加）会导致细胞凋亡，但细胞色素 c 从线粒体到细胞质的易位也已被证明在细胞凋亡途径中发挥着关键作用。 Bcl-2 相关蛋白家族（即 Bcl-xL 和 Bax）在细胞凋亡中发挥重要作用，但其对细胞内 Ca 信号转导的功能影响仍然很大程度上未知。 这里提出的实验将检验以下假设：ER 和线粒体相互通讯，将细胞色素 c 释放到胞质溶胶中，Ca 作为细胞凋亡启动的信使，细胞内 Ca 和 Bax（或其伙伴）的运动发挥重要作用在决定细胞凋亡或坏死的命运中发挥作用。 该提案的目标 1 将涉及开发表达绿色荧光蛋白标记的细胞色素 c (cyt.c-GFP) 和兰尼碱受体，作为细胞内 Ca 释放通道。 通过激活兰尼碱受体控制Ca的释放，同时测量线粒体的Ca摄取和线粒体中cyt.c-GFP的易位，我们将建立ER Ca运动和线粒体细胞色素c易位之间的时空关系，并测试细胞内 Ca 释放和细胞外 Ca 进入在细胞凋亡和坏死细胞死亡途径中的作用。 细胞凋亡中的一个关键早期事件与 Bax 从细胞质到线粒体和/或 ER 膜的重新分布有关。 我们的初步数据表明，Bax 的过度表达可以影响 ER Ca 稳态，细胞内 Ca 的扰动可以反馈到 Bax 的易位过程。 该提案的目标2将探讨细胞凋亡中Ca稳态变化与Bax运动之间的关系，并研究Bcl-xL和Bax在细胞凋亡Ca信号传导过程中的细胞和分子功能。 NRP-154 细胞对 TGF-β 介导的细胞凋亡敏感，但对 Bax 过表达介导的细胞凋亡具有抵抗力。 这与大多数其他已知细胞相反，在大多数其他已知细胞中，Bax 被描述为普遍存在的细胞死亡刺激剂。该提案的目标3将寻找可能构成Bax诱导NRP-154细胞凋亡的拮抗辅助因子的推定基因，以期进一步了解Bcl-xL、Bax和Ca在TGF-β-中的作用。介导前列腺组织的细胞凋亡。 我们该项目的长期目标是了解细胞凋亡中 Ca 信号传导的细胞和分子机制，并为前列腺癌的治疗提供替代干预措施。