Functional analysis of a novel apoptosis-inducing gene upregulated in atherosclerotic lesions

细胞凋亡是一种程序性细胞死亡，调节着许多生理稳态。已知其失调与许多人类疾病如癌症、神经变性疾病和动脉粥样硬化有关（Science 258：468，1992）。诱导细胞凋亡有两种主要途径。一种是以Fas和肿瘤坏死因子α受体为代表的外源性或受体介导的途径。另一种是内源性途径，其中线粒体通过将细胞色素C从膜间隙释放到细胞质中发挥关键作用。在这项研究中，我们已经确定了一个新的基因上调，在动脉粥样硬化病变的载脂蛋白E缺陷小鼠，这是一个模型，动脉粥样硬化引起的高脂血症。我们比较了这些小鼠动脉粥样硬化斑块（P）和非斑块培养的血管平滑肌细胞（VSMC）的基因表达谱。我们成功克隆了这个新基因，并将其命名为Apop-1。为了阐明该基因的功能，A ...更多信息 将pop-1表达载体pcDNA 3Apop-1导入培养的VSMC中。有趣的是，引入该基因通过从线粒体释放细胞色素C诱导VSMC凋亡。转染细胞的条件培养基没有诱导凋亡的能力，表明Apop-1诱导的凋亡不是通过分泌因子介导的。我们还证明了Apop-1基因具有线粒体定位信号，并且Apop-1蛋白在Apop-1转染的细胞中实际上定位于线粒体。已知Bcl-2家族蛋白调节细胞色素C从线粒体的释放。重要的是，与Bcl-2家族成员不同，Apop-1既不具有Bcl-2同源性（BH），也不具有羧基末端跨膜（TM）结构域。此外，Bcl-2的过表达不抑制Apop-1诱导的细胞凋亡，表明Apop-1是一种新的促凋亡蛋白，从线粒体释放细胞色素C。我们现在正在产生Apop-1敲除小鼠和腺病毒基因转染系统，以分析这种新基因的功能。少

Apoptosis, a programmed cell death, regulates many physiological homeostasis. Its deregulation is known to link to many human diseases such as cancer, neurodegenerative disorders, and atherosclerosis (Science 258:468,1992). There are two major pathways for induction of apoptosis. One is the extrinsic or receptor-mediated pathway represented by Fas and tumor necrosis factor α receptor. The other is the intrinsic pathway, in which mitochondria play a crucial role by releasing cytochrome C from the inter-membrane space into the cytoplasm. In this study, we have identified a novel gene upregulated in atherosclerotic lesions in apolipoprotein E-deficient mice, which are a model of atherosclerosis caused by hyperlipidemia. We compared the gene expression profile of vascular smooth muscle cells(VSMC) cultured from atherosclerotic plaque (P) to that from non-plaque in these mice. We succeeded to clone this novel gene and designated it as Apop-1. In order to clarify the function of this gene, A … More pop-1 expression vector, pcDNA3Apop-1, was introduced into cultured VSMC. Interestingly, introduction of this gene induces apoptosis of VSMC by releasing cytochrome C from mitochondria. The conditioned medium of the transfected cells had no capability to induce apoptosis, indicating that Apop-1-induced apoptosis is not mediated through secretary factors. We also demonstrated that Apop-1 gene has a mitochondria localization signal and Apop-1 protein is actually localized in mitochondria in Apop-1-transfected cells. Bcl-2 family proteins are known to regulate the release of cytochrome C from mitochondria. Importantly unlike Bcl-2 family members, Apop-1 possesses neither Bcl-2 homology (BH) nor carboxy terminal transmembrane (TM) domains. In addition, overexpression of Bcl-2 did not suppress Apop-1-induced apoptosis, suggesting that Apop-1 is a novel pro-apoptotic protein that release cytochrome C from mitochondria. We are now generating Apop-1 knockout mice and an adenoviral gene transfection system to analyze the function of this novel gene. Less