Regulation of Apoptosis by Integrin

整合素介导的细胞粘附至细胞外基质（ECM）的最重要的细胞功能之一是通过介导从ECM至细胞的抗凋亡信号来促进细胞存活。大多数类型的细胞依赖于ECM附着生存;如果拒绝它，它们会发生凋亡（失巢凋亡）。恶性细胞比正常细胞更少依赖于整合素介导的存活信号。由一些但不是全部整合素激活的途径上调抗凋亡蛋白Bcl-2。在筛选调节该途径的cDNA时，我们分离了一种新蛋白质的eDNA，我们将其命名为Bit 1。Bit 1是一种线粒体蛋白，当释放到细胞质中时，会诱导细胞凋亡。酵母双杂交筛选和生化数据 揭示了Bit 1和转录调节蛋白氨基末端分裂增强子（AES）之间的相互作用。AES的强制表达也引起细胞凋亡。细胞凋亡的程度与细胞质中Bit 1-AES复合物的水平相关，表明该复合物是促凋亡因子。Bcl-2、Bcl-xL、各种半胱天冬酶抑制剂或活化的PI 3-K、Akt或H-Ras转染不阻断AES或胞质Bit 1诱导的凋亡。将细胞接种到纤连蛋白上是迄今为止唯一确定的抵消Bit 1和AES诱导骨坏死作用的治疗方法。使用替代粘合表面 抗体抑制实验表明，Bit 1/AES的抗凋亡作用受特异性整合素的调控。Bit 1/AES通路可能至少部分地负责整合素介导的细胞粘附的抗凋亡作用。本申请提出了建立Bit 1/AES途径的整合素调节并鉴定调节它的整合素的子集的实验。整合素调节研究将集中于Bit 1/AES复合物（假定的促凋亡因子）的形成。并对配合物进行了晶体学研究。最后，我们将分析在正常情况下Bit 1的体内功能， 生理学和肿瘤发生的条件Bit 1基因敲除，这是已经在手。这些研究的结果可能描绘了一个信号通路，是在正常细胞的锚定依赖性的根本重要性。变成恶性的细胞可以绕过该途径而变得不依赖于锚定和转移。

One of the most important cellular functions of integrin-mediated cell adhesion to extracellular matrix (ECM) is to promote cell survival by mediating anti-apoptotic signals from ECM to cells. Most types of cells depend on ECM attachment for survival; if denied it, they undergo apoptosis (anoikis). Malignant cells are less dependent on integrin-mediated survival signals than normal cells. A pathway activated by some, but not all, integrins up-regulates the anti-apoptotic protein Bcl-2. In screening for cDNAs that regulate this pathway, we isolated a eDNA for a novel protein, which we have named Bit1. Bit1 is a mitochondrial protein which, when released into the cytoplasm, induces apoptosis. Yeast two-hybrid screening and biochemical data revealed an interaction between Bit1 and the transcriptional regulator protein Amino-terminal Enhancer of Split (AES). Forced expression of AES also causes apoptosis. The degree of apoptosis correlates with the level of Bit1-AES complexes in the cytoplasm, suggesting that the complex is the pro-apoptotic factor. Transfection with Bcl-2, Bcl-xL, various caspase inhibitors, or activated PI3-K, Akt or H-Ras, does not block apoptosis induced by AES or cytoplasmic Bit1. Plating cells onto fibronectin is the only treatment identified so far that counteracts the apoptosis-inducing effect of Bit1 and AES. The use of alternative adhesion surfaces and antibody inhibition experiments suggests that the anti-apoptotic effect of Bit1/AES is regulated by specific integrins. The Bit1/AES pathway may be, at least in part, responsible for the anti-apoptotic effect of integrin-mediated cell adhesion. This application proposes experiments to establish the integrin-regulation of the Bit1/AES pathway and to identify the subset of integrins that regulate it, The integrin regulation studies will focus on the formation of the Bit1/AES complex, the presumed pro-apoptotic factor. A crystallographic study on the complex is also proposed. Finally, we will analyze the in vivo function of Bit1 in normal physiology and in tumorigenesis by using a conditional Bit1 gene knockout, which is already at hand. The results of these studies may delineate a signaling pathway that is of fundamental importance in the anchorage dependence of normal cells. Cells that become malignant may bypass this pathway in becoming anchorage independent and metastatic.