Regulation of Apoptosis by Integrins

描述（由申请人提供）：整联蛋白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附着来存活，并且如果拒绝ECM附着，则进行凋亡（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。由一些但不是所有整合素激活的途径上调抗凋亡蛋白Bcl-2并减少凋亡。在筛选调节这种抗凋亡途径的cDNA时，我们分离了编码179个残基的线粒体蛋白的cDNA，我们将其命名为Bit 1。这种蛋白质似乎是以前未知的细胞凋亡途径的一部分，该途径由整合素介导的细胞附着调节。Bit 1是一种线粒体蛋白，当细胞从ECM脱离时从线粒体释放。在细胞质中与转录调节蛋白氨基末端分裂增强子（AES）形成复合物。Bitl/AES复合物是活性凋亡部分;细胞质Bitl的强制表达导致表达AES的细胞凋亡，AES导致表达Bitl的细胞凋亡。将细胞接种到纤连蛋白上减少了Bitl/AES复合物的形成，并抵消了Bitl和AES的骨坏死诱导作用。相反，转染半胱天冬酶抑制剂，或活化的H-Ras，PI 3-K或Akt，不阻断由Bit 1或AES诱导的细胞凋亡。Bcl-2对Bit 1诱导的凋亡也没有影响，但部分阻断AES诱导的凋亡，可能是因为它稳定线粒体并阻止Bit 1释放到细胞质中。在缺乏Bit 1的细胞中恢复线粒体Bit 1的表达增强了对失巢凋亡的易感性。这些结果表明，Bitl/AES途径可能是，至少部分负责整合素介导的细胞粘附的抗凋亡作用。本申请提出了鉴定调节Bitl/AES失巢凋亡的整联蛋白信号传导途径的实验，并确定Bitl/AES途径在恶性细胞中是否失调。这些研究的结果可能描绘的信号通路，可能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。

DESCRIPTION (provided by applicant): 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, and, if denied ECM attachment, 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 and reduces apoptosis. In screening for cDNAs that regulate this anti-apoptotic pathway, we isolated a cDNA that encodes a 179-residue mitochondrial protein, which we have named Bit1. This protein appears to be part of a previously unknown apoptosis pathway that is regulated by integrin-mediated cell attachment. Bit1 is a mitochondrial protein that is released from mitochondria when cells have detached from ECM. It forms a complex with the transcriptional regulator protein Amino-terminal Enhancer of Split (AES) in the cytoplasm. The Bitl/AES complex is the active apoptotic moiety; forced expression of cytoplasmic Bit1 causes apoptosis in cells that express AES, and AES causes apoptosis in cells that express Bit1. Plating cells onto fibronectin reduces Bitl/AES complex formation and counteracts the apoptosis-inducing effect of Bit1 and AES. In contrast, transfection with caspase inhibitors, or activated H-Ras, PI3-K or Akt, does not block apoptosis induced by Bit1 or AES. Bcl-2 also has no effect on apoptosis induced by Bit1, but partially blocks apoptosis induced by AES, presumably because it stabilizes mitochondria and prevents Bit1 release into the cytoplasm. Restoring the expression of mitochondrial Bit1 in cells that lack Bit1 enhances susceptibility to anoikis. These results suggest that the Bitl/AES pathway may be, at least in part responsible for the anti-apoptotic effect of integrin-mediated cell adhesion. This application proposes experiments to identify the integrin signaling pathways that regulate the Bitl/AES anoikis, and to determine whether the Bitl/AES pathway is dysregulated in malignant cells. The results of these studies may delineate a signaling pathway that could be of fundamental importance in the anchorage dependence of normal cells. Cells that become malignant may bypass this pathway in becoming anchorage independent and metastatic.