Regulation of Apoptosis by Integrins

描述（由申请人提供）：整合素介导的细胞粘附到细胞外基质（ECM）最重要的细胞功能之一是通过介导ECM向细胞传递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附着来生存，如果拒绝ECM附着，就会发生凋亡（anoikis）。恶性细胞比正常细胞更少依赖整合素介导的生存信号。一些（但不是全部）整合素激活的途径上调抗凋亡蛋白Bcl-2并减少细胞凋亡。在筛选调节这种抗凋亡途径的cDNA时，我们分离了一个编码179个残基线粒体蛋白的cDNA，我们将其命名为Bit1。这种蛋白似乎是先前未知的细胞凋亡途径的一部分，该途径受整合素介导的细胞附着调节。Bit1是一种线粒体蛋白，当细胞与ECM分离时从线粒体中释放出来。它在细胞质中与转录调节蛋白氨基酸末端分裂增强子（AES）形成复合体。Bitl/AES复合物是活性凋亡片段；胞质Bit1的强迫表达导致AES细胞的凋亡，AES导致表达Bit1细胞的凋亡。将细胞镀在纤维连接蛋白上可减少Bit1 /AES复合物的形成，抵消Bit1和AES诱导细胞凋亡的作用。相比之下，转染caspase抑制剂或活化的H-Ras、PI3-K或Akt并不会阻止Bit1或AES诱导的细胞凋亡。Bcl-2对Bit1诱导的细胞凋亡也没有影响，但可以部分阻断AES诱导的细胞凋亡，这可能与Bcl-2稳定线粒体，阻止Bit1释放到细胞质中有关。在缺乏Bit1的细胞中恢复线粒体Bit1的表达可增强对疾病的易感性。这些结果表明，Bitl/AES通路可能至少部分负责整合素介导的细胞粘附的抗凋亡作用。本申请拟通过实验确定调节Bitl/AES信号通路的整合素信号通路，并确定Bitl/AES通路在恶性细胞中是否失调。这些研究的结果可能描绘了一个信号通路，可能是对正常细胞的锚定依赖性的根本重要性。恶性细胞可能绕过这一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.