Elucidating the in vivo Role of the p53 Apoptosis-Specific Target Gene Siva

描述(由申请人提供)：P53是一种关键的肿瘤抑制基因，其在人类癌症中的频繁突变和P53基因缺失小鼠的肿瘤易感性证明了这一点。在应激反应中，P53通过诱导细胞周期停滞或细胞凋亡而发挥肿瘤抑制作用。为了了解P53驱动不同细胞反应的基础，我们先前进行了一项筛选，以确定在细胞凋亡期间选择性激活的P53靶基因，而不是细胞周期停滞。利用基因表达谱，我们鉴定了促凋亡基因SIVA(CD27结合蛋白)，它编码一种先前显示的与死亡受体相互作用的蛋白质。我们对SIVA的初步研究表明，SIVA对于依赖于P53的原代小脑颗粒神经元的凋亡是必要的，也是充分的。有趣的是，在我们对P53转录激活突变体的研究中，我们发现SIVA是该反式激活突变体诱导的一小部分与肿瘤抑制相关的P53靶基因之一。该突变体不能有效地激活绝大多数已知的P53靶基因，但完全可以作为肿瘤抑制因子。这些发现表明，SIVA可能是P53-肿瘤抑制的关键介质。此外，我们使用我们培育的湿疹病毒缺失小鼠进行的研究揭示了湿疹病毒在早期胚胎发育中的关键作用。SIVA缺乏导致胚胎死亡，与胚胎外血管重建和神经管关闭缺陷相关，这些表型与转化生长因子-β途径成分敲除小鼠中看到的表型相似。此外，我们对SIVA缺失型胚胎的特征表明，在转化生长因子-β家族受体下游的Smad5通路中，信号的调节被解除。这些发现，结合我们和其他人的观察，表明SIVA可以与转化生长因子-β信号转导的介体--转化生长因子-β激活激酶-1(Tak1)相互作用，提示SIVA在转化生长因子-β超家族信号转导中起着至关重要的作用。然而，SIVA在转化生长因子-β途径中对SIVA凋亡功能的信号作用的重要性尚不清楚。此外，SIVA在P53细胞的凋亡、G1期停滞和衰老中的作用以及在体内P53介导的肿瘤抑制中的作用仍不完全清楚。我们将用我们用ROSA-26-CRE-ER小鼠产生的SIVA条件基因敲除小鼠(Sivafl/fl)通过他莫昔芬治疗来普遍删除SIVA，并评估SIVA缺乏对辐射敏感组织中细胞凋亡水平的影响，这些组织已知在电离辐射后表现出P53依赖的细胞凋亡。这些研究将揭示SIVA在P53介导的细胞凋亡中起重要作用的背景。此外，我们还将利用小鼠胚胎成纤维细胞模型系统评估SIVA在细胞周期停滞和衰老的P53细胞功能中的未知作用。综上所述，这些方法将提供对SIVA在p53下游的功能的完整了解。为了研究SIVA在肿瘤抑制中的未知作用，我们将把SIVA条件基因敲除小鼠转移到E？myc转基因小鼠，这些小鼠容易发生B细胞淋巴瘤，其中P53介导的细胞凋亡是肿瘤抑制的中心。胎肝造血干细胞(HSCs)将被提取，用逆转录病毒MSCV-CRE-ER-荧光素酶转导，并注射到致死性照射的受体小鼠中，并将监测淋巴瘤的发生。这些研究将揭示SIVA是否具有肿瘤抑制活性。为了阐明SIVA-Tak1相互作用在SIVA和P53介导的细胞凋亡中的重要性，我们将定义SIVA对Tak1相互作用的关键结构域，并评估SIVA的E3连接酶活性在泛素化Tak1中的作用。到时候我们会的 评估SIVA-Tak1相互作用和SIVA泛素化活性对SIVA诱导E1a；P53-/-MEF凋亡的需求。这些研究将确定SIVA在转化生长因子-β信号转导中的作用是否在SIVA诱导的细胞凋亡中起重要作用，从而为深入了解转化生长因子-β信号转导在p53依赖的细胞凋亡中的作用提供依据。总之，这些实验将阐明SIVA在P53依赖的效应器功能和肿瘤抑制中的作用，并提供对SIVA和P53介导的细胞凋亡中转化生长因子-β信号的意义的洞察。

DESCRIPTION (provided by applicant): p53 is a critical tumor suppressor, as evidenced by its frequent mutation in human cancers and the completely penetrant tumor predisposition of p53 null mice. p53 acts as a tumor suppressor by inducing cell cycle arrest or apoptosis in response to stress signals. To understand the basis for p53 driving different cellular responses, we previously conducted a screen to identify target genes of p53 activated selectively during apoptosis but not cell cycle arrest. Using gene expression profiling, we identified the pro-apoptotic gene Siva (CD27-binding protein), which encodes a protein shown previously to interact with death receptors. Our initial characterization of Siva showed that it is both necessar and sufficient for p53-dependent apoptosis in primary cerebellar granular neurons. Interestingly, in our studies of a p53 transcriptional activation mutant that is unable to efficiently activate th vast majority of known p53 target genes, yet is completely competent as a tumor suppressor, we found that Siva is one of the small set of tumor-suppression-associated p53 target genes induced by this transactivation mutant. These findings suggest Siva could be a critical mediator of p53-tumor suppression. In addition, our studies using Siva null mice we generated have revealed a critical role for Siva during early embryonic development. Siva-deficiency results in embryonic lethality associated with defects in extra-embryonic vasculature remodeling and neural tube closure, phenotypes that resemble those seen in TGF-beta pathway component knockout mice. Moreover, our characterization of Siva-null embryos revealed a deregulation of signaling in the SMAD5 pathway downstream of TGF-beta family receptors. These findings, combined with the observation of ours and others that Siva can interact with TGF-Beta Activating Kinase-1 (Tak1), a mediator of TGF-beta signaling, suggest a vital role for Siva in TGF-beta superfamily signaling. However, the importance of Siva's signaling role in the TGF-beta pathway for Siva apoptotic function is unclear. Moreover, Siva's role in the p53 cellular functions of apoptosis, G1 arrest and senescence and in p53- mediated tumor suppression in vivo remain incompletely understood. We will cross Siva conditional knockout mice (Sivafl/fl) that we generated with Rosa-26-Cre-ER mice to ubiquitously delete Siva through tamoxifen treatment and assess the effect of Siva-deficiency on apoptosis levels in radiosensitive tissues known to display p53-dependent apoptosis following ionizing radiation. These studies will reveal the contexts in which Siva is important for p53-mediated apoptosis. In addition, we will also evaluate the as yet unknown role of Siva in the p53 cellular functions of cell cycle arrest and senescence using mouse embryonic fibroblast model systems. Together, these approaches will provide a complete understanding of Siva's function downstream of p53. To investigate the unknown role of Siva in tumor suppression, we will cross the Siva conditional knockout mice to E¿-myc transgenic mice, which are prone to B-cell lymphomagenesis and in which p53-mediated apoptosis is central for tumor suppression. Fetal liver hematopoetic stem cells (HSCs) will be derived, transduced with retroviral MSCV-Cre-ER-Luciferase, and injected into lethally irradiated recipient mice and lymphomagenesis will be monitored. These studies will reveal whether Siva has tumor suppressor activity. To elucidate the importance of the Siva-Tak1 interaction in Siva- and p53-mediated apoptosis, we will define domains of Siva critical for Tak1 interaction and evaluate the role of Siva's E3 ligase activity in ubiquitylating Tak1. We will then assess the requirement for the Siva-Tak1 interaction and Siva ubiquitylation activity for Siva-induced apoptosis in E1A;p53-/- MEFs. These studies will determine whether Siva's function in TGF-beta signaling is important for Siva-induced apoptosis and thus provide insight into the role of TGF-beta signaling in p53-dependent apoptosis. Together, these experiments will elucidate the role of Siva in p53-dependent effector functions and tumor suppression as well as providing insight into the significance of TGF-beta signaling in Siva- and p53-mediated apoptosis.