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

阐明 p53 细胞凋亡特异性靶基因 Siva 的体内作用

• 批准号: 8312445
• 负责人: Jeanine L Van Nostrand
• 金额: $ 4.22万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8312445
• 关键词: 4-Hydroxy-Tamoxifen; Apoptosis; Apoptotic; Automobile Driving; B-Lymphocytes; Binding Proteins; Biochemical; Biological; Biological Assay; Biological Models; Cancer Diagnostics; Cell Cycle Arrest; Cell Death Process; Cell physiology; Cells; Cessation of life; DNA Damage; Defect; Development; Embryo; Embryonic Development; Family; Fetal Liver; Fibroblasts; G1 Arrest; Gene Expression Profiling; Gene Targeting; Genes; Genetic; Human; Ionizing radiation; Knockout Mice; Knowledge; Luciferases; Lymphoma; Lymphomagenesis; MAP kinase kinase kinase 7; Malignant Neoplasms; Measures; Mediating; Mediator of activation protein; Monitor; Mus; Mutant Strains Mice; Mutation; Neural Tube Closure; Neurons; Pathway interactions; Phenotype; Phosphotransferases; Predisposition; Protein p53; Proteins; Role; Rosa; Signal Transduction; Small Intestines; Stem cells; Stress; TP53 gene; Tamoxifen; Techniques; Testing; Thymus Gland; Tissues; Transactivation; Transcriptional Activation; Transforming Growth Factor beta; Transgenic Mice; Tumor Suppression; Tumor Suppressor Proteins; base; cancer therapy; cell type; design; human MAP3K7 protein; in vivo; insight; mutant; protein protein interaction; receptor; research study; response; senescence; tumor; tumorigenesis; ubiquitin-protein ligase

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 Eu-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.

描述（由申请人提供）：p53是一种重要的肿瘤抑制因子，其在人类癌症中的频繁突变和p53缺失小鼠的完全渗透性肿瘤倾向证明了这一点。p53通过响应应激信号诱导细胞周期停滞或细胞凋亡而作为肿瘤抑制因子。为了了解p53驱动不同细胞反应的基础，我们先前进行了筛选，以确定p53的靶基因在凋亡过程中选择性激活，但不是细胞周期停滞。使用基因表达谱，我们确定了促凋亡基因Siva（CD 27结合蛋白），它编码一种蛋白质，以前显示与死亡受体相互作用。我们对Siva的初步研究表明，它对p53依赖的原代小脑颗粒神经元凋亡是必要的，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是这种转录激活突变体诱导的一小部分肿瘤抑制相关的p53靶基因之一，该突变体不能有效激活绝大多数已知的p53靶基因，但完全有能力作为肿瘤抑制基因。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Siva裸小鼠的研究揭示了Siva在早期胚胎发育中的关键作用。Siva缺乏导致胚胎死亡，与胚胎外血管重塑和神经管闭合缺陷相关，表型类似于TGF-β途径组分敲除小鼠中观察到的表型。此外，我们对Siva无效胚胎的表征揭示了TGF-β家族受体下游SMAD 5通路中的信号转导失调。这些发现，结合我们和其他人的观察，Siva可以与TGF-β激活激酶-1（Tak 1）相互作用，TGF-β信号传导的介质，表明Siva在TGF-β超家族信号传导中起着至关重要的作用。然而，Siva在TGF-β通路中的信号作用对Siva凋亡功能的重要性尚不清楚。此外，Siva在p53细胞凋亡、G1期阻滞和衰老功能中的作用以及在p53介导的体内肿瘤抑制中的作用仍然不完全清楚。我们将Siva条件性基因敲除小鼠（Sivafl/fl）与Rosa-26-Cre-ER小鼠杂交，通过他莫昔芬治疗普遍删除Siva，并评估Siva缺乏对已知在电离辐射后显示p53依赖性凋亡的辐射敏感组织中凋亡水平的影响。这些研究将揭示Siva对p53介导的细胞凋亡的重要性。此外，我们还将使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。总之，这些方法将提供一个完整的理解湿婆的功能下游的p53。为了研究Siva在肿瘤抑制中未知的作用，我们将Siva条件性敲除小鼠与Eu-myc转基因小鼠杂交，Eu-myc转基因小鼠容易发生B细胞淋巴瘤，其中p53介导的细胞凋亡是肿瘤抑制的核心。将衍生胎肝造血干细胞（HSC），用逆转录病毒MSCV-Cre-ER-荧光素酶转导，并注射到致死性辐照的受体小鼠中，并监测淋巴瘤发生。这些研究将揭示西瓦是否具有肿瘤抑制活性。为了阐明Siva和p53介导的细胞凋亡中Siva-Tak 1相互作用的重要性，我们将定义Siva对Tak 1相互作用至关重要的结构域，并评估Siva的E3连接酶活性在泛素化Tak 1中的作用。然后我们将 评估E1 A;p53-/- MEFs中Siva诱导凋亡对Siva-Tak 1相互作用和Siva泛素化活性的需求。这些研究将确定Siva在TGF-β信号传导中的功能是否对Siva诱导的细胞凋亡很重要，从而深入了解TGF-β信号传导在p53依赖性细胞凋亡中的作用。总之，这些实验将阐明Siva在p53依赖性效应功能和肿瘤抑制中的作用，并深入了解TGF-β信号在Siva和p53介导的细胞凋亡中的意义。