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: 8613476
• 负责人: Jeanine L Van Nostrand
• 金额: $ 2.48万
• 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: 2012-03-15 至 2014-07-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8613476
• 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 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.

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以及 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凋亡来充当肿瘤抑制因子。为了了解 p53 驱动不同细胞反应的基础，我们之前进行了筛选，以确定在细胞凋亡而非细胞周期停滞期间选择性激活的 p53 靶基因。通过基因表达谱分析，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与死亡受体相互作用的蛋白质。我们对 Siva 的初步表征表明，它对于原代小脑颗粒神经元中 p53 依赖性细胞凋亡是必要且充分的。有趣的是，在我们对 p53 转录激活突变体的研究中，该突变体无法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因子，我们发现 Siva 是由这种反式激活突变体诱导的一小部分肿瘤抑制相关 p53 靶基因之一。这些发现表明 Siva 可能是 p53 肿瘤抑制的关键介质。此外，我们使用我们生成的 Siva 缺失小鼠进行的研究揭示了 Siva 在早期胚胎发育过程中的关键作用。湿婆缺陷导致胚胎致死，与胚胎外脉管系统重塑和神经管闭合缺陷相关，其表型类似于 TGF-β 途径成分敲除小鼠中所见的表型。此外，我们对 Siva 缺失胚胎的表征揭示了 TGF-β 家族受体下游 SMAD5 通路中信号传导的失调。这些发现，结合我们和其他人的观察，即 Siva 可以与 TGF-β 信号传导介质 TGF-β 激活激酶-1 (Tak1) 相互作用，表明 Siva 在 TGF-β 超家族信号传导中发挥着至关重要的作用。然而，Siva 在 TGF-β 途径中的信号传导作用对于 Siva 凋亡功能的重要性尚不清楚。此外，Siva 在 p53 细胞凋亡、G1 停滞和衰老功能以及 p53 介导的体内肿瘤抑制中的作用仍未完全了解。我们将用 Rosa-26-Cre-ER 小鼠培育的 Siva 条件敲除小鼠 (Sivafl/fl) 进行杂交，通过他莫昔芬治疗普遍删除 Siva，并评估 Siva 缺陷对已知在电离辐射后表现出 p53 依赖性细胞凋亡的放射敏感组织中细胞凋亡水平的影响。这些研究将揭示 Siva 对 p53 介导的细胞凋亡的重要作用。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，这些方法将提供对 Siva p53 下游功能的完整理解。为了研究 Siva 在肿瘤抑制中的未知作用，我们将 Siva 条件敲除小鼠与 E¿-myc 转基因小鼠杂交，后者易于发生 B 细胞淋巴瘤，并且 p53 介导的细胞凋亡是肿瘤抑制的核心。将获得胎肝造血干细胞 (HSC)，用逆转录病毒 MSCV-Cre-ER-荧光素酶转导，然后注射到经致死照射的受体小鼠中，并监测淋巴瘤的发生。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，我们将定义对 Tak1 相互作用至关重要的 Siva 结构域，并评估 Siva 的 E3 连接酶活性在泛素化 Tak1 中的作用。我们随后将 评估 Siva-Tak1 相互作用和 Siva 泛素化活性对 Siva 诱导的 E1A;p53-/- MEF 细胞凋亡的要求。这些研究将确定 Siva 在 TGF-β 信号传导中的功能对于 Siva 诱导的细胞凋亡是否重要，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。这些实验将共同阐明 Siva 在 p53 依赖性效应器功能和肿瘤抑制中的作用，并深入了解 TGF-β 信号传导在 Siva 和 p53 介导的细胞凋亡中的重要性。