NON-APOPTOTIC ACTION OF CASPASES IN PLURIPOTENT EMBRYONIC STEM CELLS

半胱天冬酶在多能胚胎干细胞中的非凋亡作用

• 批准号: 8073463
• 负责人: Thomas P. Zwaka
• 金额: $ 30.09万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8073463
• 关键词: Address; Affect; Apoptosis; Apoptotic; Caspase; Cell Death; Cells; Cleaved cell; Clinical; Development; Developmental Biology; Disease; Enzymes; Goals; Human body; In Vitro; Knock-out; Link; Measures; Mediating; Mediator of activation protein; Molecular; Pathway interactions; Phenotype; Pluripotent Stem Cells; Process; Proteins; Regenerative Medicine; Regulation; Resistance; Role; Signal Transduction; Small Interfering RNA; System; Testing; Undifferentiated; Work; caspase-3; caspase-9; cell type; drug discovery; embryonic stem cell; genetic regulatory protein; insight; loss of function; mutant; pluripotency; public health relevance; regenerative therapy; research study; self-renewal; small hairpin RNA; stem cell differentiation; stem cell division; stem cell fate; tool; transcription factor; transplantation medicine

DESCRIPTION (provided by applicant): Embryonic stem (ES) cells are pluripotent and can expand in vitro without any apparent limits, while retaining their ability to become any type of cell in the body. The long-term goal of this proposal is to link the molecular mechanisms of programmed cell death to those underlying ES cell renewal and differentiation, with a view toward accelerating the clinical introduction of ES cell regenerative medicine. In my preliminary studies, I found that caspase-3, an important mediator of programmed cell death, has an unexpected role in controlling ES cell fate. I demonstrate an increase of caspase-3 activity upon induction of differentiation and show that caspase-3 can directly cleave the Nanog transcription factor, leading to rapid loss of this core pluripotency- related protein and subsequent ES cell differentiation it typically mediates. These results suggest that caspase- 3 and perhaps other key components of the programmed cell death pathway may have an integral role in the regulation of ES cell renewal/differentiation. The central hypothesis of this proposed work is that classical mediators of programmed cell death, especially caspase-3, also mediate the fate decisions affecting pluripotent stem cells. In Aim 1 I will dissect the functional roles of caspase-3 and it's activating caspase in the fate of ES cells. In Aim 2 I will modulate caspase activity in ES cells and assess the effects on self-renewal, differentiation and programmed cell death. I will also address the question of whether the differentiation-promoting activity of caspase-3 in ES cells is due to an instructive or selective signaling and elucidate if caspase activity provides a specific signal to differentiate or simply promotes differentiation in general. In Aim 3 I will assess the importance of caspase-3-mediated cleavage of Nanog in ES cell differentiation. I consider the transcription factor Nanog to be a paradigm for other potential caspase targets in ES cells, so that my findings for this regulatory protein could well extend to other transcriptional pathways involved in ES cell differentiation. Results of the experiments described in this proposal are expected to provide insight into the pleiotropic effects of caspases in pluripotent stem cells. Thus, specific pharmacological alteration of caspases may be useful not only for modulating apoptosis, but also for directing stem cell fate. The involvement of caspases in nonapoptotic pathways suggests that efforts to block apoptosis via caspase inhibition could have much broader consequences than initially thought. Public Health Relevance: The ability of embryonic stem cells to remain undifferentiated in culture while retaining the ability to become any cell within the human body make them an invaluable tool for use in transplant medicine, drug discovery, and understanding basic developmental biology. Results of the experiments described in this proposal are expected to provide insight into the pleiotropic effects of the cell death enzyme caspase on the differentiation process of embryonic stem cells. Specific pharmacological alteration of caspases may be useful not only for modulating programmed cell death, but also for directing stem cell fate.

描述（由申请人提供）：胚胎词干（ES）细胞具有多能性，可以在体外扩展而无需任何明显的限制，同时保持其成为体内任何类型的细胞的能力。该提案的长期目标是将程序性细胞死亡的分子机制与基本ES细胞更新和分化的人联系起来，以加速ES细胞再生医学的临床引入。在我的初步研究中，我发现CASPASE-3是编程细胞死亡的重要介质，在控制ES细胞命运方面具有出乎意料的作用。我证明了caspase-3活性在诱导分化后的增加，并表明caspase-3可以直接裂解纳米转录因子，从而导致该核心多能相关蛋白的迅速损失，并随后进行ES细胞分化IT通常介导。这些结果表明，caspase-3以及程序性细胞死亡途径的其他关键组成部分可能在ES细胞更新/分化的调节中具有不可或缺的作用。这项提出的工作的中心假设是，程序性细胞死亡的经典介体，尤其是caspase-3，也介导了影响多能干细胞的命运决定。在AIM 1中，我将剖析caspase-3的功能作用，并在ES细胞的命运中激活caspase。在AIM 2中，我将调节ES细胞中的caspase活性，并评估对自我更新，分化和程序性细胞死亡的影响。我还将解决ES细胞中caspase-3的分化促进活性是否是由于具有启发性或选择性的信号引起的问题，并阐明了caspase活性是否提供了特定的信号以区分或简单地促进分化。在AIM 3中，我将评估caspase-3介导的Nanog裂解在ES细胞分化中的重要性。我认为转录因子Nanog是ES细胞中其他潜在caspase靶标的范式，因此我对这种调节蛋白的发现可以很好地扩展到与ES细胞分化有关的其他转录途径。预计本提案中描述的实验结果将提供对胱天蛋白酶在多能干细胞中的多效作用的见解。因此，胱天蛋白酶的特定药理改变不仅可用于调节细胞凋亡，还可用于指导干细胞命运。胱天蛋白酶在非凋亡途径中的参与表明，通过caspase抑制阻止凋亡的努力可能比最初想象的要多得多。 公共卫生相关性：胚胎干细胞在培养中保持未分化的能力，同时保留成为人体中任何细胞的能力，使其成为用于移植医学，药物发现和了解基本发展生物学的宝贵工具。预计该提案中描述的实验结果将洞悉细胞死亡酶caspase对胚胎干细胞分化过程的多效作用。胱天蛋白酶的特定药理改变不仅可用于调节程序性细胞死亡，还可用于指导干细胞命运。