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）：胚胎干（ES）细胞是多能的，可以在体外不受任何明显限制地扩增，同时保留其在体内成为任何类型细胞的能力。该提案的长期目标是将程序性细胞死亡的分子机制与ES细胞更新和分化的分子机制联系起来，以期加速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作为程序性细胞死亡的重要介质，在控制ES细胞命运方面具有意想不到的作用。我证明了在诱导分化时 caspase-3 活性增加，并表明 caspase-3 可以直接裂解 Nanog 转录因子，导致这种核心多能性相关蛋白的快速丢失以及随后它通常介导的 ES 细胞分化。这些结果表明 caspase-3 以及程序性细胞死亡途径的其他关键成分可能在 ES 细胞更新/分化的调节中发挥着不可或缺的作用。这项工作的中心假设是程序性细胞死亡的经典介质，尤其是 caspase-3，也介导影响多能干细胞的命运决定。在目标 1 中，我将剖析 caspase-3 的功能作用以及它在 ES 细胞命运中激活 caspase 的作用。在目标 2 中，我将调节 ES 细胞中的 caspase 活性，并评估其对自我更新、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归因于指导性或选择性信号传导的问题，并阐明 caspase 活性是否提供了特定的分化信号或只是一般性地促进分化。在目标 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 靶标的范例，因此我对这种调节蛋白的发现可以很好地扩展到 ES 细胞分化中涉及的其他转录途径。该提案中描述的实验结果预计将有助于深入了解半胱天冬酶在多能干细胞中的多效性作用。因此，半胱天冬酶的特定药理学改变不仅可用于调节细胞凋亡，还可用于指导干细胞命运。半胱天冬酶参与非凋亡途径表明，通过半胱天冬酶抑制来阻止细胞凋亡的努力可能会产生比最初想象的更广泛的后果。 公共健康相关性：胚胎干细胞在培养物中保持不分化的能力，同时保留成为人体内任何细胞的能力，使其成为移植医学、药物发现和理解基本发育生物学的宝贵工具。该提案中描述的实验结果有望深入了解细胞死亡酶半胱天冬酶对胚胎干细胞分化过程的多效性影响。半胱天冬酶的特异性药理学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。