NON-APOPTOTIC ACTION OF CASPASES IN PLURIPOTENT EMBRYONIC STEM CELLS

描述（由申请人提供）：胚胎干（ES）细胞是多能的，可以在体外扩增而没有任何明显的限制，同时保留它们成为体内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细胞更新和分化的基础机制联系起来，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，程序性细胞死亡的重要介质，在控制ES细胞命运中具有意想不到的作用。我证明了在诱导分化后半胱天冬酶-3活性的增加，并表明半胱天冬酶-3可以直接切割Nanog转录因子，导致这种核心多能性相关蛋白的快速丧失和随后的ES细胞分化，其通常介导。这些结果表明，caspase- 3和程序性细胞死亡途径的其他关键组分可能在ES细胞更新/分化的调节中具有不可或缺的作用。这项工作的核心假设是程序性细胞死亡的经典介质，特别是caspase-3，也介导影响多能干细胞的命运决定。目的一：探讨caspase-3及其激活caspase在ES细胞命运中的作用。在目标2中，我将调节ES细胞中的半胱天冬酶活性，并评估其对自我更新、分化和程序性细胞死亡的影响。我还将解决的问题，是否分化促进活性的caspase-3在ES细胞是由于一个指导性或选择性的信号和阐明，如果caspase活性提供了一个特定的信号，分化或只是促进分化一般。在目标3中，我将评估caspase-3介导的Nanog切割在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靶点的范例，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。本提案中描述的实验结果有望为多能干细胞中半胱天冬酶的多效性效应提供见解。因此，特定的药理学改变半胱天冬酶可能是有用的，不仅用于调节细胞凋亡，而且用于指导干细胞的命运。半胱天冬酶参与非凋亡途径表明，通过抑制半胱天冬酶来阻断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，同时保留成为人体内任何细胞的能力，使其成为移植医学，药物发现和理解基础发育生物学的宝贵工具。本实验的结果有望为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提供参考。特异性的半胱天冬酶的药理学改变不仅可用于调节程序性细胞死亡，而且可用于指导干细胞的命运。

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.