NON-APOPTOTIC ACTION OF CASPASES IN PLURIPOTENT EMBRYONIC STEM CELLS

描述(申请人提供)：胚胎干细胞(ES)是多能的，可以在体外扩张，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。这一建议的长期目标是将程序性细胞死亡的分子机制与潜在的ES细胞更新和分化联系起来，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是细胞程序性死亡的重要中介，在控制ES细胞命运方面具有意想不到的作用。我证明了caspase-3在诱导分化时活性增加，并证明caspase-3可以直接切割Nanog转录因子，导致这种与核心多能性相关的蛋白迅速丧失，并随后它通常介导的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调节ES细胞的更新/分化中起着不可或缺的作用。这项拟议工作的中心假设是，程序性细胞死亡的经典调节因子，特别是caspase-3，也调节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caspase的活性，并评估其对自我更新、分化和细胞程序性死亡的影响。我还将讨论caspase-3在ES细胞中促进分化的活性是由于指导性信号还是选择性信号，并阐明caspase活性是否提供了分化的特定信号，还是只是总体上促进了分化。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与ES细胞分化有关的其他转录途径。这项建议中描述的实验结果有望为了解半胱氨酸天冬氨酸酶在多能干细胞中的多效性提供帮助。因此，半胱氨酸天冬氨酸酶的特异性药理学改变可能不仅有助于调节细胞凋亡，而且还有助于指导干细胞的命运。半胱氨酸天冬氨酸酶参与非凋亡途径表明，通过抑制半胱氨酸天冬氨酸酶来阻止细胞凋亡的努力可能会产生比最初认为的更广泛的后果。 公共卫生相关性：胚胎干细胞在培养中保持未分化的能力，同时保持成为人体内任何细胞的能力，使它们成为移植医学、药物发现和理解基本发育生物学的宝贵工具。这项建议中描述的实验结果有望为深入了解细胞死亡酶caspase在胚胎干细胞分化过程中的多效性作用提供依据。半胱氨酸天冬氨酸酶的特殊药理学改变可能不仅有助于调节细胞程序性死亡，而且还有助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.