NON-APOPTOTIC ACTION OF CASPASES IN PLURIPOTENT EMBRYONIC STEM CELLS

胚胎干细胞（ES细胞）是多能性的，并且可以在体外扩增而没有任何明显的限制，同时保留 它们成为身体中任何类型细胞的能力。这项提案的长期目标是将分子 程序性细胞死亡的机制，那些潜在的ES细胞更新和分化，以期 加速胚胎干细胞再生医学的临床应用。在我的初步研究中， 发现caspase-3是程序性细胞死亡的重要介质，在控制细胞凋亡中具有意想不到的作用。 ES细胞命运。我证明了诱导分化后caspase-3活性的增加，并表明， caspase-3可以直接切割Nanog转录因子，导致这种核心多能性的快速丧失。 它通常介导相关蛋白和随后的ES细胞分化。这些结果表明，caspase- 3和程序性细胞死亡途径的其他关键成分可能在细胞死亡中起着不可或缺的作用。 ES细胞更新/分化的调节。 这项工作的中心假设是，程序性细胞死亡的经典介质，特别是 半胱天冬酶-3也介导影响多能干细胞的命运决定。在目标1中，我将剖析功能 caspase-3及其活化caspase在ES细胞命运中的作用。在目标2中，I将调节半胱天冬酶活性 在ES细胞中，并评估对自我更新、分化和程序性细胞死亡的影响。我也会 解决了这样一个问题，即在ES细胞中caspase-3的分化促进活性是否是由于 指导性或选择性信号传导，并阐明胱天蛋白酶活性是否提供特异性信号以分化或 只是促进了总体上的差异化。在目标3中，我将评估caspase-3介导的 Nanog在ES细胞分化中的裂解。我认为转录因子Nanog是一个范例， 其他潜在的caspase靶点，所以我对这种调节蛋白的发现可以很好地扩展到 其他转录途径参与ES细胞分化。 本提案中所描述的实验结果有望提供对多效性效应的深入了解 多能干细胞中的半胱天冬酶。因此，特异性的半胱天冬酶药理学改变可能是有用的， 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，通过半胱天冬酶抑制来阻断凋亡的努力可能具有很大的意义。 比最初想象的更广泛的后果。胚胎干细胞在培养中保持未分化的能力，同时保持成为 人体内的任何细胞都使它们成为移植医学、药物发现 了解基本的发育生物学。本提案中描述的实验结果如下： 预期提供深入了解细胞死亡酶半胱天冬酶对分化的多效性作用 胚胎干细胞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，还用于指导干细胞命运。

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 its 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. 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.