NON-APOPTOTIC ACTION OF CASPASES IN PLURIPOTENT EMBRYONIC STEM CELLS

描述（由申请人提供）：胚胎干细胞（ES）具有多能性，可以在体外无任何明显限制地扩增，同时保留其成为体内任何类型细胞的能力。本研究的长期目标是将程序性细胞死亡的分子机制与胚胎干细胞的更新和分化联系起来，以期加速胚胎干细胞再生医学的临床应用。在我的前期研究中，我发现caspase-3作为一种重要的程序性细胞死亡介质，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时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相关蛋白的快速丧失，并导致其通常介导的胚胎干细胞分化。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ES细胞更新/分化的调控中起着不可或缺的作用。这项工作的中心假设是程序性细胞死亡的经典介质，特别是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的功能作用它在胚胎干细胞的命运中激活caspase。在目标2中，我将调节胚胎干细胞中的caspase活性，并评估对自我更新、分化和程序性细胞死亡的影响。我还将讨论caspase-3在胚胎干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活性是提供特定的分化信号还是仅仅促进一般的分化。在Aim 3中，我将评估caspase-3介导的Nanog切割在胚胎干细胞分化中的重要性。我认为转录因子Nanog是胚胎干细胞中其他潜在caspase靶点的典范，因此我对这种调节蛋白的发现可以很好地扩展到胚胎干细胞分化的其他转录途径。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提供见解。因此，半胱天冬酶的特定药理改变可能不仅对调节细胞凋亡有用，而且对指导干细胞命运也有用。caspase参与非凋亡途径表明，通过caspase抑制来阻止凋亡的努力可能比最初想象的具有更广泛的后果。

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.