Cell Death in Normal and Diseased Human Epidermis

人体皮肤的表皮隔室战略性地位于与环境的交界面，保护身体免受可能扰乱体内平衡的有害物理、化学和感染因子的影响。表皮通过产生角质层形成这种屏障或表面密封，角质层是由角化细胞（KCs）形成的，这些细胞经历了终末分化和细胞死亡。细胞死亡的适当调节不仅要平衡细胞增殖，以维持一致的和生理上可接受的表皮厚度，协调细胞死亡的分子事件也必须在适当的时间内以空间和时间协调的方式发生，以产生有效的屏障。这种有序的表皮过程被称为“计划性细胞死亡”，调控表皮细胞死亡的分子事件是本研究的重点。我们的假设是，一组独特的生化反应是这一重要凋亡过程的基础，涉及各种关键分子参与者，包括：主要属于肿瘤坏死家族的特异性死亡受体，以及诱饵受体，死亡配体，半胱天冬酶级联反应和转录因子。我们建议进行系统和全面的一系列实验，初步确定正常人类皮肤中这种计划的细胞死亡途径，然后利用这一新知识来研究几种皮肤疾病，这些疾病以失调和/或过早的KC细胞死亡为特征。初步证据表明，与生化凋亡机制相关的独特分子变化可能定位于特定的表皮层，并且上述皮肤病的特定死亡途径也异常。此外，越来越多的证据强调了我们的实验设计，因为越来越清楚的是，对表皮细胞死亡的全面解释需要作为细胞生物学和生物化学的一个问题来解决。这项资助的特点是实验方案的过渡，从传统的组织培养技术开始，然后转向使用含有KCs分层层的活表皮等效物，通过将培养物提升到空气/液体界面，可以触发KCs进行最终分化和角化。利用这些离体表皮等效物将有助于解剖产生终端分化/角化的分子事件。通过成功完成提出的目标，治疗策略将出现，这对于开发新的和改进的方法非常重要，这些方法可能在各种临床环境中有用，包括：加速屏障功能的形成；防止细胞过早凋亡；减少或延缓皮肤癌的发生。

The epidermal compartment of human skin is strategically located at the interface with the environment, shielding the body from harmful physical, chemical, and infectious agents that could perturb homeostasis. The epidermis creates this barrier, or surface seal, by production of a stratum corneum formed by keratinocytes (KCs) that have undergone terminal differentiation and cell death. Not only must proper regulation of cell death balance cell proliferation to maintain a consistent and physiologically acceptable thickness of epidermis, the molecular events that orchestrate cell death must also be properly timed to occur in both a spatially and temporally coordinated fashion to produce an effective barrier. This orderly epidermal process has been termed "planned cell death", and the molecular events that regulate epidermal cell death are the focus of this proposal. Our hypothesis is that a distinct set of biochemical reactions underlie this vital apoptotic process, involving a variety of key molecular participants including: specific death receptors that primarily belong to the tumor necrosis family, as well as decoy receptors, death ligands, caspase cascades, and transcription factors. We propose to perform a systematic and comprehensive series of experiments to initially define this planned cell death pathway in normal human skin, and then utilizing this new knowledge to investigate several skin disorders that feature either dysregulated and/or premature KC cell death. Preliminary evidence indicates distinctive molecular changes related to the biochemical apoptotic machinery can be localized to specific layers of epidermis, and that specific death pathways are also abnormal in the aforementioned skin diseases. Moreover, a growing body of evidence highlights our experimental design, as it is becoming clear that a full explanation for cell death in the epidermis needs to be approached as a problem in cell biology as well as one in biochemistry. This grant features a transition of experimental protocols beginning with conventional tissue culture techniques, and then moving to the use of living epidermal equivalents that contain stratified layers of KCs that can be triggered to undergo terminal differentiation and cornification by lifting the culture to an air/liquid interface. Utilizing these ex-vivo epidermal equivalents will facilitate dissection of molecular events that produce terminal differentiation/cornification. By successfully completing the proposed objectives, therapeutic strategies will emerge that are important to develop new and improved methods that may be useful in a variety of clinical settings including: accelerating barrier function formation; preventing premature apoptosis; reducing or delaying the onset of skin cancer.