Cell Death in Normal and Diseased Human Epidermis

人类皮肤的表皮隔层位于与环境的交界处，保护身体免受可能扰乱体内平衡的有害物理、化学和感染性因素的影响。表皮通过角质形成细胞(KCs)形成角质层形成这种屏障或表面封闭，角质形成细胞经历了末端分化和细胞死亡。细胞死亡的适当调节不仅必须平衡细胞增殖，以维持一致的和生理上可接受的表皮厚度，而且协调细胞死亡的分子事件也必须适当地定时，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细胞死亡，调节表皮细胞死亡的分子事件是这一提议的重点。我们的假设是，一系列独特的生化反应是这一重要的凋亡过程的基础，涉及到各种关键的分子参与者，包括：主要属于肿瘤坏死家族的特定死亡受体，以及诱骗受体、死亡配体、半胱氨酸天冬氨酸酶级联反应和转录因子。我们建议进行一系列系统和全面的实验，以初步确定正常皮肤中这一计划中的细胞死亡途径，然后利用这一新知识来研究几种以失调和/或过早死亡为特征的皮肤疾病。初步证据表明，与生化凋亡机制相关的独特分子变化可定位于特定的表皮层，并且特定的死亡途径在上述皮肤病中也是异常的。此外，越来越多的证据突出了我们的实验设计，因为越来越明显的是，需要将表皮细胞死亡的完整解释作为细胞生物学和生物化学中的一个问题来处理。这笔赠款的特点是实验方案的过渡，从传统的组织培养技术开始，然后转移到使用活的表皮等价物，其中包含分层的KCs，可以通过将培养物提升到空气/液体界面来触发进行末端分化和角化。利用这些体外表皮替代物将有助于剖析产生终末分化/角化的分子事件。通过成功完成建议的目标，将出现对开发新的和改进的方法非常重要的治疗策略，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；防止过早的细胞凋亡；减少或推迟皮肤癌的发病。

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.