Regulation and Function of Keratins in the Epidermis

描述(申请人提供)：随着14种不同角蛋白基因的突变可以导致8种不同的上皮脆性疾病的发现，人们意识到角蛋白细丝细胞骨架对于暴露在机械应力下的上皮组织的结构完整性至关重要。导致这些疾病的突变的鉴定提高了我们对角蛋白结构和功能的理解，特别是关于中央杆状结构域的高度保守的区域。然而，角蛋白生物学中的两个核心问题仍然存在。第一个问题涉及单个角蛋白基因的调控，因为这些蛋白质的合成受到严格调控是表皮正常发育的先决条件。为了解决这个问题，我们将重点研究在表皮发育的不同阶段表达的两个角蛋白基因，角蛋白K14和K1。K14是承诺层化后最早在表面外胚层表达的基因之一。我们最近已经证明，转录因子p63是启动表皮层化程序的分子开关。这一发现为我们提供了对K14在表皮发育过程中诱导表达的分子机制的新见解。我们还发现，p63亚型表达的转换是允许基底层角质形成细胞退出细胞周期并致力于终末分化所必需的。由于K1是角质形成细胞致力于终末分化后在表皮中表达的首批基因之一，我们将利用这一发现来深入了解表皮成熟过程中K1表达的分子机制。第二个问题涉及角蛋白N-末端和C-末端结构域的作用。这些结构域对于每种角蛋白来说都是不同的，但在不同物种之间保存得非常好，可能具有功能意义。体外研究表明，这些结构域与桥粒和/或细胞被膜相互作用，但对这些结构域在体内的细胞类型特异性功能知之甚少。为了解决这个问题，提出了两种转基因方法。最后，表皮松解性角化过度(EHK)是一种主要由有丝分裂后表达的角蛋白K1或K10突变引起的遗传性皮肤疾病，对基因治疗提出了挑战。在这笔赠款的最后一次资助期间，我们建立了一个在遗传和表型水平上模拟EHK的小鼠模型。这个小鼠模型为研究EHK的分子和细胞基础提供了新的见解，我们建议使用这个模型来测试新的基因治疗策略。这项研究的结果将产生有价值的信息，为影响有丝分裂后角质形成细胞的其他疾病提供新的治疗方法。

DESCRIPTION (provided by applicant): With the discovery that mutations in fourteen different keratin genes can cause eight distinct epithelial fragility disorders, comes the realization that the keratin filament cytoskeleton is crucial to the structural integrity of epithelial tissues exposed to mechanical stress. The identification of mutations causing these disorders has improved our understanding of keratin structure and function, particularly with regard to the highly conserved regions of the central rod domain. However, two central questions in keratin biology still remain. The first question concerns the regulation of individual keratin genes, since the tightly regulated synthesis of these proteins is a pre-requisite for the normal development of the epidermis. To address this question, we will focus on two keratin genes that are expressed at different stages of epidermal development, keratins K14 and K1. K14 is one of the first genes expressed in the surface ectoderm after commitment to stratification. We have recently demonstrated that the transcription factor p63 is the molecular switch for initiation of the epidermal stratification program. This discovery has provided us with novel insights into the molecular mechanisms responsible for the induction of K14 expression during epidermal development. We have also discovered that a switch in p63 isoform expression is required to allow basal keratinocytes to withdraw from the cell cycle and commit to terminal differentiation. Since K1 is one of the first genes expressed in the epidermis after keratinocytes have committed to terminal differentiation, we will exploit this discovery to gain insight into the molecular mechanisms regulating K1 expression during maturation of the epidermis. The second question concerns the roles of the keratin N- and C-terminal end domains. These domains are distinctive for each keratin protein but are remarkably well conserved across species and presumably have functional significance. In vitro studies have suggested that these domains interact with desmosomes and/or the cell envelope, however very little is known regarding cell type-specific functions of these domains in vivo. Two transgenic approaches are proposed to address this question. Finally, epidermolytic hyperkeratosis (EHK), the dominantly inherited skin disorder caused by mutations in the post-mitotically expressed keratins, K1 or K10, presents a challenge for gene therapy. During the last funding period of this grant, we generated a mouse model that mimics EHK at both the genetic and phenotypic level. This mouse model has provided new insights into the molecular and cellular basis of EHK and we propose to use this model to test new gene therapy strategies. The results of this research will yield valuable information that can lead to novel therapeutic approaches for other diseases affecting post-mitotic keratinocytes.