CORNIFIED ENVELOPE ASSOCIATED PROTEINS AND PERMEABILITY BARRIER FUNCTION

虽然结构蛋白在表皮机械屏障中的作用是明确的，但它们在通透性屏障中的作用是未知的。我们假设选择的结构蛋白和酶蛋白；即，那些既受ca2 +调控又与冠状包膜（CE）相关的蛋白：天花素、氯丙氨酸、谷氨酰胺转酶1 （TG1）、前填充蛋白和K1/10，是屏障稳态所必需的。该提案将评估这些蛋白质中的哪些受渗透性屏障要求的调节，以及这些ce相关蛋白如何与脂质结合包膜（LBE）和富含脂质的细胞外片层合作，从渗透性屏障中分离出来。具体而言，在Aim #1中，我们将确定哪些CE相关蛋白受到屏障需求改变的调节，以及Ca++是否调节其表达，方法是：a)测量无毛小鼠急性屏障破坏（丙酮，胶带剥离）和必需脂肪酸缺乏（EFAD）后CE相关蛋白mRNA/蛋白表达的时间过程。b)确定细胞外Ca++的变化（通过声泳和浸泡在体内）是否调节被调节的CE蛋白的表达；钙受体（CaR）和电压敏感的钙离子通道参与了这些变化。c)判断ce相关蛋白表达的短暂下降是否有利于屏障恢复。利用三种转基因过表达（天花素、K6/16和前聚丝蛋白）的小鼠模型，其中一种蛋白干扰通透性屏障稳态和板层体分泌。在Aim #2中，我们将确定哪些ce相关蛋白是正常屏障内稳态所必需的，以及这些蛋白的改变导致屏障异常的机制，方法是：a)评估具有ce相关蛋白特异性缺失或突变的人和小鼠模型的内稳态；即板层状鱼鳞病（LI； TG1缺失）、表皮松解性角化过度病（EHK；K1或10突变）、西门子大疱性鱼鳞病（IBS； K2e突变）、掌足底角化病（NSPPK； K1突变）、鱼鳞病、角化病（NSPPK； K1突变）、寻常型鱼鳞病（前聚丝蛋白缺乏）、Vohwinkle病（loricrin突变）、外加天花素和K10小鼠敲除动物。b)评估ce相关蛋白影响通透性屏障功能的机制。

Although the role of structural proteins in the epidermal mechanical barrier is clear, their role in the permeability barrier is unknown. We hypothesize that selected structural and enzymatic proteins; i.e., those that are both regulated by Ca++ and associated with the cornified envelope (CE): involucrin, loricrin, transglutaminase 1 (TG1), pro-fillaggrin, and K1/10, are regulated/required for barrier homeostasis. This proposal will assess which of these proteins are regulated by permeability barrier requirements, and how these CE-associated proteins, in partnership with the lipid bound envelope (LBE) and the lipid-enriched extracellular lamellae, from the permeability barrier. Specifically, in Aim #1 we will determine which CE- associated proteins are regulated by altered barrier requirements, and whether Ca++ regulates their expression, by: a) measuring the time course of mRNA/protein expression for the CE-associated proteins after acute barrier disruption (acetone, tape stripping) and in essential fatty acid deficiency (EFAD) in hairless mice. b) Ascertaining whether changes in extracellular Ca++ (in vivo by sonophoresis and by immersion) modulate expression of the regulated CE protein(s); and the participation of the calcium receptor (CaR) and voltage-sensitive Ca++ channels in these changes. c) Determining whether the transient decrease in CE-associated protein expression facilitates barrier recovery. Using three transgenic over- expressing (involucrin, K6/16, and pro-filaggrin) murine models, and one of these proteins interferes with permeability barrier homeostasis and lamellar body secretion. In Aim #2, we will determine which CE-associated proteins are required for normal barrier homeostasis, and the mechanisms by which alterations in these proteins lead to barrier abnormalities, by: a) Assessing homeostasis in human and murine models with specific deletions or mutations of CE-associated proteins; i.e., in patients with lamellar ichthyosis (LI; TG1 deletion), epidermolytic hyperkeratoses (EHK;K1 or 10 mutation), ichethyosis bullosa of Siemens (IBS; K2e mutation), palmo-plantar keratoderma (NSPPK; K1 mutation), ichthyosis, Keratoderma (NSPPK; K1 mutation), ichthyosis vulgaris (pro-filaggrin deficiency), Vohwinkle's disease (loricrin mutation), plus involucrin and K10 murine knockout animals. b) Assessing the mechanisms whereby CE-associated proteins influence permeability barrier function.