Breaking down barriers: defining the role of EphA2 in building epidermal tight junctions in atopic dermatitis

Perez白色，Bethany 5 K 01 AR 072773 -03行政补充申请 项目摘要 紧密连接是表皮屏障的重要组成部分，是皮肤功能所必需的。虽然我们知道 这些结构在皮肤病如特应性皮炎（AD）中被破坏，我们还不完全了解这些结构， 正常的信号传导有助于它们的功能。这种知识差距使得寻找恢复紧张的疗法 交通活动困难。因此，确定负责建立紧密连接的信号通路 不仅有助于促进我们对皮肤生物学的基本理解，而且可能为药物治疗提供线索。 针对性和疾病干预。我们的初步证据表明EphA 2受体酪氨酸激酶 (RTK)有助于2D培养中紧密连接的形成和功能。基因操作这些 去除EphA 2的培养物导致层中紧密连接蛋白的定位丧失 他们居住的地方。此外，通过测量，我们发现了紧密连接功能的丧失。 电阻和2D角质形成细胞培养物中大分子的渗透性。基于这些 在EphA 2缺失后表皮完整性的变化中，我们假设EphA 2是EphA 2表达的正调节因子， 表皮中的紧密连接，并且可以靶向改善紧密连接屏障功能。我们 将确定EphA 2如何调节紧密连接，以及EphA 2是否可以靶向 增强皮肤屏障功能。我们最初的研究表明结构蛋白afadin 在EphA 2信号传导的下游，作为紧密连接的形成和功能的正调节剂， 表皮角质形成细胞我们的研究已经发现EphA 2缺失导致afadin从 细胞边界到细胞内部区域，以及afadin和紧密连接蛋白的损失。因此，我们将首先 测试EphA 2和afadin损失对3D重构细胞中紧密连接的组装和功能性的影响 人表皮（3D RHE）。我们接下来将测试EphA 2改变afadin表达的机制， 导致紧密连接缺陷。我们还发现了新的EphA 2/EGFR RTK串扰，其是 TJ形态发生最后，我们将使用3D RHE和AD动物模型来确定EphA 2配体是否 肝配蛋白-A1的激活可以增强紧密连接屏障功能， 这种疾病的表现。AD是一种常见的皮肤病，主要影响婴幼儿 其特征在于与皮肤炎症相关的表皮屏障缺陷。这种慢性疾病 严重降低了患者及其家属的生活质量，并造成重大的终身经济损失。 负担通过了解紧密连接的生物学，这是皮肤的关键结构成分， 我们可以寻求新的疗法来缓解这种疾病。我们建议， EphA 2/ephrin-A1信号传导轴可用于使AD中的紧密连接缺陷正常化。

Perez White, Bethany 5K01AR072773-03 Administrative Supplement Request PROJECT SUMMARY Tight junctions are a vital component of the epidermal barrier and are required for skin function. While we know these structures are disrupted in skin diseases such as atopic dermatitis (AD), we do not fully understand the normal signaling contributing to their function. This knowledge gap makes finding therapies to restore tight junction activity difficult. Therefore, defining the signaling pathways responsible for establishing tight junctions would serve not only to advance our basic understanding of skin biology, but also may provide leads for drug targeting and disease intervention. Our preliminary evidence indicates that EphA2 receptor tyrosine kinase (RTK) contributes to the formation and function of tight junctions in 2D cultures. Genetic manipulation of these cultures to remove EphA2 results in a loss of the localization of tight junction proteins from the stratum granulosum in which they reside. In addition, we have found a loss of tight junction function by measuring electrical resistance and the permeability of large molecules in 2D keratinocyte cultures. Based on these changes in epidermal integrity after loss of EphA2, we hypothesize that EphA2 is a positive regulator of tight junctions in the epidermis and can be targeted to improve tight junction barrier function. We specifically will determine how EphA2 is regulating tight junctions and if EphA2 could be targeted pharmacologically to enhance skin barrier function. Our initial studies implicate the structural protein afadin, downstream of EphA2 signaling, as a positive regulator of the formation and function of tight junctions in epidermal keratinocytes. Our studies have found that EphA2 loss leads to the mislocalization of afadin from cell borders to the cell's interior regions, and a loss in afadin and tight junction proteins. Therefore, we will first test the effect of EphA2 and afadin loss on the assembly and functionality of tight junctions in 3D reconstituted human epidermis (3D RHE). We will next test the mechanism by which EphA2 alters afadin expression, leading to tight junction defects. We have also uncovered novel EphA2/EGFR RTK crosstalk that is integral to TJ morphogenesis. Finally, we will use 3D RHE and animal models of AD to determine if EphA2 ligand activation with ephrin-A1 can enhance tight junction barrier function and either prevent or reverse the manifestations of this disease. AD is a prevalent skin disease primarily affecting infants and young children characterized by an epidermal barrier defect in association with skin inflammation. This chronic condition severely decreases the quality of life of patients and their families, and causes a significant lifelong economic burden. By understanding the biology of tight junctions, a critical structural component of the skin that is dysfunctional in AD, we can seek new therapies to alleviate this disease. We have proposed that the EphA2/ephrin-A1 signaling axis can be harnessed to normalize tight junction defects in AD.