EphA/Ephrin-A signaling in epidermal differentiation and disease

描述（由申请人提供）：表皮分化对包括牛皮癣在内的常见皮肤病的生存和受损至关重要。角质形成细胞通过细胞表面蛋白与邻近细胞相互作用和交流；这有助于确定表皮细胞是否在基底层分裂，在上基底层分化，在角化层死亡。本提案的长期目标是确定细胞-细胞通信途径（称为EphA/ephrin-A信号轴）在表皮[分化]和疾病中的作用。这个受体酪氨酸激酶大家族在胚胎发育和癌症进展中起着关键作用，但尚未在正常成人上皮组织，特别是皮肤中进行深入研究。表皮中EphA受体的特征和分子组成的物种差异需要应用利用人类细胞的实验方法。因此，我们将采用一个适应长期基因沉默和重组的人表皮有机型筏模型来确定EphA1和EphA2在表皮分化和形态发生中的相互作用。我们将验证EphA1和EphA2在角质形成细胞分化中表现出离散功能的创新假设，这些功能是由它们对ephrin配体结合的反应控制的。在第一个目标中，EphA2维持角化细胞处于未分化状态的机制将被阐明，直到该受体被ephrin-A1参与并下调。EphA2细胞质结构域在抑制角质形成细胞分化中的重要性[通过与EGFR信号通路的调节剂SHIP2相互作用]将通过去除该区域内关键信号元件的遗传方法来确定。EphA2对配体结合的反应将使用基因诱变方法来定义[重点关注Notch信号的下游激活以促进分化]。第二个目的是验证新的假设，即EphA1仅在EphA2从细胞表面丢失后才能促进角质形成细胞分化，以响应ephrin-A1的结合。用EphA2序列突变和替换EphA1的胞外和胞质亚结构域将揭示触发角质细胞分化的关键信号元件，重点是EphA1 SAM结构域调节整合素连接激酶- rhoa -[血清反应因子]途径的能力。在炎症性皮肤条件下，利用表皮EphA/ephrin-A轴的能力增强了该提案的翻译潜力，在炎症性皮肤条件下，它们的相对表达改变和角化细胞分化受损。[西北大学皮肤病研究中心，Robert M. Lavker；[西北大学-蛋白质组学核心，Dhaval Nanavati])和外部(王炳成，Case Western Reserve;]保罗Dotto;哈佛大学;莉娜Dagnino;研究上皮生物学和Eph/ephrin信号传导的西方研究小组提高了该提案成功的可能性和影响。

DESCRIPTION (provided by applicant): Epidermal differentiation is essential for survival and impaired in common skin diseases, including psoriasis. Keratinocytes interact and communicate with their neighbors via cell surface proteins; this helps determine whether epidermal cells divide in the basal layer, differentiate in the suprabasal layers and die in the cornified layer. Te long-term goal of this proposal is to define the role of a cell-cell communication pathway, termed the EphA/ephrin-A signaling axis, in epidermal [differentiation] and disease. This large family of receptor tyrosine kinases plays a key role in embryonic development and cancer progression but has not been thoroughly investigated in normal adult epithelial tissues, especially the skin. Species differences in the character and molecular composition of EphA receptors in the epidermis necessitate the application of experimental approaches that take advantage of human cells. Consequently, an organotypic raft model of human epidermis that has been adapted for long-term gene silencing and reconstitution will be employed to define the reciprocal roles for EphA1 and EphA2 in epidermal differentiation and morphogenesis. We will test the innovative hypothesis that EphA1 and EphA2 exhibit discrete functions in keratinocyte differentiation that are controlled by their responses to ephrin ligand binding. In the first aim, the mechanism(s) by which EphA2 maintains keratinocytes in an undifferentiated state until this receptor is engaged and downregulated by ephrin-A1 will be elucidated. The importance of the EphA2 cytoplasmic domain in inhibiting keratinocyte differentiation [by interacting with SHIP2, a modulator of the EGFR signaling pathway,] will be determined using genetic approaches that remove key signaling elements within this region. The response of EphA2 to ligand binding will be defined using gene mutagenesis approaches [that focus on the downstream activation of Notch signaling to promote differentiation.] The second aim will test the novel hypothesis that EphA1 promotes keratinocyte differentiation in response to ephrin-A1 binding only after EphA2 is lost from the cell surface. Mutating and replacing the extracellular and cytoplasmic subdomains of EphA1 with EphA2 sequences will reveal the key signaling elements that trigger keratinocyte differentiation, focusing on the ability of the EphA1 SAM domain to regulate the integrin-linked kinase-RhoA-[serum response factor] pathway. [The translational potential of the proposal is strengthened by the ability to harness the epidermal EphA/ephrin-A axis in inflammatory skin conditions where their relative expression changes and keratinocyte differentiation is impaired.] Important interactions with local (Northwestern U.-Skin Disease Research Center, Robert M. Lavker; [Northwestern U.-Proteomics Core, Dhaval Nanavati]) and outside (Bing-Cheng Wang, Case Western Reserve; [G.-Paolo Dotto; Harvard; Lina Dagnino; Western U.]) groups studying epithelial biology and Eph/ephrin signaling enhance the likelihood of success and impact of the proposal. PUBLIC HEALTH RELEVANCE: Information from this project will lead to a better understanding of how a cell-cell communication pathway, termed the EphA/ephrin-A signaling axis, governs the differentiation of keratinocytes in [the human epidermis. Insight gained from our studies on the reciprocal roles of EphA1 and EphA2 in keratinocyte signaling and differentiation will lead to the development of novel treatments for inflammatory skin diseases where their expression is altered, such as psoriasis.]