Epha/Ephrin - A Signaling in Epidermal Differentiation and Disease

描述（由申请人提供）：表皮分化是生存所必需的，在常见皮肤病（包括银屑病）中受损。角质形成细胞通过细胞表面蛋白与它们的邻居相互作用和交流;这有助于确定表皮细胞是否在基底层中分裂，在基底上层中分化并在皮质层中死亡。该提案的长期目标是确定细胞间通讯途径（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关键作用，但尚未在正常成人上皮组织，特别是皮肤中进行彻底研究。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，一个器官型筏模型的人表皮，已适应长期的基因沉默和重建将被用来定义EphA 1和EphA 2在表皮分化和形态发生的相互作用。我们将测试的创新假设，EphA 1和EphA 2表现出离散的角质形成细胞分化的功能，控制他们的反应肝配蛋白配体结合。在第一个目的中，将阐明EphA 2维持角质形成细胞处于未分化状态直到该受体被肝配蛋白-A1接合和下调的机制。EphA 2胞质结构域在抑制角质形成细胞分化中的重要性[通过与SHIP 2（EGFR信号传导途径的调节剂）相互作用]将使用去除该区域内的关键信号传导元件的遗传方法来确定。EphA 2对配体结合的响应将使用基因诱变方法来定义[其关注Notch信号传导的下游激活以促进分化]。第二个目的将测试新的假设，即EphA 1促进角质形成细胞分化的反应肝配蛋白-A1结合后，EphA 2是从细胞表面丢失。用EphA 2序列突变和替换EphA 1的细胞外和细胞质亚结构域将揭示触发角质形成细胞分化的关键信号传导元件，重点关注EphA 1 SAM结构域调节整联蛋白连接激酶-RhoA-[血清反应因子]途径的能力。[The该提议的翻译潜力通过在炎性皮肤状况中利用表皮EphA/肝配蛋白-A轴的能力而得到加强，在炎性皮肤状况中它们的相对表达变化和角质形成细胞分化受损。与当地的重要互动（西北大学-皮肤病研究中心，罗伯特M。[Northwestern U. Proteomics Core，Dhaval Nanavati]）和外部（Bing-Cheng Wang，Case Western Reserve; [G.- Paolo Dotto;哈佛; Lina Dagnino;美国西部]）研究上皮生物学和Eph/ephrin信号传导的小组增强了该提案成功和影响的可能性。

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.