Epha/Ephrin - A Signaling in Epidermal Differentiation and Disease

描述(申请人提供)：表皮分化是生存所必需的，在包括牛皮癣在内的常见皮肤病中受损。角质形成细胞通过细胞表面蛋白与相邻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，在基底层分化，在角化层死亡。这项建议的长期目标是确定一个细胞-细胞交流途径，称为EphA/ePhin-A信号轴，在表皮[分化]和疾病中所起的作用。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，但在正常的成人上皮组织，特别是皮肤中还没有得到彻底的研究。表皮中EphA受体的特性和分子组成的物种差异，使得利用人类细胞的实验方法的应用是必要的。因此，一个适应于长期基因沉默和重建的人表皮器官型RAW模型将被用来确定EPHA1和EphA2在表皮分化和形态发生中的相互作用。我们将检验这一创新假设，即EPHA1和EphA2在角质形成细胞分化过程中发挥不同的功能，这是由它们对ephin配体结合的反应控制的。在第一个目的中，将阐明EphA2维持角质形成细胞处于未分化状态的机制(S)，直到该受体被ePhin-A1激活并下调。EphA2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信号通路的调节器SHIP2相互作用]将通过移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突变方法来定义[该方法侧重于Notch信号的下游激活以促进分化。]第二个目的将检验新的假设，即只有在EphA2从细胞表面丢失后，EPHA1才能促进角质形成细胞的分化。用EphA2序列突变和替换EPHA1的胞外和胞内亚结构域将揭示触发角质形成细胞分化的关键信号元件，重点在于EPHA1 SAM结构域调节整合素连接的激酶-RhoA-[血清反应因子]途径的能力。[该提案的翻译潜力得到了加强，因为它能够在炎症皮肤条件下利用表皮EphA/eaffin-A轴，在这些情况下，它们的相对表达发生变化，角质形成细胞分化受损。]与当地(西北大学-皮肤病研究中心，罗伯特·M·拉夫克；[西北大学-蛋白质组学核心，达瓦尔·纳纳瓦蒂])和外部(Bing-程望，凯斯西部保护区；[G.-Paolo Dotto；哈佛大学；Lina Dagnino；西部大学])的重要互动研究上皮生物学和Eph/EPhin信号的小组增加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.