Department of Dermatology, Northwestern University, Chicago, Illinois, United States.
Department of Anatomy and Regenerative Biology, The George Washington University Medical Center, Washington, District of Columbia, United States.
Invest Ophthalmol Vis Sci. 2018 Jan 1;59(1):393-406. doi: 10.1167/iovs.17-22941.
Progenitor cells of the limbal epithelium reside in a discrete area peripheral to the more differentiated corneal epithelium and maintain tissue homeostasis. What regulates the limbal-corneal epithelial boundary is a major unanswered question. Ephrin-A1 ligand is enriched in the limbal epithelium, whereas EphA2 receptor is concentrated in the corneal epithelium. This reciprocal pattern led us to assess the role of ephrin-A1 and EphA2 in limbal-corneal epithelial boundary organization.
EphA2-expressing corneal epithelial cells engineered to express ephrin-A1 were used to study boundary formation in vitro in a manner that mimicked the relative abundance of these juxtamembrane signaling proteins in the limbal and corneal epithelium in vivo. Interaction of these two distinct cell populations following initial seeding into discrete culture compartments was assessed by live cell imaging. Immunofluoresence and immunoblotting was used to evaluate the contribution of downstream growth factor signaling and cell-cell adhesion systems to boundary formation at sites of heterotypic contact between ephrin-A1 and EphA2 expressing cells.
Ephrin-A1-expressing cells impeded and reversed the migration of EphA2-expressing corneal epithelial cells upon heterotypic contact formation leading to coordinated migration of the two cell populations in the direction of an ephrin-A1-expressing leading front. Genetic silencing and pharmacologic inhibitor studies demonstrated that the ability of ephrin-A1 to direct migration of EphA2-expressing cells depended on an a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and epidermal growth factor receptor (EGFR) signaling pathway that limited E-cadherin-mediated adhesion at heterotypic boundaries.
Ephrin-A1/EphA2 signaling complexes play a key role in limbal-corneal epithelial compartmentalization and the response of these tissues to injury.
边缘上皮的祖细胞位于更分化的角膜上皮的周边离散区域内，并维持组织内稳态。调节边缘-角膜上皮边界的因素是一个主要的未解决的问题。 边缘上皮中富含 Ephrin-A1 配体，而角膜上皮中集中表达 EphA2 受体。这种相互的模式使我们评估 Ephrin-A1 和 EphA2 在边缘-角膜上皮边界组织中的作用。
我们构建了表达 Ephrin-A1 的角膜上皮细胞，以研究体外边界形成，这种方法模拟了这些跨膜信号蛋白在活体边缘和角膜上皮中的相对丰度。通过活细胞成像评估这两种不同细胞群在初始接种到离散培养隔室后相互作用的情况。免疫荧光和免疫印迹用于评估下游生长因子信号转导和细胞-细胞黏附系统对 Ephrin-A1 和 EphA2 表达细胞异质接触部位边界形成的贡献。
Ephrin-A1 表达细胞在形成异质接触时阻碍并逆转 EphA2 表达的角膜上皮细胞的迁移，导致这两种细胞群在 Ephrin-A1 表达的前缘方向上协调迁移。基因沉默和药理学抑制剂研究表明，Ephrin-A1 指导 EphA2 表达细胞迁移的能力依赖于 a disintegrin 和金属蛋白酶域蛋白 10（ADAM10）和表皮生长因子受体（EGFR）信号通路，该信号通路限制了异质边界处 E-钙黏蛋白介导的黏附。
Ephrin-A1/EphA2 信号复合物在边缘-角膜上皮区室化和这些组织对损伤的反应中发挥关键作用。
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