Purpose

To understand the relationship between ciliogenesis and autophagy in the corneal epithelium.

Methods

siRNAs for EphA2 or PLD1 were used to inhibit protein expression in vitro. Morpholino-anti-EphA2 was used to knockdown EphA2 in Xenopus skin. An EphA2 knockout mouse was used to conduct loss of function studies. Autophagic vacuoles were visualized by contrast light microscopy. Autophagy flux, was measured by LC3 turnover and p62 protein levels. Immunostaining and confocal microscopy were conducted to visualize cilia in cultured cells and in vivo.

Results

Loss of EphA2 (i) increased corneal epithelial thickness by elevating proliferative potential in wing cells, (ii) reduced the number of ciliated cells, (iii) increased large hollow vacuoles, that could be rescued by BafA1; (iv) inhibited autophagy flux and (v) increased GFP-LC3 puncta in the mouse corneal epithelium. This indicated a role for EphA2 in stratified epithelial assembly via regulation of proliferation as well as a positive role in both ciliogenesis and end-stage autophagy. Inhibition of PLD1, an EphA2 interacting protein that is a critical regulator of end-stage autophagy, reversed the accumulation of vacuoles, and the reduction in the number of ciliated cells due to EphA2 depletion, suggesting EphA2 regulation of both end-stage autophagy and ciliogenesis via PLD1. PLD1 mediated rescue of ciliogenesis by EphA2 depletion was blocked by BafA1, placing autophagy between EphA2 signaling and regulation of ciliogenesis.

Conclusion

Our findings demonstrate a novel role for EphA2 in regulating both autophagy and ciliogenesis, processes that are essential for proper corneal epithelial homeostasis.

中文翻译：

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纤毛发生和自噬由角膜中的 EphA2 协调调节，以维持适当的上皮结构

目的

了解角膜上皮纤毛发生与自噬之间的关系。

方法

EphA2 或 PLD1 的 siRNA 用于体外抑制蛋白质表达。Morpholino - anti -EphA2 用于敲低非洲爪蟾皮肤中的 EphA2。使用 EphA2 敲除小鼠进行功能丧失研究。通过对比光学显微镜观察自噬泡。通过 LC3 转换和 p62 蛋白水平测量自噬通量。进行免疫染色和共聚焦显微镜以观察培​​养细胞和体内的纤毛。

结果

EphA2 的缺失（i）通过提高翼细胞的增殖潜能来增加角膜上皮的厚度，（ii）减少纤毛细胞的数量，（iii）增加大的中空液泡，这可以通过 BafA1 来挽救；(iv) 抑制自噬通量和 (v) 增加小鼠角膜上皮中的 GFP-LC3 斑点。这表明 EphA2 通过调节增殖在分层上皮组装中的作用以及在纤毛发生和终末期自噬中的积极作用。PLD1 是一种 EphA2 相互作用蛋白，它是终末期自噬的关键调节因子，抑制 PLD1 可逆转液泡的积累，以及由于 EphA2 耗竭导致纤毛细胞数量减少，这表明 EphA2 调节终末期自噬和纤毛发生通过PLD1。

结论

我们的研究结果证明了 EphA2 在调节自噬和纤毛发生中的新作用，这些过程对于适当的角膜上皮稳态至关重要。