PAX6-WNK2 Axis Governs Corneal Epithelial Homeostasis.

IF 5 2区 医学 Q1 OPHTHALMOLOGY Investigative ophthalmology & visual science Pub Date : 2024-10-01 DOI:10.1167/iovs.65.12.40
Liqiong Zhu, Chaoqun Chen, Siqi Wu, Huizhen Guo, Lingyu Li, Li Wang, Dongmei Liu, Yu Zhan, Xinyue Du, Jiafeng Liu, Jieying Tan, Ying Huang, Kunlun Mo, Xihong Lan, Hong Ouyang, Jin Yuan, Xiangjun Chen, Jianping Ji
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Abstract

Purpose: Limbal stem/progenitor cells (LSCs) continuously proliferate and differentiate to replenish the corneal epithelium and play a vital role in corneal function and normal vision. A previous study revealed that paired box 6 (PAX6) is a master transcription factor involved in determining the fate of corneal epithelial cells (CECs). However, the molecular events downstream of PAX6 remain largely unknown. In this study, we aimed to clarify the regulation network of PAX6 in driving CEC differentiation.

Methods: An air-liquid culture system was used to differentiate LSCs into mature CECs. Specific targeting PAX6 short-hairpin RNAs were used to knock down PAX6 in LSC. RNA sequencing (RNA-seq) was used to analyze shPAX6-transfected CECs and CEC differentiation-associated genes to identify the potential downstream targets of PAX6. RNA-seq analysis, quantitative real-time PCR, and immunofluorescence staining were performed to clarify the function of WNK lysine deficient protein kinase 2 (WNK2), a downstream target of PAX6, and its relationship with corneal diseases.

Results: WNK2 expression increased during CEC differentiation and decreased upon PAX6 depletion. The distribution of WNK2 was specifically limited to the central corneal epithelium and suprabasal layer of the limbus. Knockdown of WNK2 impaired the expression of CEC-specific markers (KRT12, ALDH3A1, and CLU), disrupted the corneal differentiation process, and activated the terms of keratinization, inflammation, and cell proliferation, consistent with PAX6-depleted CEC and published microbial keratitis. Thus, aberrant expression of WNK2 was linked to corneal ulcers.

Conclusions: As a downstream target of PAX6, WNK2 plays an essential role in corneal epithelial cell differentiation and maintenance of corneal homeostasis.

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PAX6-WNK2 轴控制角膜上皮细胞的稳态。
目的:角膜缘干细胞/祖细胞(LSCs)不断增殖和分化,以补充角膜上皮,在角膜功能和正常视力中发挥着重要作用。先前的一项研究发现,配对盒 6(PAX6)是参与决定角膜上皮细胞(CECs)命运的主转录因子。然而,PAX6 下游的分子事件在很大程度上仍不为人知。本研究旨在阐明 PAX6 在驱动 CEC 分化过程中的调控网络:方法:采用气液培养系统将 LSCs 分化为成熟的 CECs。方法:采用气液培养系统将 LSCs 分化为成熟的 CECs。利用 RNA 测序(RNA-seq)分析 shPAX6 转染的 CECs 和 CEC 分化相关基因,以确定 PAX6 的潜在下游靶标。为了明确 PAX6 下游靶标 WNK 赖氨酸缺陷蛋白激酶 2(WNK2)的功能及其与角膜疾病的关系,研究人员进行了 RNA-seq 分析、定量实时 PCR 和免疫荧光染色:结果:WNK2的表达在CEC分化过程中增加,并在PAX6耗竭时减少。WNK2的分布特别局限于角膜中央上皮和角膜缘基底上层。WNK2的敲除损害了角膜上皮细胞特异性标记物(KRT12、ALDH3A1和CLU)的表达,破坏了角膜分化过程,激活了角质化、炎症和细胞增殖,这与PAX6耗竭的角膜上皮细胞和已发表的微生物性角膜炎一致。因此,WNK2的异常表达与角膜溃疡有关:结论:作为 PAX6 的下游靶标,WNK2 在角膜上皮细胞分化和维持角膜稳态中发挥着重要作用。
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来源期刊
CiteScore
6.90
自引率
4.50%
发文量
339
审稿时长
1 months
期刊介绍: Investigative Ophthalmology & Visual Science (IOVS), published as ready online, is a peer-reviewed academic journal of the Association for Research in Vision and Ophthalmology (ARVO). IOVS features original research, mostly pertaining to clinical and laboratory ophthalmology and vision research in general.
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