State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangdong Provincial Key Laboratory of Ophthalmology and Visual Science, Guangzhou, China.
Department of Experimental Research, Bioinformatics Platform, State Key Laboratory of Oncology in South China, Guangdong Provincial Clinical Research Center for Cancer, Sun Yat-Sen University Cancer Center, Guangzhou, China.
Invest Ophthalmol Vis Sci. 2024 Oct 1;65(12):40. doi: 10.1167/iovs.65.12.40.
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.
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.
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.
As a downstream target of PAX6, WNK2 plays an essential role in corneal epithelial cell differentiation and maintenance of corneal homeostasis.
角膜缘干细胞/祖细胞（LSCs）持续增殖和分化，以补充角膜上皮，并在角膜功能和正常视力中发挥重要作用。先前的研究表明，配对盒 6（PAX6）是一种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清楚。在这项研究中，我们旨在阐明 PAX6 驱动 CEC 分化的调控网络。
采用气液培养系统将 LSCs 分化为成熟的 CECs。使用特定靶向 PAX6 的短发夹 RNA 敲低 LSC 中的 PAX6。使用 RNA 测序（RNA-seq）分析 shPAX6 转染的 CECs 和与 CEC 分化相关的基因，以鉴定 PAX6 的潜在下游靶标。进行 RNA-seq 分析、定量实时 PCR 和免疫荧光染色，以阐明 PAX6 的下游靶标 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。
WNK2 的表达在 CEC 分化过程中增加，而在 PAX6 耗尽时减少。WNK2 的分布特异性局限于中央角膜上皮和角膜缘的基底上层。WNK2 的敲低会损害 CEC 特异性标记物（KRT12、ALDH3A1 和 CLU）的表达，破坏角膜分化过程，并激活角化、炎症和细胞增殖等术语，与 PAX6 耗尽的 CEC 和已发表的微生物角膜炎一致。因此，WNK2 的异常表达与角膜溃疡有关。
作为 PAX6 的下游靶标，WNK2 在角膜上皮细胞分化和维持角膜内稳态中发挥重要作用。
