PAX6-WNK2 axis governs corneal epithelial homeostasis

PURPOSE. Limbal stem/progenitor cells (LSC) continuously proliferate and differentiate to replenish the corneal epithelium and play a vital role in corneal function and normal vision. Previous study revealed that Paired box 6 (PAX6) is a master transcription factor involved in determining the fate of corneal epithelial cells (CEC). 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 LSC into mature CEC. Specific targeting PAX6 short-hairpin shRNAs were used to knock down PAX6 in LSC. RNA-seq was used to analyze shPAX6-transfected CEC 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. To elucidate the regulatory mechanisms of PAX6 in driving corneal epithelial cells (CEC) differentiation, human primary limbal stem/progenitor cells (LSC) were cultured. A serum-free air-liquid interface system was established to obtain mature CEC in vitro. Short-hairpin shRNAs were used to knock down the expression of PAX6 or WNK2 in LSC, which were then differentiated into CEC, with scramble shRNA serving as control for each group. RNA samples frpm LSC, CEC, scramble-transfected CEC, shPAX6-transfected CEC, and shWNK2-transfected CEC were extracted and subjected to RNA-seq analysis. Differential gene expression analysis was performed to compare the gene expression: LSC versus CEC, scramble-transfected CEC versus shPAX6-transfected CEC, and scramble-transfected CEC versus shWNK2-transfected CEC.