PAX6ΔPD modulates the balance between proliferation and differentiation programs in corneal epithelial cells through protein-protein interactions.

PAX6 is a key regulator of corneal epithelial differentiation, expressed as multiple variants that result either from alternative splicing or by the use of an alternative internal promoter. These variants include the canonical PAX6, PAX6(5a), and the paired-less PAX6ΔPD variant. This study evaluated the functional roles of these variants in rabbit RCE1(5T5) corneal epithelial cells. Endogenous PAX6 and PAX6(5a) proteins were detected in a 2:1 ratio, consistent with previously reported mRNA expression, while PAX6ΔPD was not clearly observed. Overexpression of each variant revealed distinct biological effects. PAX6 and PAX6(5a) did not enhance proliferation and led to reduced growth upon serial passage; in contrast, PAX6ΔPD significantly increased cell proliferation and sustained growth capacity. Consistently, PAX6 and PAX6(5a) promoted differentiation, as evidenced by increased expression of the terminal differentiation marker KRT3, while PAX6ΔPD inhibited differentiation and led to an increase in ΔNp63α expression, a marker associated with proliferative and stem/progenitor-like states. Mechanistically, bimolecular fluorescence complementation (BiFC) assays demonstrated that all variants could form homo- and heterodimers, with PAX6ΔPD exhibiting enhanced dimerization potential, particularly with PAX6(5a). Mutation of a conserved motif in PAX6ΔPD reduced these interactions, suggesting that protein-protein interactions underlie its functional effects. Overall, these findings suggest that PAX6 and PAX6(5a) promote corneal epithelial differentiation, whereas PAX6ΔPD antagonizes this process by sustaining proliferation, likely through competitive interactions with other PAX6 variants.