Transcription factor profiling identifies Sox9 as regulator of proliferation and differentiation in corneal epithelial stem/progenitor cells

Understanding transcription factor (TF) regulation of limbal epithelial stem/progenitor cells (LEPCs) may aid in using non-ocular cells to regenerate the corneal surface. This study aimed to identify and characterize TF genes expressed specifically in LEPCs isolated from human donor eyes by laser capture microdissection. Using a profiling approach, preferential limbal expression was found for SoxE and SoxF genes, particularly for Sox9, which showed predominantly cytoplasmic localization in basal LEPCs and nuclear localization in suprabasal and corneal epithelial cells, indicating nucleocytoplasmic translocation and activation during LEPC proliferation and differentiation. Increased nuclear localization of Sox9 was also observed in activated LEPCs following clonal expansion and corneal epithelial wound healing. Knockdown of SOX9 expression in cultured LEPCs by RNAi led to reduced expression of progenitor cell markers, e.g. keratin 15, and increased expression of differentiation markers, e.g. keratin 3. Furthermore, SOX9 silencing significantly suppressed the proliferative capacity of LEPCs and reduced levels of glycogen synthase kinase 3 beta (GSK-3ß), a negative regulator of Wnt/ß-catenin signaling. Sox9 expression, in turn, was significantly suppressed by treatment of LEPCs with exogenous GSK-3ß inhibitors and enhanced by small molecule inhibitors of Wnt signaling. Our results suggest that Sox9 and Wnt/ß-catenin signaling cooperate in mutually repressive interactions to achieve a balance between quiescence, proliferation and differentiation of LEPCs in the limbal niche. Future molecular dissection of Sox9-Wnt interaction and mechanisms of nucleocytoplasmic shuttling of Sox9 may aid in improving the regenerative potential of LEPCs and the reprogramming of non-ocular cells for corneal surface regeneration.