Abstract
The Pax6 gene encodes the Pax6 transcription factor, whose sequence and expression pattern are highly conserved in evolution. Pax6 resides at the top of a genetic hierarchy controlling development and morphogenesis of the eye. The gene is expressed in various tissues of the anterior segment and retina during development. Pax6 is also expressed in specific tissues of the postnatal eye, including the corneal epithelium, and protein levels and DNA binding activity of Pax6 are increased at the front of corneal epithelium migrating to close a wound. This suggests that Pax6 is also involved in corneal epithelial dynamics in the adult eye. This study investigated the mechanism whereby Pax6 modulates corneal epithelial cell growth by studying the effects of over-expression.To address the hypothesis, cultures of an immortalized corneal epithelial cell line (SIRC) were stably-transformed with Pax6 and individual transformed cells were cloned to obtain pure populations for study. To avoid any confounding effects on cell growth or survival due to Pax6 expression during clonal expansion, the tetracycline-inducible gene expression (Tet-On) system was used. To further assess biological relevance, Pax6 over-expression was also performed in primary cultures of rabbit corneal epithelial cells using the efficient adenoviral-mediated transfection method. Over-expression of a truncated form of Pax6 (Pax6Delta286) from which the transcriptional activation domain had been deleted was also examined in both cell lines for comparison.The Tet-On system was fully functional in the stably-transformed SIRC clones and the level of Pax6 was tightly regulated by doxycycline. Pax6 over-expression retarded cell proliferation by inhibiting entry into S phase. The truncated Pax6 protein Pax6Delta286 had no effects on cell proliferation. Over-expression of Pax6 induced caspase-3 independent apoptosis. Recombinant mPax6 adenovirus transduced primary cultures of rabbit corneal epithelium cells also resulted in retarded entry into S phase. Overexpression of Pax6 in SIRC cells also inhibited corneal epithelial cell migration. The mechanisms whereby Pax6 modulates cell migration will be further investigated in a follow-up study.In summary, the feature of cell cycle retardation and cell migration inhibition in Pax6 over-expressing cells supports the hypothesis that Pax6 plays a role in the maintenance of adult corneal epithelial cell homeostasis.