Epidermal growth factor-induced proliferation requires down-regulation of Pax6 in corneal epithelial cells

Growth factors play important roles in regulating corneal epithelial cell proliferation/ differentiation during wound healing. It is suggested that PAX6 involves corneal epithelium lineage-specific differentiation ( Liu, J. J., Kao, W. W., and Wilson, S. E. ( 1999) Exp. Eye Res. 68, 295 - 301); however, the regulatory mechanism and function of Pax6 in growth factor- induced corneal epithelial responses is still unknown. In the present study, we found that the mitogenic effect of epidermal growth factor ( EGF) in corneal epithelial cells required suppression of PAX6 activity through cellular mechanisms involving Erk-signaling pathway- mediated increase in CTCF expression. EGF- induced CCCTC binding factor ( CTCF) activation subsequently inhibited Pax6 expression by interacting with a CTCF- specific region upstream of the pax6 P0 promoter. Suppression of EGF-induced Erk activation by specific inhibitor or by the dominant expression of a silent Erk mutant effectively abolished the effects of EGF stimulation on regulations of CTCF and pax6. Apparently, down-regulation of Pax6 expression induced by EGF is required for corneal epithelial proliferation, because overexpression of pax6 in these cells attenuated EGF- induced proliferation. In contrast, knockdown of mRNA expression with pax6- or CTCF- specific small interfering RNA in corneal epithelial cells significantly promoted or attenuated EGF- induced proliferation, respectively. Thus, our results revealed a new regulatory mechanism that involves cellular signaling events and pax6 transcription regulation in growth factor- mediated proliferation. In corneal epithelial cells, this suggests that inhibition of pax6 expression is a prerequisite for EGF to elicit controls of cell growth and fate.