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.