Activation of ATF2 During Healing of Corneal Epithelium Debridement

Abstract

Purpose: : To examine whether ATF2 involved in healing of corneal epithelium debridement.

Methods: : TGF–ß type II receptor (Tbr2) floxed mice were bred with Krt12–Cre mice to generate bitransgenic mice in which the TGF–ß receptor 2 gene (Tbr2) was disrupted selectively in the corneal epithelial cells. Corneal epithelial debridement (2 mm diameter) was created in 2–month–old bitransgenic Tbr2^ceΔ/ceΔ (Krt12^Cre/CreTbR2^f/f) mice and their littermates as controls Tbr2^ceΔ/w (Krt12^Cre/CreTbR2^f/w) and Tbr2^w/w (Krt12^Cre/CreTbR2^w/w) The healing of corneal epithelium defect was assessed by fluorescein staining at different intervals of debridement. The experimental mice were administrated by i.p. injection of BrdU two hours prior to sacrifice to determine cell proliferation. Immunohistochemistry with rabbit monoclonal anti–phospo–ATF2 antibody and other antibodies directed against other signaling molecules was performed to elucidate their roles in wound healing.

Results: : The corneal epithelium of Tbr2^ceΔ/ceΔ mice lost TbR2 as judged by western blot analysis with anti–Tbr2 antibodies. The naïve uninjured corneal epithelium of Tbr2^ceΔ/ceΔ mice exhibited higher cell proliferative activities than controls. Tbr2^ceΔ/ceΔ mice exhibited delayed healing of debridement in comparison to control littermates Tbr2^ceΔ/w and Tbr2^w/w mice. Corneal epithelium debridement caused cessation of epithelial cell proliferation in all three groups of experimental mice in 6–12 h. Immunohistochemistry using anti–phospho–p38 and phosphoATF2 revealed that no phosphor–p38 nor phosphor–ATF2 was detected in naïve uninjured corneas. A significant delayed phosphorylation of p38 was observed at 48 h of debridement of Tbr2^ceΔ/ceΔ mice, whereas in control littermates p38 phosphorylation was seen in 6 h. In contrast, no difference in activation of ATF2 was observed at 6–24 h of epithelium debridement in all three genotypes of mice. There was a second phase of ATF2 activation at 48–72 h of debridement in corneas of Tbr2^ceΔ/ceΔ mice, which was not seen in control littermates.

Conclusions: : Our results indicate that there is an alternative pathway for the activation of ATF2 independent of TGF–ß signaling pathways. The activation of ATF2 may account for the cessation of epithelial cell proliferation at early stages of corneal epithelial wound healing.

Keywords: cornea: epithelium • wound healing • growth factors/growth factor receptors