C-terminal cleavage of δNp63α Is associated with TSA-induced apoptosis in immortalized corneal epithelial cells

Abstract

PURPOSE. In the central human corneal epithelium, loss of ΔNp63 occurs in all surface epithelial cells preparing to undergo desquamation, suggesting a potential role for ΔNp63 isoforms in mediating surface cell apoptotic shedding. In this study, the authors investigated a role for ΔNp63 isoforms in caspase-mediated apoptosis in a telomerase-immortalized corneal epithelial cell line. METHODS. For in vitro studies, hTCEpi cells were cultured in KGM-2 serum-free culture media containing 0.15 mM calcium. To assess dynamic protein interactions among individual ΔNp63 isoforms, ΔNp63-EGFP expression plasmids were transiently expressed in hTCEpi cells and evaluated by FRAP. Trichostatin-A (TSA; 3.31 μM) was used to induce cell death as measured by caspase activity. Cleavage and loss of endogenous ΔNp63 α ΔNp63-EGFP expression plasmids, and p53 were assessed after treatment with TSA and siRNA. RESULTS. Transient expression of ΔNp63-EGFP α and β isoforms resulted in the formation of a smaller isoform similar in size to ΔNp63 α -EGFP. FRAP demonstrated that ΔNp63 α -EGFP has greater immobile fraction than α or β. TSA induced caspasemediated apoptotic pathways; caspase induction was accompanied by a decrease in endogenous ΔNp63 α and p53. TSA upregulated ΔNp63-EGFP plasmid expression; this was accompanied by a selective increase in cleavage of ΔNp63 α -EGFP. siRNA knockdown of ΔNp63 α correlated with a reduction in p53 independently of TSA. CONCLUSIONS. ΔNp63 α is the dominant active isoform in corneal epithelial cell nuclei. Loss of ΔNp63 α occurs during apoptotic signaling by cleavage at the C terminus. The corresponding loss of p53 suggests that a significant relationship appears to exist between these two regulatory proteins. © Association for Research in Vision and Ophthalmology.