Abstract
Purpose: :
Three ΔNp63 isoforms (, β, γ) are expressed throughout the corneal epithelium; however the exact function of these isoforms is unknown. The purpose of this study was to investigate a role for ΔNp63 isoforms in regulating apoptosis through interactions with the apoptotic regulatory proteins p53 and BCL2 in telomerase-immortalized corneal epithelial cells (hTCEpi).
Methods: :
hTCEpi cells were cultured in KGM-2 serum-free culture media. BCL2 was examined as a function of confluence. Levels of ΔNp63, p53 and BCL2 were assessed by western blot following over-expression of ΔNp63-EGFP isoforms and siRNA knockdown. Protein expression was further assessed following incubation in 3.31 µM and 331 nM Trichostatin-A (TSA), an inhibitor of histone deacetylase. Caspase 3 activity was measured using the Image-iT LIVE Red Caspase 3 Detection Kit.
Results: :
Exogenous expression of ΔNp63-EGFP isoforms decreased BCL2 but failed to alter p53; increased levels of endogenous ΔNp63 as a function of confluence did not alter BCL2. siRNA knockdown and TSA-stimulated reduction of endogenous ΔNp63 resulted in a decrease in p53 and upregulated BCL2. While TSA decreased endogenous levels of ΔNp63, there was an increase in ΔNp63-EGFP expression accompanied by simultaneous cleavage of ΔNp63 and β to γ and significant caspase 3 activation. A p53-regulated increase in BCL2 following TSA treatment failed to abrogate caspase 3-mediated apoptosis.
Conclusions: :
Taken together, these results demonstrate that selective cleavage of ΔNp63 and β at the N-terminus to ΔNp63γ, which is not affected by EGFP, occurs during caspase 3-mediated apoptosis, suggesting that loss of these isoforms is necessary for cell death. The concomitant upregulation of BCL2 in response to loss of ΔNp63 appears to be unable to block apoptosis suggesting that the pro-survival effects of BCL2 function upstream of ΔNp63, potentially through p53. As these cells are K3/K12 negative and ABCG2 positive and can be induced to differentiate, these interactions likely represent signaling events in the basal compartment and not desquamative events at the ocular surface.
Keywords: cornea: epithelium • apoptosis/cell death