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D. M. Robertson, S. I. Ho, H. D. Cavanagh; Interactive Regulation of Cell Survival in Corneal Epithelial Cells by Np63/β:Np63. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4300.
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© ARVO (1962-2015); The Authors (2016-present)
The nuclear transcription factor ΔNp63 is subject to alternative splicing at the C terminus producing three distinct isoforms: α, β, and γ. It has been well established that all three isoforms are present in the human corneal epithelium; however, the exact function is unknown. The purpose of this study was to characterize the relationship between isoforms in corneal epithelial cells and to establish the role each of these isoforms play in mediating cell survival.
hTCEpi cells were cultured in KGM-2 serum-free culture media. cDNA encoding α, β, and γ isoforms were generated by PCR and cloned into pEGFP expression plasmids. For all experiments, hTCEpi cells were transfected using FuGene 6 transfection reagent and isoform expression was measured by western blot using mouse monoclonal antibodies directed against the N-terminus of ΔNp63 (4A4), the anti-apoptotic protein BCL2, and EGFP. An empty vector was used as a control. Isoforms were further assessed following incubation in 3.31 µM Trichostatin-A (TSA), an inhibitor of histone deacetylase.
Enhanced expression of ΔNp63α and ΔNp63β resulted in an increase in ΔNp63γ; whereas enhanced expression of each individual isoform (α, β, γ) down-regulated BCL2 expression. Interestingly, over-expression of each isoform also resulted in an increase in non-adherent cells compared to the EGFP control. Incubation in TSA, which has been previously reported to induce robust cell death in hTCEpi cells, potentiated the increase in ΔNp63γ seen following ΔNp63α and ΔNp63β over-expression and produced a concurrent decrease in endogenous ΔNp63α levels.
Taken together, these findings suggest that hTCEpi cells are sensitive to small changes in ΔNp63γ and that an alteration in the ratio of ΔNp63α/β to γ may play a role in apoptotic signaling through BCL2 in corneal epithelial cells.
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