Characterization of ΔNp63 isoforms in normal cornea and telomerase-immortalized human corneal epithelial cells

Abstract

Previous reports have suggested that specific isoforms of the potential stem cell marker p63 may regulate corneal epithelial homeostatic renewal through control of cell proliferation. In this study, we characterized the presence of ΔNp63 isoforms in telomerase-immortalized human corneal epithelial cells (hTCEpi) in comparison to normal human corneal epithelium to validate the hTCEpi cell line as a viable model for the study of p63 isoforms. We further examined roles for ΔNp63 in proliferation and differentiation. For in vitro studies, hTCEpi cells were cultured in serum-free culture media and grown under 0.15mM calcium or sequential 1.15mM calcium/air-lifted culture. Fresh donor human corneal tissue was used to assess expression and localization in situ. mRNA and protein levels were assessed by real-time PCR, Immunofluorescence (IF) and Western blotting (WB). ΔNp63 expression levels throughout the cell cycle were assessed by double-labeling with ΔNp63 and Ki-67. In situ, ΔNp63 localized to nuclei throughout the human corneal epithelium and was lost only in superficial cells. WB confirmed the presence of all three ΔNp63 isoforms in the central corneal epithelium and in hTCEpi cells. ΔNp63 mRNA levels decreased when grown on collagen substrate and under increased calcium/air-lifted culture. mRNA and protein levels increased as cells approached confluence, with a significant decrease in post-confluent culture. ΔNp63 expression levels did not vary with the cell cycle, as assessed by Ki-67 labeling. Collectively, the presence of all three ΔNp63 isoforms in hTCEpi cells and in intact cornea validates the use of this cell line for the study of individual isoforms in the corneal epithelium; and these data suggest that expression of ΔNp63 isoforms are not altered as a function of the cell cycle or cell division in subconfluent hTCEpi cells cultured in serum-free media, but demonstrate reduced expression upon contact-inhibited growth down-regulation and differentiation. Significantly, the localization of ΔNp63 in central corneal epithelial cells with a loss of expression in superficial cells suggests that ΔNp63 may play a role in mediating desquamative events at the ocular surface.