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J. Shankardas, S. Dimitrijevich; Intracellular Distribution of Np63 During Corneal Epithelial Proliferation and Differentiation. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2959.
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To study the role of ΔNp63 in proliferation and differentiation of human corneal epithelial cellsIntroduction: p63, a p53 homolog, was proposed as putative marker of epithelial stem cells found in specific niches of skin & cornea but was later found to be expressed in transient amplifying cells. Six splice variants of p63 are now known comprising two TA and ΔN groups, each of which consists of α, β and γ isoforms. In epithelial cell homeostasis TA and ΔNp63 have been shown to have opposing functions. The ΔNp63 isoform suppresses the expression of p53 and TAp63, and controls the fate of stem cell fate by competing for the p53 binding site on 14-3-3 σ promoter thereby suppressing its expression. In the corneal epithelia, it has been shown by RT-PCR, that ΔNp63α is the predominant isoform, which plays a major role in epithelial cell proliferation. The expression and localization of the p63 isoforms in response to differentiation in the human corneal epithelium has not been studied.
Indirect immunofluorescence was used to determine the localization of TAp63 and ΔNp63 in the central and limbal epithelial cells of the corneal epithelium. Primary CEC and hTERT CEC were cultured under proliferation and differentiation conditions. Localization of ΔNp63 was determined by indirect IF and compared with proliferating controls. The quantitated expression levels of ΔNp63 were derived from western blot analysis.
TAp63 expression was observed in the superficial layers of the central cornea and was absent in the limbus. ΔNp63 localization is nuclear in the basal cells of the corneal epithelium.ΔNp63 localization is cytosolic in the superficial/differentiated cells of both the central cornea and the limbus. Western blot analysis showed that ΔNp63 expression decreases under differentiation conditions. Translocation of ΔNp63 from the nucleus to the cytoplasm also takes place as shown by indirect immunofluorescence.
Primary cultures of hCEC contain a heterogeneous population of proliferating and differentiated/differentiating cells. The presence of differentiated cells may be characterized by the cytosolic localization of ΔNp63. The translocation of ΔNp63 is more prominent in the hTERT CEC cells in which ΔNp63 in completely nuclear, but after 2-weeks under differentiation conditions; small populations of cells begin to show cytosolic localization of ΔNp63. Since p53 may target ΔNp63α for degradation, it may be one of the upstream regulators involved in the differentiation cascade. Since there is no known cytosolic function for ΔNp63, its translocation to the cytosol could be an indication of targeted degradation.
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