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H. D. Cavanagh, D. M. Robertson, S. I. Ho, B. S. Hansen, W. M. Petroll; Transcriptional Activity of Np63 Isoforms in Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2744.
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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, ß, and γ. The specific function of these isoforms in the human corneal epithelium is unknown. The purpose of this study was to characterize the presence and transcriptional activity of ΔNp63 isoforms in telomerase-immortalized human corneal epithelial cells (hTCEpi).
hTCEpi cells were cultured in KGM-2 serum-free culture media. Relative levels of mRNA and protein were assessed by Real-Time PCR (RT PCR) and Western blotting (WB). Antibodies recognizing pan-ΔNp63 isoforms, and alpha and gamma specific termini were used. cDNA encoding α, ß, 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 assessed by WB and RT PCR at 0, 24, 48, and 72 hours. To establish dynamic interactions with nuclear chromatin, relative mobility was analyzed by FRAP on a Leica SP2 laser scanning confocal microscope.
WB confirmed the presence of ΔNp63α, ß, and γ in hTCEpi cells. ΔNp63α was the predominant isoform present, followed by ß, and trace amounts of γ. RT PCR detected the presence of all three isoforms, with highest mRNA levels seen with γ. The high levels of γ mRNA did not appear to correlate with protein levels. RT PCR and WB confirmed high expression of all three isoforms. Over-expression of ΔNp63α led to an upregulation of ß, and a decrease in γ. Over-expression of ΔNp63ß also led to a reduction in γ. Interestingly, over-expression of γ had no effect on α, but upregulated ß. FRAP analysis of proteins within the nuclear compartment showed a significantly higher immobile fraction for α and ß, than for γ (P<0.001).
All three ΔNp63 isoforms are present and possess the ability to regulate transcriptional activity in hTCEpi cells. Further, ΔNp63 isoforms demonstrate the ability to auto-regulate one another in vitro, suggesting a second level of modulation of gene function in the human corneal epithelium in vivo.
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