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D. M. Robertson, B. S. Hansen, S. I. Ho, W. M. Petroll, H. D. Cavanagh; Np63 Modulates IGFBP3 Activity in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2743.
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© ARVO (1962-2015); The Authors (2016-present)
Insulin-like Growth Factor Binding Protein-3 (IGFBP3) is a high affinity binding protein shown to regulate cell growth, differentiation, and apoptosis in a variety of cellular systems. The purpose of this study was to characterize IGFBP3 mediated apoptotic signaling in the corneal epithelium and to establish a link between the transcription factor ΔNp63 and IGFBP3 transcriptional control in human corneal epithelial cells.
Human telomerase-immortalized corneal epithelial cells (hTCEpi) were cultured in serum-free KGM2 culture media. cDNA encoding ΔNp63α, ß, and γ isoforms were generated by PCR and cloned into pEGFP expression plasmids. hTCEpi cells were then transfected and IGFBP3 was assessed by Western Blotting and Real time PCR. IGFBP3 levels were further measured following treatment with Trichostatin (TSA), an inhibitor of histone deacetylation. To establish apoptotic potential, hTCEpi cells were treated with either recombinant human IGFBP3 (rhIGFBP3) or (TSA) for up to 24 hours. Apoptosis was assayed using a Calcein-AM Ethidium-1 Viability/Cytotoxicity Assay or triple-labeling with Calcein-AM, Annexin V, and PI. All cells were imaged on a Leica SP2 laser scanning confocal microscope.
Independent over-expression of ΔNp63α, ß, and γ appeared to repress already low levels of IGFBP3 mRNA compared to controls. Addition of TSA to the culture media resulted in a differential upregulation of IGFBP3, dependent on the specific ΔNp63 isoform expressed. The largest increase in IGFBP3 mRNA was seen following over-expression of ΔNp63gamma + TSA. Treatment of cells with TSA alone resulted in a significant increase in Annexin V positive cells at 18 and 24 hours (P<0.001). Apoptosis was also induced by the addition of rhIGFBP3 to cell culture media.
These results demonstrate, for the first time, that ΔNp63 isoforms independently function to repress IGFBP3 transcription in corneal epithelial cells and that these effects are modulated in part, by histone acetylation. Taken together, these findings suggest that ΔNp63 isoforms may play a role in mediating differentiation and apoptosis in the corneal epithelium.
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