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H.D. Cavanagh, D.M. Robertson, S.I. Ho, J. Mei, K.C. Dimiceli, W.M. Petroll; Characterization of IGFBP3 in Telomerase–Immortalized Human Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4922.
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© ARVO (1962-2015); The Authors (2016-present)
Insulin–like Growth Factor Binding Protein–3 (IGFBP3) has known functional roles in inducing apoptotic cell death in mammalian epithelial cells. The purpose of this study was to characterize the presence and subcellular localization of IGFBP3 in a telomerase–immortalized human corneal epithelial cell line (hTCEpi).
hTCEpi cells were grown in KGM–2 culture medium containing 0.15mM calcium. For RT–PCR, RNA was extracted using RNA STAT60 and cDNA generated by SuperScript First Round Synthesis using random primers. Primers specific for both full–length isoforms, differing only within an intronic splice site, were used. For immunofluorescence analysis, hTCEpi cells were plated on collagen–coated glass coverslips and probed with an IGFBP3 goat polyclonal antibody and a FITC–conjugated secondary antibody. Cells were double–labeled with Propidium Iodide. Cell lystates were further analyzed by Western blotting. To further characterize the possibility of a transient upregulation of IGFBP3 during the apoptotic process, cells were incubated in 1µM Staurosporine and imaged from 0–60 minutes at 5 minute intervals. All cells were imaged on a Leica SP2 laser scanning confocal microscope.
RT–PCR revealed the presence of both isoforms of IGFBP3; 893 bp variant 1 and 875 bp variant 2. While the majority of hTCEpi cells were negative for IGFBP3 by immunofluorescence, occasional IGFBP3 positive cells were found. Western blotting confirmed protein expression. Following treatment with Staurosporine, we saw an early, transient, upregulation of IGFBP3 in the cytoplasm, followed by translocation to the nucleus.
These findings demonstrate for the first time, the presence of IGFBP3 in human corneal epithelial cells. These findings further suggest a potential apoptotic signaling role for IGFBP3 in corneal epithelial cells. Additional studies to elucidate the function of IGFBP3 in the human cornea are needed.
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