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Han Peng, Spiro Getsios, Wending Yang, Robert M. Lavker; Factor-inhibiting Hypoxia-inducible Factor 1 (FIH-1) Is A Positive Regulator Of Corneal Epithelial Migration Under Normoxic Conditions. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2208.
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© ARVO (1962-2015); The Authors (2016-present)
Previously we demonstrated that the hydroxylase, FIH-1, a relatively unrecognized component of keratinocytes, plays a central role in negatively regulating corneal epithelial differentiation by attenuating Notch signaling. Increasing FIH-1 levels in human corneal epithelial keratinocytes (HCEKs) also decreased AKT signaling and activated GSK-3β, which plays an important role in membrane polarization and directional migration of epithelial cells. Since proper corneal epithelial migration is essential for maintaining corneal homeostasis and critical during re-epithelialization following wounding, we investigated whether FIH-1 plays a role in corneal epithelial migration.
The telomerase immortalized human corneal epithelial cell line (hTCEpi) has high levels of FIH-1. Therefore, we used dimethyloxalyglycine (DMOG) to inhibit FIH-1 or siRNA technology to reduce the levels of FIH-1 in this cell line. Such loss of function studies were combined with immunoblotting and linear scratch wound assays to study migratory activity. These studies were complemented with gain of function experiments using HCEKs, which have relatively low levels of FIH-1. HCEKs were transduced with either an empty vector or FIH-1-cds, in conjunction with live cell imaging to assess single cell migration.
FIH-1 is abundantly expressed in hTCEpi cells and these cells rapidly seal linear scratch wounds. Treatment of hTCEpi cells with DMOG or knock-down of FIH-1 in these cells resulted in a: (i) significant delay in sealing linear scratch wounds (DMOG - 50%, shFIH - 20%) compared with controls; (ii) decrease in phosphorylation of focal adhesion kinase (FAK) ; and (iii) inactivation of GSK3β. In contrast, increasing the levels of FIH-1 in HCEKs enhanced the directional migration (distance/path length) of single corneal keratinocytes by 50% and activated GSK3β.
Collectively, these observations demonstrate that FIH-1 acts as a motogen in corneal epithelial keratinocytes by increasing GSK3β-mediated directional cell migration.
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