FIH-1/c-kit signaling: a novel regulator of corneal epithelial glycogen metabolism

Abstract

Purpose: Corneal epithelium has large glycogen stores, which serve as a primary energy source. Recently, we demonstrated that Factor-inhibiting hypoxia-inducible factor 1 (FIH-1) reduces corneal epithelial glycogen, in part, by diminishing Akt signaling. Interestingly, such an inhibitory effect did not appear to involve the mTOR pathway. Because c-kit signaling is known to regulate the Akt/GSK-3β pathway, we investigated the relationship between FIH-1 and c-kit as it relates to limbal and corneal epithelial glycogen metabolism.

Methods: We evaluated the corneal and limbal epithelia from mice that were either null for FIH-1 or deficient in c-kit signaling ability. Limbal and corneal epithelia from wild-type, FIH-1^-/- and Kit^W/Wv mice were stained with Periodic Acid-Schiff (PAS) to detect glycogen. Quantification was carried out using computer-assisted image analysis. RNA samples prepared from laser-capture microdissected populations of limbal epithelium were subjected to real-time qPCR to determine c-kit ligand (stem cell factor) expression . Submerged cultures of primary human corneal epithelial keratinocytes (HCEKs) transduced with FIH-1 were treated with c-kit ligand to establish further a FIH-1/c-kit interaction via Western analysis.

Results: FIH-1 is constitutively present in the limbal epithelium and markedly diminished in the corneal epithelium of wild-type mice. In these mice, a reciprocal relationship is observed with respect to FIH-1 and glycogen, with more glycogen present in the corneal epithelium compared with the limbal epithelium. Mice null for FIH-1 had increased amounts of limbal epithelial glycogen as well as increased c-kit ligand mRNA compared with wild-type controls. Consistent with a FIH-1/c-kit association, the diminished Akt signaling observed in FIH-1-overexpressing HCEKs could be restored by the addition of c-kit ligand. Conversely, in Kit^W/Wv mice that have impaired c-kit signaling, glycogen content and phosphorylation of Akt (T308, S473) were significantly decreased in the corneal epithelium compared with wild-type mice.

Conclusions: Collectively, these observations demonstrate that FIH-1’s effect on corneal epithelial glycogen stores involves, in part, an interaction between c-kit/Akt/GSK-3β signaling pathways.

Keywords: 482 cornea: epithelium • 592 metabolism • 543 growth factors/growth factor receptors