MicroRNA‐31 targets FIH‐1 to positively regulate corneal epithelial glycogen metabolism

Abstract

Corneal epithelium relies on abundant glycogen stores as its primary energy source. MicroRNA-31 (miR-31), a corneal epithelial-preferred miRNA, negatively regulates factor inhibiting hypoxia-inducible factor-1 (FIH-1). Since HIF-1α is involved in anaerobic energy production, we investigated the role that miR-31 and FIH-1 play in regulating corneal epithelial glycogen. We used antagomirs (antago) to reduce the level of miR-31 in primary human corneal epithelial keratinocytes (HCEKs), and a miR-31-resistant FIH-1 to increase FIH-1 levels. Antago-31 raised FIH-1 levels and significantly reduced glycogen stores in HCEKs compared to irrelevant-antago treatment. Similarly, HCEKs retrovirally transduced with a miR-31-resistant FIH-1 had markedly reduced glycogen levels compared with empty vector controls. In addition, we observed no change in a HIF-1α reporter or known genes downstream of HIF-1α indicating that the action of FIH-1 and miR-31 on glycogen is HIF-1α-independent. An enzyme-dead FIH-1 mutation failed to restore glycogen stores, indicating that FIH-1 negatively regulates glycogen in a hydroxylase-independent manner. FIH-1 overexpression in HCEKs decreased AKT signaling, activated GSK-3β, and inactivated glycogen synthase. Treatment of FIH-1-transduced HCEKs with either a myristolated Akt or a GSK-3β inhibitor restored glycogen stores, confirming the direct involvement of Akt/GSK-3β signaling. Silencing FIH-1 in HCEKs reversed the observed changes in Akt-signaling. Glycogen regulation in a HIF-1α-independent manner is a novel function for FIH-1 and provides new insight into how the corneal epithelium regulates its energy requirements.

microRNA-31/factor-inhibiting hypoxia-inducible factor 1 nexus regulates keratinocyte differentiation

“…Interestingly, expression profiling indicated that with respect to the ocular anterior segmental epithelium, miR-31 is a corneal epithelial-preferred miRNA (32). As described previously, FIH-1 protein is abundantly detected in human limbal epithelium but not in human corneal epithelium (12). We propose that low levels of miR-31 and correspondingly high levels of FIH-1, which attenuates Notch signaling, may be one mechanism that maintains the relatively undifferentiated phenotype characteristic of limbal epithelial basal cells (20).…”

“…The limbal epithelium, which is the preferential site of the corneal epithelial stem cells (20,28), interestingly has high levels of FIH-1 expression compared with the corneal epithelium (12). In concert with a lack of FIH-1 staining in the limbal epithelium (Fig.…”

“…These in vivo results confirm our in vitro findings that FIH-1 attenuates Notch signaling and negatively regulates keratinocyte differentiation. (12). To investigate the relevance of this miR-31/FIH-1 relationship to keratinocyte differentiation, we altered miR-31 levels in submerged cultures of HCEKs (Fig.…”

“…Furthermore, only recently has FIH-1 been recognized to play a role in regulating stratified squamous epithelia. For example, we have recently demonstrated that FIH-1 negatively regulates corneal epithelial glycogen metabolism in a HIF-1α-independent manner, which is a unique function for this hydroxylase (12).…”

microRNA-182 Mediates Sirt1-Induced Diabetic Corneal Nerve Regeneration

“…microRNAs (miRNAs) are small, noncoding RNA molecules found in multicellular eukaryotes and play essential regulatory roles in translational repression (15). Several wound healing-related miRNAs, such as miR-146a, were identified in the corneas of people with diabetes (14,16,17). Abnormal miR-146a upregulation may be an important mechanism of delayed wound healing in the diabetic cornea (17,18).…”