Abstract
Purpose:
Factor inhibiting hypoxia-inducible factor-1 (FIH-1), a previously unknown component of keratinocytes, is now recognized as a hydroxylase that has a positive role in epithelial proliferation and is a negative regulator of epithelial differentiation. Furthermore, FIH-1 expression in corneal epithelium was significantly increased following wounding. The mechanisms by which FIH-1 enhances keratinocyte migration and facilitates wound healing were not clear.
Methods:
We used genetic approaches to alter FIH-1 expression in vitro and in vivo in conjunction with linear scratch wound and in vivo wound healing assays to study migratory activity. To investigate the downstream pathway involved in the FIH-1-induced migration, immunoprecipitation was combined with immunoblotting. To confirm the downstream target pathway modulated by FIH-1, we conducted rescue experiments using a series of pharmacological inhibitors.
Results:
Loss of FIH-1 in vivo and in vitro significantly delayed epithelial wound healing. Conversely, ectopic expression of FIH-1 in primary human corneal epithelial keratinocytes (HCEKs) promoted keratinocyte migration. This increase in keratinocyte migration required the enhancement of EGFR/ERK signaling by FIH-1. Moreover, our studies reveal that leucine rich repeat kinase 1 (LRRK1), which facilitates EGFR endosomal trafficking via interacting with EGFR, is a novel downstream target of FIH-1. Loss of LRRK1 in keratinocytes increased EGFR signaling activity and consequently enhanced the ability of these cells to seal linear scratch wounds. This suggests that the LRRK1/EGFR interaction is fundamental to the maintenance of proper epithelial cell migration. Importantly, the interaction of FIH-1/LRRK1 prevented the formation of the EGFR/LRRK1 complex required for attenuation of downstream signaling. This blocked the transport of the phosphorylated EGFR from endosomes to lysosomes resulting in a prolonged activation of EGFR signaling, which ultimately promoted migration.
Conclusions:
Here we identify that FIH-1 is a novel regulator of EGFR signaling. The interaction of FIH-1 with LRRK1 sustains EGFR signaling by altering LRRK1-mediated EGFR endocytic trafficking resulting in enhanced keratinocyte migration. The identification of LRRK1 as a novel target for FIH-1 provides new insights into how EGFR signaling can be modulated during corneal epithelial repair.
Keywords: 482 cornea: epithelium •
765 wound healing •
714 signal transduction