GCN2 EIF2 Kinase is Critical for Keratinocyte Collective Migration and Wound Healing

Abstract

A critical factor in the healing of a cutaneous wound is the closure of the wound bed that is accomplished by collectively migrating keratinocytes. Wounds that fail to heal appropriately are a significant burden on patient well-being as well as healthcare systems. Further understanding of the molecular pathways involved in wound healing is needed to find new treatments to accelerate healing and reduce treatment costs. Previously, we demonstrated that the integrated stress response (ISR) is critical for keratinocyte response to multiple stresses. Because wounding and repair mechanisms can induce stresses in the skin, we hypothesized that the ISR plays a central role in wound healing. The ISR features a family of stress-activated protein kinases phosphorylate the translation factor eIF2 (eIF2α-P), resulting in diminished global protein synthesis coincident with preferential translation of gene transcripts that lead to the remedy of the stress. Wounding of immortalized NTERT human keratinocyte monolayers led to rapid activation of the eIF2 kinase GCN2, and subsequent eIF2α-P and translational control. Deletion of GCN2 in wounding assays diminished eIF2α-P and translational control during wound healing. Global transcriptome analysis of wounded keratinocytes revealed that deletion of GCN2 induced a compensatory unfolded protein response and dysregulation of mRNAs important for cellular migration. Pathway analysis suggested that GCN2 is necessary for proper activation of key signaling networks and subsequent coordination of RAC-GTP driven reactive oxygen species (ROS) generation following wounding. Additionally, amino acid control of cysteine is regulated by GCN2. We therefore investigated ROS levels following wounding and observed that GCN2 was required for proper ROS induction and actin reorganization in leading edge keratinocytes and that these changes were coincident with reduced RAC and RHO activation and cysteine depletion. The loss of leading-edge ROS in GCN2-deleted cells can be phenocopied with NOX inhibition. Lastly, mice deleted for GCN2 exhibited delayed wound healing compared to WT controls in an excisional wound healing model. These results indicate that GCN2 is required for the induction of collective cell migration and plays a critical role in coordinating the re-epithelialization of cutaneous wounds. We propose the ISR is a potential therapeutic target in chronic wounds.