The eIF2 kinase GCN2 directs keratinocyte collective cell migration during wound healing via coordination of reactive oxygen species and amino acids

Rebecca R Miles,Parth H Amin,Miguel Barriera Diaz,Jagannath Misra,Erica Aukerman,Amitava Das,Nandini Ghosh,Tanner Guith,Michael D Knierman,Sashwati Roy,Dan F Spandau,Ronald C Wek

doi:10.1016/j.jbc.2021.101257

Rebecca R Miles, Parth H Amin + Show 10 more

Open Access

https://doi.org/10.1016/j.jbc.2021.101257

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Abstract

Healing of cutaneous wounds requires the collective migration of epithelial keratinocytes to seal the wound bed from the environment. However, the signaling events that coordinate this collective migration are unclear. In this report, we address the role of phosphorylation of eukaryotic initiation factor 2 (eIF2) and attendant gene expression during wound healing. Wounding of human keratinocyte monolayers in vitro led to the rapid activation of the eIF2 kinase GCN2. We determined that deletion or pharmacological inhibition of GCN2 significantly delayed collective cell migration and wound closure. Global transcriptomic, biochemical, and cellular analyses indicated that GCN2 is necessary for maintenance of intracellular free amino acids, particularly cysteine, as well as coordination of RAC1-GTP-driven reactive oxygen species (ROS) generation, lamellipodia formation, and focal adhesion dynamics following keratinocyte wounding. In vivo experiments using mice deficient for GCN2 validated the role of the eIF2 kinase during wound healing in intact skin. These results indicate that GCN2 is critical for appropriate induction of collective cell migration and plays a critical role in coordinating the re-epithelialization of cutaneous wounds.

Highlights

Healthy individuals heal cutaneous wounds without complication, there are many patients whose wounds do not resolve and require long-term care
Using a combination of biochemical, genetic, and cellular approaches in cell culture and mouse model systems, we show that GCN2 and its attendant gene expression are important for appropriate management of amino acids, reactive oxygen species (ROS) generation, lamellipodia formation, and focal adhesion dynamics that are central for keratinocyte collective cell migration (KCCM) and optimal wound healing
We addressed the role of GCN2 in KCCM by using CRISPR/CAS9 and guide RNAs targeting exon 2, which encodes the N-terminal RWD domain of GCN2, or exon 12, a region encoding the critical lysine reside in the kinase domain (Fig. 1C)

Summary

Introduction

Healthy individuals heal cutaneous wounds without complication, there are many patients whose wounds do not resolve and require long-term care. An important process during the proliferation phase is re-epithelialization that involves keratinocyte collective cell migration (KCCM) where sheets of connected cells migrate in concert to cover the wound bed [4] If these phases are dysregulated or do not occur in an orderly fashion, a wound will not properly heal. Accompanying the global reduction in protein synthesis, eIF2α-P directs preferential translation of key genes that are critical for stress remediation, such as the transcription factor ATF4, which directs adaptive reprogramming of GCN2 regulates collective cell migration in wounding gene expression. Using a combination of biochemical, genetic, and cellular approaches in cell culture and mouse model systems, we show that GCN2 and its attendant gene expression are important for appropriate management of amino acids, ROS generation, lamellipodia formation, and focal adhesion dynamics that are central for KCCM and optimal wound healing

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