Activation of the Unfolded Protein Response in Wound Healing

Abstract

Introduction: The unfolded protein response (UPR) is an adaptive response centered in the endoplasmic reticulum (ER) to protect cells against the accumulation of misfolded proteins due to increased cellular stresses or protein synthesis. Activation of the UPR triggers cell signaling through three pathways: IRE1-XBP1, PERK-ATF4, and ATF6. Dysregulation of the UPR has been implicated in diseases as diverse as type I diabetes mellitus and cancer. We have previously demonstrated increased UPR activation in keloid-derived fibroblasts. We wished to examine the normal activation of the UPR in wound healing and its potential role. Methods: A construct was generated containing a luciferase (Luc) gene driven by XBP1 enhancer sites (XBP1-Luc). XBP1-Luc was transfected into the HT1080 pancreatic adenocarcinoma cell line to confirm responsiveness of the construct to UPR activation. Transfected cells were subjected to hypoxia (<0.1% O2) or ER stress conditions (tunicamycin, no glucose). Transgenic mice were generated with the construct and subjected to excisional skin wounding. In vivo imaging was performed using an IVIS charge-coupled device camera system (Xenogen, Alameda, CA). Results: XBP1-Luc reporter activity in HT1080 cells was strongly induced after 16-24 h of incubation in hypoxic conditions (<0.1% O2) or ER stress conditions, known UPR inducers, and reflected endogenous XBP1 activation. Furthermore, reporter activity decayed to near-background levels within 24 hours after reoxygenation, indicating that cellular luminescence reflects recent or ongoing ER stress. Following excisional wounding, we began seeing an initial increase in XBP1-Luc signal in the transgenic animals 8-10 days after wounding with nearly 10-fold increased activity seen at day 14. Conclusions: The UPR has been demonstrated to be an important pathway in a variety of pathologies. This represents the first description of UPR activation in wound healing. By defining the temporal and spatial activation of the UPR during wound healing, we hope it will provide insights into its biological role. We have already identified enhanced activation of the UPR in keloid cells compared to normal cells suggesting that modulation of the UPR can offer a new approach to wound healing therapies.