Interferon regulatory factor 6: a novel regulator of keratinocyte differentiation and migration

Cutaneous wound healing is an inherent biological process to every injury, surgical procedure, and cases of trauma. Wound healing is loosely categorized into three overlapping phases: inflammation, epithelialization, and maturation. Dysregulation of any of these steps leads to poor wound healing, as do conditions such as diabetes, obesity, and advanced age. We recently identified the clefting syndrome, Van der Woude (VWS), as another condition resulting in poor healing. VWS and the allelic syndrome, popliteal pterygium (PPS) are caused by mutations in Interferon Regulatory Factor 6 (IRF6). In the mouse, removal of Irf6 leads to major developmental defects, including limb, craniofacial and cutaneous anomalies. As embryonic development and wound healing share common biological processes, we hypothesized that Irf6 is critical to the regulation of differentiation, proliferation, and migration of keratinocytes, processes essential to wound healing. Upon in vitro differentiation, wild type murine keratinocytes exhibited increased Irf6 expression. Fittingly, Irf6^-/- keratinocytes exhibited a defect in terminal differentiation and increased capacity for long-term proliferation. We also determined that a craniofacial enhancer of Irf6 is functional in differentiating keratinocytes in vitro and in vivo. As cellular migration is a critical process to wound epithelialization, we tested the role of Irf6 in keratinocyte migration. We report a significant delay in closing an in vitro scratch wound in the absence of Irf6. Analysis of time-lapse microscopy revealed that this delay was due to a reduction in keratinocyte velocity. We report a substrate non-specific reduction in adhesion in Irf6-/- keratinocytes compared to wild type cells. In addition,Irf6^-/- keratinocytes exhibited longer and more prominent actin stress fibers that were rescued by the addition of the ROCK inhibitor, Y27632. The active form of RhoA, a GTPase regulating stress fiber formation upstream of ROCK was increased in Irf6^-/- keratinocytes, suggesting a role for Irf6 in regulating RhoA-dependent stress fibers. We further identified Arhgap29, a GTPase activating protein deactivator of RhoA, as a downstream effector of Irf6 in skin and keratinocytes. Finally, we used a conditional strategy to delete Irf6 in adult murine epidermis to study in vivo wound healing. We report a significantly smaller macroscopically visible wound that was not confirmed by wound stereological analysis. We also utilized an embryonic model to exclusively test epithelialization in the absence of Irf6. Our results indicate a trend toward faster migration in Irf6^-/- e10.5 wounds, yet the difference in wound closure after 6 hours of healing was not significant. Thus we conclude that Irf6 regulates the balance between keratinocyte proliferation and differentiation. Furthermore, it regulates stress fiber formation and speed of migrating keratinocytes in vitro, while Irf6 is dispensable for cutaneous wound epithelialization in vivo.