ARHGAP29 Expression and Localization in Cutaneous Wound Healing

Tanner Reeb; Martine Dunnwald

doi:10.1096/fasebj.2018.32.1_supplement.645.9

Despite every person incurring acute wounds on a regular basis, the genetics and molecular mechanisms regulating wound healing remain to be elucidated. Wound closure requires the concerted action of cellular proliferation, differentiation, and migration. Interferon Regulatory Factor 6 (IRF6) has been shown to regulate all of these processes, with IRF6‐deficient keratinocytes displaying both a decrease in the expression of Rho GTPase Activating Protein 29 (ARHGAP29) as well as an increase in stress fibers. ARHGAP29 is a Rho GTPase Activating Protein with a high affinity for the small GTPase RhoA, a known regulator of stress fibers. However, despite our current knowledge about IRF6 and RHOA, little is known about how IRF6 regulates ARHGAP29 and the role of ARHGAP29 in cellular migration, adhesion, and wound healing. We hypothesize that ARHGAP29 is transcriptionally regulated by IRF6 and perturbation of this system will result in impaired wound healing. To test our hypothesis, we first characterized the expression and subcellular localization of ARHGAP29 in keratinocytes. Using immunofluorescence, ARHGAP29 was detected at the lamellipodia and filopodia as well as in fine puncta throughout the cytoplasm with the strongest expression surrounding the nucleus. In vivo, ARHGAP29 was observed throughout all layers the epidermis, with the highest epidermal expression observed in basal keratinocytes. ARHGAP29 was also detected in dermal fibroblasts, myocytes, endothelial cells and sebocytes. Following wounding, ARHGAP29 was observed at the wound margin in keratinocytes and in inflammatory cells in the granulation tissue throughout the process of wound healing. Preliminary immunofluorescent staining of ARHGAP29 throughout the process of wound healing suggest an increase in the variability of ARHGAP29 expression in the keratinocytes of the leading edge of the migratory tongue when compared to unwounded tissue and closed wounds. To determine if ARHGAP29 is required for proper wound healing in vivo, we took advantage of a murine allele harboring a K326X orofacial cleft patient‐derived mutation. Because homozygotes are embryonic lethal, we performed full thickness excisional wounds on heterozygotes and harvested them 4 and 7 days post‐injury. Morphometric analyses revealed no significant differences at 7 days following wounding, suggesting that at that time point, 50% reduction of ARHGAP29 is sufficient for proper healing. Collectively, these results indicate that ARHGAP29 is expressed during wound healing in a number of cell types relevant to tissue repair. The reduced level of ARHGAP29 observed in our ARHGAP29 K326X mice is, however, still sufficient to heal a cutaneous wound. A keratinocyte‐specific knockout of ARHGAP29 will be essential to understand the role of ARHGAP29 in cutaneous wound healing and to test the interaction between ARHGAP29 and IRF6. These findings will provide key information on the mechanism underlying wound healing and fill the gap linking the previously observed relationship between IRF6 and the RHOA pathway. Support or Funding Information NIH/NIAMS AR067739 This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.

ARHGAP29 Expression and Localization in Cutaneous Wound Healing

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