831 ARHGAP29 regulates keratinocyte migration in vitro but not in vivo

Abstract

ARHGAP29 is a Rho GTPase Activating Protein with a high affinity for the small GTPase RhoA. In endothelial cells, ARHGAP29 regulates tubulogenesis and endothelial barrier function. However, very little is know about the role of ARHGAP29 in keratinocytes. We previously demonstrated that ARHGAP29 was reduced in Irf6-deficient skin and keratinocytes. Beause IRF6 is required for keratinocyte migration, we hypothesize that ARHGAP29 also regulates keratinocyte migration. To test this hypothesis, we used CRISPR-Cas9 technology to generate keratinocyte cell lines possessing two mutant alleles for ARHGAP29. ARHGAP29 mutant keratinocytes had more rounded cell morphology, irregular cellular edges and a disorganized actin cytoskeleton. We also observed an increased incidence of cellular blebbing, a phenomenon previously associated with increased RhoA activity. Using these cells, we performed in vitro scratch assays and observed that ARHGAP29 mutant keratinocytes were significantly delayed in their ability to close the scratch. Live imaging analysis of individual cells at the scratch edge showed that ARHGAP29 mutant keratinocytes migrated with significantly reduced migration speed, directionality, and persistence. To test a similar role for ARHGAP29 in vivo, we generated a keratinocyte-specific knockout of Arhgap29. These animals were viable and did not exhibit macroscopic cutaneous defects. Following excisional wounds, keratinocyte migration was not altered 7 days post-injury. Together, these data demonstrate that ARHGAP29 is required for proper keratinocyte morphology and migration in vitro, and additional wound healing time points will be needed to fully investigate the function of ARHGAP29 in in vivo keratinocyte migration.