GRHL3 activates FSCN1 to relax cell-cell adhesions between migrating keratinocytes during wound reepithelialization
Ghaidaa Kashgari, Sanan Venkatesh, Samuel Refuerzo, Brandon Pham, Anita Bayat, Rachel Herndon Klein, Raul Ramos, Albert Paul Ta, Maksim V. Plikus, Ping H. Wang, Bogi Andersen
Ghaidaa Kashgari, Sanan Venkatesh, Samuel Refuerzo, Brandon Pham, Anita Bayat, Rachel Herndon Klein, Raul Ramos, Albert Paul Ta, Maksim V. Plikus, Ping H. Wang, Bogi Andersen
View: Text | PDF
Research Article Dermatology Genetics

GRHL3 activates FSCN1 to relax cell-cell adhesions between migrating keratinocytes during wound reepithelialization

Abstract

The migrating keratinocyte wound front is required for skin wound closure. Despite significant advances in wound healing research, we do not fully understand the molecular mechanisms that orchestrate collective keratinocyte migration. Here, we show that, in the wound front, the epidermal transcription factor Grainyhead like-3 (GRHL3) mediates decreased expression of the adherens junction protein E-cadherin; this results in relaxed adhesions between suprabasal keratinocytes, thus promoting collective cell migration and wound closure. Wound fronts from mice lacking GRHL3 in epithelial cells (Grhl3-cKO) have lower expression of Fascin-1 (FSCN1), a known negative regulator of E-cadherin. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on wounded keratinocytes shows decreased wound-induced chromatin accessibility near the Fscn1 gene in Grhl3-cKO mice, a region enriched for GRHL3 motifs. These data reveal a wound-induced GRHL3/FSCN1/E-cadherin pathway that regulates keratinocyte-keratinocyte adhesion during wound-front migration; this pathway is activated in acute human wounds and is altered in diabetic wounds in mice, suggesting translational relevance.

Authors

Ghaidaa Kashgari, Sanan Venkatesh, Samuel Refuerzo, Brandon Pham, Anita Bayat, Rachel Herndon Klein, Raul Ramos, Albert Paul Ta, Maksim V. Plikus, Ping H. Wang, Bogi Andersen

×

Figure 3

GRHL3 mediates decreased E-cadherin expression and loosening of cell-cell adhesion in the migrating wound front.

Options: View larger image (or click on image) Download as PowerPoint
GRHL3 mediates decreased E-cadherin expression and loosening of cell-cel...
(A) Toluidine blue staining of resin embedded semi-thin (100 nm) wound sections at day 3 after wounding. Dotted lines indicate the basis of the migrating wound epithelium. Scale bar: 36 μm. (B) Transmission electron microscopy (TEM) of the wound front at day 3 in WT and Grhl3-cKO mice. Dotted lines indicate the basement membrane. Scale bar: 10 μm. (C) Higher-magnification TEM images showing individual cells at the wound front in WT and Grhl3-cKO mice 3 days after wounding. Scale bar: 2 μm. Light blue filling indicates the intercellular spaces between 2 migrating keratinocytes at the wound front. (D) The area covered by intercellular spaces (data are presented as mean ± SD) between migrating keratinocytes at the wound front in C (n = 2/genotype). (E) Higher-magnification TEM images showing desmosomal junctions between migrating keratinocytes at the wound front in WT and Grhl3-cKO mice. Scale bar: 100 nm. (F) Immunofluorescence analysis of E-cadherin (anti–E-cadherin) in day 3 wound sections collected from WT and Grhl3-cKO mice. Dotted lines indicate the basis of the wound front epithelium. Right panels show higher magnification of migrating keratinocytes at the wound front. Arrows show cell surface expression of E-cadherin in WT mice and increased accumulation of E-cadherin in Grhl3-cKO mice. Scale bar: 30 μm. (G) Western blot analysis of E-cadherin protein and loading control (GAPDH) in lysates isolated from wound margins at day 3 in WT and Grhl3-cKO mice (n = 2–3/genotype). (H) Quantifications of Western blot band intensity (data are the mean ± SEM) of E-cadherin and GAPDH proteins in G. (I) Immunofluorescence analysis of Claudin-1 (anti-CLDN1) expression in day 3 wound sections collected from WT and Grhl3-cKO mice. Scale bar: 33 μm. Dotted lines indicate the basis of the wound epithelium (left) and the basement membrane of the unwounded epidermis (right). Statistical significance was determined using Student’s t test (*P < 0.05).