β-Catenin-Dependent Wnt Signaling: A Pathway in Acute Cutaneous Wounding

Abstract

Background: Acute wound healing is a dynamic process that results in the formation of scar tissue. The mechanisms of this process are not well understood; numerous signaling pathways are thought to play a major role. Here, the authors have identified β-catenin-dependent Wnt signaling as an early acute-phase reactant in acute wound healing and scar formation.

Methods: The authors created 6-mm full-thickness excisional cutaneous wounds on adult β-catenin-dependent Wnt signal (BAT-gal) reporter mice. The expression of canonical Wnt after wounding was analyzed using X-gal staining and quantitative real-time polymerase chain reaction. Next, recombinant mouse Wnt3a (rmWnt3a) was injected subcutaneously to the wound edge, daily. The mice were killed at stratified time points, up to 15 days after injury. Histologic analysis, quantitative real-time polymerase chain reaction, and Western blot were performed.

Results: Numerous individual Wnt ligands increased in expression after wounding, including Wnt3a, Wnt4, Wnt10a, and Wnt11. A specific pattern of Wnt activity was observed, localized to the hair follicle and epidermis. Mice injected with rmWnt3a exhibited faster wound closure, increased scar size, and greater expression of fibroblast growth factor receptor-2 and type I collagen.

Conclusions: The authors' data suggest that β-catenin-dependent Wnt signaling expression increases shortly after cutaneous wounding, and exogenous rmWnt3a accelerates reepithelialization, wound matrix maturation, and scar formation. Future experiments will focus on the intersection of Wnt signaling and other known profibrotic cytokines.

Figure 1. Wnt expression in fibroblasts is driven by hypoxia

Postnatal dermal fibroblasts were cultured in normoxia (21% oxygen) or hypoxia (1% oxygen). (A-B) Proliferation was assessed by cell counting (A) and bromodeoxyuridine (BrdU) incorporation assays (B). (C) After 48 hrs in normoxic or hypoxic conditions, gene expression for Axin2 and Wnt4 by qRT-PCR. (n=3 for cell counting and gene assays; n=6 for BrdU incorporation; *p<0.01)

Figure 2. Endogenous Wnt expression post adult cutaneous injury

(A-B) X-gal staining in BAT-gal reporter mice, 24 hrs post-wounding. (C) Uninjured adult mouse skin for comparison. (D) PCNA staining on adjacent slide, demonstrating similar distribution of staining. (E) Wnt gene expression 24 hrs post-injury (red) in comparison to uninjured adult skin (grey), by qRT-PCR. (n=3; * p<0.01)

Figure 3. Recombinant Wnt3a speeds wound closure and increases TGF-ß1 expression

(A) Representative photographs of adult cutaneous wounds injected daily with a rmWnt3a suspension or control. Differential healing was observed beginning POD 5. (n=7) (B) Quantification of wound area in rmWnt3a or control wounds from 0-15 days. (C) Protein expression of nuclear ß-catenin, cytoplasmic TGF-ß1, and ß-actin at 0, 1, and, 5 days postoperatively, by Western blot. (n=3 for each time point)

Figure 4. Recombinant Wnt3a increases re-epithelization

(A) Routine H&E (left) and picrosirius red staining of control wound at POD 5. (B) H&E (left) and picrosirius red staining of rmWnt3a-treated wound at POD 5. A substantially thicker layer of re-epithelization was noted in Wnt-treated wounds on H&E. Greater picrosirius red staining, corresponding to increased collagen content was observed among rmWnt3a-treated groups. (n=3)

Figure 5. Recombinant Wnt3a increases scar formation

(A) Quantification of collagen pixels in Mallory’s Trichome stain per 5x field in control (blue) as compared to rmWnt3a-treated wounds (red) at POD 5 and 10. (n=3 for each group) (B) Relative Type I Collagen expression in control (blue) as compared to rmWnt3a-treated wounds (red) through POD 10, as assessed by qRT-PCR. (n=3 for all gene assays; *p<0.01)