Cutaneous wound reepithelialization is compromised in mice lacking functional Slug (Snai2)

Abstract

Background: Keratinocytes at wound margins undergo partial epithelial to mesenchymal transition (EMT). Based on previous in vitro and ex vivo findings, Slug (Snai2), a transcriptional regulator of EMT in development, may play an important role in this process.

Objectives: This study was designed to validate an in vivo role for Slug in wound healing.

Methods: Excisional wounds in Slug null and wild type mice were examined histologically at 6, 24, 48, and 72h after wounding; reepithelialization was measured and immunohistochemistry for keratins 8, 10, 14, and 6 and E-cadherin was performed. In 20 Slug null and 20 wild type mice exposed three times weekly to two minimal erythemal doses of UVR, the development of non-healing cutaneous ulcers was documented. Ulcers were examined histologically and by immunohistochemistry.

Results: The reepithelialization component of excisional wound healing was reduced 1.7-fold and expression of the Slug target genes keratin 8 and E-cadherin was increased at wound margins in Slug null compared to wild type mice. In contrast, no differences in expression of keratins 10 or 14 or in markers of proliferation K6 and Ki-67 were observed. Forty per cent of Slug null mice but no wild type mice developed non-healing cutaneous ulcers in response to chronic UVR. Keratinocytes at ulcer margins expressed high levels of keratin 8 and retained E-cadherin expression, thus resembling excisional wounds.

Conclusion: Slug is an important modulator of successful wound repair in adult tissue and may be critical for maintaining epidermal integrity in response to chronic injury.

Fig. 1

Histology of 72-h wounds in wild type (A) and Slug null (B) mice (scale bar = 200 μm). Arrows indicate edge of migrating epithelium. Note the thin and elongated ingrowth of migrating epithelium in the wild type wound which covers the defect to the level of the panniculus carnosus. In contrast, the epithelial ingrowth in the 72-h Slug null wound is short and ends bluntly, covering the defect to a level somewhat above the level of the hypodermis, which overlies the panniculus carnosus. Asterisks indicate the panniculus carnosus.

Fig. 2

Immunohistochemistry for keratin 8 in 72-h wounds of wild type (A) and Slug null (B) mice (scale bar = 100 μm). Arrows indicate edge of migrating epithelium. Note the absence of keratin 8 positivity in the wild type wound in contrast to robust staining for keratin 8 in basal keratinocytes at wound margins in Slug null mice.

Fig. 3

Immunohistochemistry for keratins 14 and 10 in 72-h wounds of wild type (A and C) and Slug null (B and D) mice (scale bar = 100 μm). Upper panels (A and B) show staining for keratin 14 and lower panels (C and D) show staining for keratin 10. Keratin 14 immunoreactivity extends to the margins of all wounds. Note that keratin 10 expression in both wild type and Slug null mice stops fairly abruptly at the initial wound margin (arrows).

Fig. 4

Immunohistochemistry for keratin 6 and Ki-67 in 72-h wounds of wild type (A and C) and Slug null (B and D) mice (scale bar = 100 μm). Upper panels (A and B) show staining for keratin 6 and lower panels show staining for Ki-67 (C and D). Keratin 6 immunoreactivity, as shown in panel A, extends from the wound margins for a moderate distance distally in both 72-h wounds; asterisks indicate the extent of distal K6 staining. As shown in Panel B, a focus of prominent Ki-67 expression in the 72-h wounds of both wild type and null mice is located at some distance from the wound margin (arrows).

Fig. 5

Immunohistochemistry for E-cadherin in 72-h wounds of wild type (A) and Slug null (B) mice (scale bar = 50 μm). Arrows indicate tip of epithelial outgrowth. Note retention of E-cadherin staining at the tip of the epithelial outgrowth in the Slug null mouse.

Fig. 6

A typical chronic ulcer on the back of a UVR-exposed Slug null mouse (arrow) is shown in the upper left panel (A). Note the smooth rounded edges on the wound and the absence of exudate. The epithelial margin of the chronic wound in the Slug null mouse does not extend into the dermis (B) (scale bar = 100 μm). There is robust expression of keratin 8 (C) and retention of E-cadherin (D) at the wound margin (scale bar = 50 μm); keratin 14 is highly expressed in all keratinocytes (E), while keratin 10 expression is not seen at the immediate wound margin (F) (scale bar = 100 μm). Keratin 6 is expressed uniformly throughout the epidermis (G), and Ki-67 is detected in many basal keratinocytes (H) (scale bar = 100 μm).