Immunolocalization of cutaneous matrix metalloproteinases during wound healing in the Ets1–/– mouse

Abstract

The E twenty‐six specific (Ets) family of eukaryotic transcription factors has been implicated in the regulation of gene expression during many biological processes, including epithelial growth control and differentiation, development, and malignant transformation. Activator protein‐1 (AP‐1) sites give several matrix metalloproteinase (MMP) genes the ability to be induced by phorbol esters and act synergistically with adjacent Ets‐binding sites in genes such as MMP‐1. The objective of this study was to assess wound closure rates and protein expression of MMP‐1, MMP‐3, MMP‐9, and urokinase‐type plasminogen activator (u‐PA) during acute dermal wound healing of wild type (Ets1+/+) and homozygous (Ets1–/–) mutant Ets1tm2Jml mice. Eight mice, two groups of two Ets1+/+ mice and two groups of two Ets1–/–mice, were used. Two identical full thickness wounds were created over the dorsoscapular region using a sterile 3.5‐mm punch biopsy. Wounds were harvested on day 5 for MMP analysis or when healed. Haematoxylin and eosin‐stained (H&E) sections were prepared by standard methods. Immunohistochemical detection of MMPs and u‐PA were performed on paraffin‐embedded sections using sheep polyclonal antibody anti‐MMP‐1, anti‐MMP‐3, anti‐MMP‐9, and anti‐u‐PA. Serial analyses of wound closure diameters were determined by calculating the average daily diameter of each wound on each mouse until complete re‐epithelialization. A mixed‐effects multiple regression model was used to determine whether significant differences were present between the diameters of the defects on each day. Differences were considered significant when P ≤ 0.05. Following wounding, an early increase in wound size, although not statistically significant, was seen in the Ets1+/+ mice; however, for all time points studied, Ets1+/+ mice did not show a significantly greater time for wound closure than their Ets1–/–counterparts. Histology of day 5 H&E‐stained wound sections showed complete re‐epithelialization in both the Ets1+/+ and the Ets1–/–mice. Although Ets1–/–mice had evidence of granulation tissue formation, the tissue had significantly more dermal edema and loosely packed collagen bundles when compared to the Ets1+/+ mice. MMP‐1 expression was detected directly in fibroblasts and endothelial cell cytoplasm of the granulation tissue. Samples from both groups possessed high levels of MMP‐3 expression in all layers of the stratified epithelium, excluding the stratum corneum. Although epithelial staining for MMP‐9 was diffuse in both groups, more intense staining was seen in the basal keratinocytes overlying the granulation tissue in Ets1–/–mice. Detection of u‐PA expression revealed no differences between Ets1+/+ and Ets1–/–mice; however, minor staining was seen in the epidermis of the Ets1+/+mice relative to the Ets1–/–mice. No consistent significant differences were found in the overall intensity of staining in the epidermis or dermis of any MMP or u‐PA protein between the groups. This study adds to the phenotypic characterization of the Ets1–/–mouse by determining that there is no delayed wound closure, no impairment in re‐epithelialization or granulation tissue formation, and finally, no altered MMP expression profiles from three subsets of the metzincin superfamily during the early stages of healing. The Ets1–/–mouse may not be ideal for studying MMP expression of acute wounds in humans. Funding: Self‐funded.