Expressed in high metastatic cells (Ehm2) is a positive regulator of keratinocyte adhesion and motility: The implication for wound healing David C. Bosanquet *, Lin Ye, Keith G. Harding, Wen G. Jiang Departments of Surgery and Wound Healing, Cardiff University School of Medicine, Heath Park, Cardiff CF14 4XW, UK 1. Introduction Successful wound healing requires the co-ordination of myriad cellular processes, including inflammation, angiogenesis, prolifer- ation and cellular migration [1]. Causes of wound healing failure are multifactorial and include systemic and local factors [2]. In recent years, an increasing volume of research has examined the cellular and molecular aberrations within cell populations surrounding chronic wounds, assuming that perturbations in gene expression account, at least in part, to the development of these wounds [3]. Of specific interest to our research collaborative is the value of examining keratinocytes at the wound edge [4,5]. In health, these keratinocytes undergo transformation from a sedentary to a migratory phenotype in response to wounding, whereas keratinocytes on the periphery of chronic wounds display a hyperproliferative, non-motile phenotype [6]. Micro-array analysis has identified a number of genes thought to be key players in promoting wound chronicity [7], whilst other authors have identified individual genes (e.g. b-catenin and c-myc) associated with delayed wound healing [8]. Since Dvorak’s landmark paper in 1986, the similarities in cellular processes between wound healing and cancer have been well recognised (i.e. cellular migration, proliferation and angio- genesis) [9]. In our department’s search for key molecular players in wound healing we have focused our attention on cancer promoters, hypothesising that these proteins would also act as wound healing promoter. Ehm2, for expressed in high metastatic cells 2, was discovered in 1996 in the highly metastatic (and notably absent in the low metastatic) K-1735 murine melanoma cell line [10], and was cloned by the same team in 2000 [11]. Ehm2 was found to belong to the FERM (protein Four.1, Ezrin, Radixin, Meosin [12]) family of proteins, and showed the greatest homology with NBL4 (Novel Band 4.1 Like protein 4). FERM proteins all contain a well conserved N-terminal 3-lobed FERM domain which Journal of Dermatological Science 71 (2013) 115–121 A R T I C L E I N F O Article history: Received 15 December 2012 Received in revised form 17 March 2013 Accepted 4 April 2013 Keywords: Wound healing HaCaT Keratinocytes ECIS Ehm2 A B S T R A C T Background: Multiple factors have been shown to delay dermal wound healing. These resultant wounds pose a significant problem in terms of morbidity and healthcare spend. Recently, an increasing volume of research has focused on the molecular perturbations underlying non-healing wounds. Objectives: This study investigates the effect of a novel cancer promoter, Ehm2, in wound healing. Ehm2 belongs to the FERM family of proteins, known to be involved in membrane–cytoskeletal interactions, and has been shown to promote cancer metastasis in melanoma, prostate cancer and breast cancer. Methods: Ehm2 mRNA levels were analysed using qRT-PCR, standardised to GAPDH, from either acute or chronic wounds, and normal skin. IHC analysis was also undertaken from wound edge biopsies. An anti- Ehm2 transgene was created and transfected into the HaCaT cell line. The effect of Ehm2 knockdown on migration, adhesion, growth, cell cycle progression and apoptosis was analysed using standard laboratory methods. Western Blot analysis was used to investigate potential downstream protein interactions. Results: Ehm2 is expressed nearly three times higher in acute wound tissues, compared to chronic wound tissues. Increased Ehm2 expression is found in wounds undergoing healing, especially at the leading wound edge. In vitro, Ehm2 knockdown reduces cellular adhesion, migration and motility, without affecting growth, cell cycle and apoptosis. Finally, Ehm2 knockdown results in reduced NWasp protein expression. Conclusion: These results suggest Ehm2 may be an important player in the wound healing process, and show that Ehm2 knockdown downregulates the expression of NWasp, through which it may have its effect on cellular migration. ß 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. * Corresponding author. Tel.: +44 2920 742316; fax: +44 2920 742896. E-mail address: davebosanquet@hotmail.com (D.C. Bosanquet). Contents lists available at SciVerse ScienceDirect Journal of Dermatological Science jou r nal h o mep ag e: w ww .elsevier .co m /jds 0923-1811/$36.00 ß 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdermsci.2013.04.008