The Connexin Mimetic Peptide Gap27 and Cx43-Knockdown Reveal Differential Roles for Connexin43 in Wound Closure Events in Skin Model Systems.

In the epidermis, remodelling of Connexin43 is a key event in wound closure. However, controversy between the role of connexin channel and non-channel functions exist. We compared the impact of SiRNA targeted to Connexin43 and the connexin mimetic peptide Gap27 on scrape wound closure rates and hemichannel signalling in adult keratinocytes (AK) and fibroblasts sourced from juvenile foreskin (JFF), human neonatal fibroblasts (HNDF) and adult dermal tissue (ADF). The impact of these agents, following 24 h exposure, on GJA1 (encoding Connexin43), Ki67 and TGF-ß1 gene expression, and Connexin43 and pSmad3 protein expression levels, were examined by qPCR and Western Blot respectively. In all cell types Gap27 (100-100 µM) attenuated hemichannel activity. In AK and JFF cells, Gap27 (100 nM-100 µM) enhanced scrape wound closure rates by ~50% but did not influence movement in HNDF or ADF cells. In both JF and AK cells, exposure to Gap27 for 24 h reduced the level of Cx43 protein expression but did not affect the level in ADF and HNDF cells. Connexin43-SiRNA enhanced scrape wound closure in all the cell types under investigation. In HDNF and ADF, Connexin43-SiRNA enhanced cell proliferation rates, with enhanced proliferation also observed following exposure of HDNF to Gap27. By contrast, in JFF and AK cells no changes in proliferation occurred. In JFF cells, Connexin43-SiRNA enhanced TGF-ß1 levels and in JFF and ADF cells both Connexin43-SiRNA and Gap27 enhanced pSmad3 protein expression levels. We conclude that Connexin43 signalling plays an important role in cell migration in keratinocytes and foreskin derived fibroblasts, however, different pathways are evoked and in dermal derived adult and neonatal fibroblasts, inhibition of Connexin43 signalling plays a more significant role in regulating cell proliferation than cell migration.

Connexin43 plays diverse roles in co-ordinating cell migration and wound closure events.

Chronic wounds are not only debilitating to patients, but also impose a huge financial burden on healthcare providers, as current treatments are not particularly effective. Wound healing is a highly co-ordinated process involving a vast array of signalling molecules and different cell types, therefore a substantial amount of research has been carried out in the quest to develop new therapies. The gap junction (GJ) protein connexin43 (Cx43) is one of the many molecules whose expression has been found to be up-regulated in chronic wounds and as a result targeting it may have therapeutic potential. Two different approaches have been adopted to investigate this: knockdown of Cx43 using antisense oligonucleotides and connexin mimetic peptides (CMPs) which inhibit the function of Cx43 without affecting gene expression. These peptides are targeted to the C-terminal domain or the extracellular loops of Cx43 and thus are likely to function by different means. However, both block channel function and have been shown to enhance cell migration rates. In recent years, non-channel functions have emerged for Cx43, many of which are linked to cytoskeletal dynamics and the extracellular matrix (ECM), showing that Cx43 plays diverse roles in co-ordinating wound closure events. It is clear that both CMPs and antisense oligonucleotides hold therapeutic potential, however maintaining Cx43 expression may be beneficial to the cell by preserving other non-channel functions of Cx43. Recent data in the field will be discussed in this article.