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

Abstract

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-β1 gene expression, and Connexin43 and pSmad3 protein expression levels, were examined by qPCR and Western Blot respectively. In all cell types Gap27 (100 nM–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-β1 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.

Connexin Communication Compartments and Wound Repair in Epithelial Tissue

“…Gap27 blocks hemichannel and gap junctional communication without influence on Cx43 gene expression in keratinocytes [ 64 , 121 , 122 , 123 ]. It enhances keratinocyte migration rates, without altering cell proliferation supporting concepts that hemichannel activity is involved in keratinocyte galvanotaixis [ 61 , 124 ].…”

“…The localised switch in phosphorylation probably changes the functional signalling parameters of Cx43 channels, further enhancing migration of wound edge keratinocytes. This may include the induction of signalling pathways, including Transforming Growth Factor-β TGF-β and ECM deposition, all of which are key events in wound closure [ 61 , 62 , 63 ]. Upon re-stratification, levels of Cx43 are re-established [ 45 , 58 , 64 , 65 ].…”

Connexin 26 and 43 play a role in regulating proinflammatory events in the epidermis

“…Both have been widely used to inhibit CX mediated signalling events in diverse tissue networks (Willebrords, Maes, Crespo Yanguas, & Vinken, ). We have recently reported that Gap27 is effective in inhibiting ATP release from keratinocytes at concentrations of 100 nM, where it promotes keratinocyte migration with limited effect on proliferation (Faniku et al, ). Gap27 is reported to be specific for CX43 and CX37 by several groups (Martin, Wall, & Griffith, ).…”

Connexin 43-Based Therapeutics for Dermal Wound Healing

“…More recently, Martin and co-workers have used an alternate gene knockdown approach, short interfering RNAs (siRNAs) targeted to Cx43, together with the Cx43 mimetic peptide Gap27, to study channel-dependent and independent effects of Cx43 in the response of dermal cells to injury [ 33 ]. Their data indicates that the response to targeting Cx43 varies between cell types (keratinocyte versus fibroblast) and between cells of the same type (skin fibroblasts), but of different tissue origins.…”