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.
Highlights
Connexin43 (Cx43) is expressed in nearly every tissue in the body where it forms hemichannels and intercellular gap junctions and plays diverse roles in coordinating cellular activities [1].The connexin mimetic peptide (CMP) Gap27, targeted to the SRPTEKTIFFI sequence on the second extracellular loop of Cx43 is a versatile inhibitor of connexin-mediated communication (CMC) in tissue networks [2,3,4]
In previous studies [22,23], we determined that while Gap27 enhanced migration rates in keratinocytes and fibroblasts isolated from juvenile foreskin discards it was less effective in fibroblasts isolated from adult dermal explants
Our findings provide new insights into the effects of Cx43 channel inhibition versus Cx43 gene expression on cell migration, and our results show that the response of such behaviour varies between cell types and between cells of the same type but of different tissue origins
Summary
The connexin mimetic peptide (CMP) Gap, targeted to the SRPTEKTIFFI sequence (amino acids 204–214) on the second extracellular loop of Cx43 is a versatile inhibitor of connexin-mediated communication (CMC) in tissue networks [2,3,4]. Connexins have reported ”non-channel” functions and controversy exists in longer term studies as to whether channel or non-channel activities play key roles in events such as cell adhesion and migration [16]. This is no less evident in the skin where dynamic changes in connexin expression occur during wound healing [17,18,19]. In previous studies [22,23], we determined that while Gap enhanced migration rates in keratinocytes and fibroblasts isolated from juvenile foreskin discards it was less effective in fibroblasts isolated from adult dermal explants
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