Cx43, IGF-I and IGFBP-5 modulate cell migration in scrape-wounded human keratinocyte and organotypic skin models

The insulin-like growth factor I: insulin-like growth factor binding protein 5 (IGF-I:IGFBP-5) ratio is perturbed in diabetic skin and may contribute to impaired cell migration in diabetic wounds. Gap junctions, and their constituent proteins the connexins, are also raised at the wound edge in diabetes. The connexin mimetic peptide Gap27 increases scrape-wound closure in human skin models. This study examined the relative roles of IGF-I, IGFBP-5 and the gap junction protein connexin43 (Cx43) in scrape-wound cell migration.
Cells were pre-treated for 5 days with 5.5 or 25 mM glucose and 1 or 10 nM insulin. Migration studies examined scrape-wound closure rates in 2D human epidermal keratinocyte and 3D organotypic skin models over 48 h in the presence or absence of 100 ng/ml IGF-I, 4 µg/ml IGFBP-5 and 100 µM Gap27. IGF-I increased cell proliferation in euglycaemia/euinsulinaemia. Addition of IGF-I increased scrape-wound closure in both 2D and 3D organotypic cultures (P < 0.001). Furthermore, IGFBP-5 counteracted this effect (P < 0.05). Gap27 increased wound closure in addition to that seen with IGF-I, and overrode inhibitory IGFBP-5 effects (P < 0.001). In euglycaemia/euinsulinaemia Cx43 and Cx26 protein were differentially expressed in organotypic skin models with basal Cx43 and Cx26 in the upper layers; with low amounts of phosphorylated Cx43 (Cx43-ser368). Gap27 increased Cx43-ser368 levels. Organotypic models exposed to or cultured in hyperglycaemia/hyperinsulinaemia showed an upregulation of Cx26 expression, with Cx43 still present in the basal layers, and an upregulation of Cx43-ser368. This was further enhanced following Gap27 treatment. Treatment with IGF-I did not alter connexin expression in organotypic models.
Gap27 blocked connexin-mediated communication and increased cell migration rates in both euglycaemia/euinsulinaemia and hyperglycaemia/hyperinsulinaemia. IGF-I increased migration of Gap27-treated cells further, while IGFBP-5 retarded them. Gap27 overcame the inhibited migration due to IGFBP-5. IGFBP-5 plays a central role in a diabetic environment and can impede IGF-I mediated effects. Thus in diabetic tissue where the IGF-I:IGFBP-5 ratio is disturbed, Gap27 can be predicted to have a positive effect on chronic wound healing.