Insulin-like growth factor 1 (IGF-I), insulin-like growth factor binding protein 5 (IGFBP-5) and connexins modulate cell migration in human keratinocyte and organotypic skin models

In diabetic skin the IGF-I:IGFBP-5 ratio is perturbed, and in other organs increased IGFBP-5 leads to fibrosis. Altered IGF-I:IGFBP-5 may contribute to impaired cell migration in non-healing diabetic wounds. We have previously shown that the connexin mimetic peptide, Gap27, directly targeted to the extracellular loop of connexin 43 (Cx43) increases rates of ‘wound’ closure in human skin organotypic models by reducing of connexin-mediated communication (CMC). Differences in Gap27-induced scrape-wound closure occur in euglycaemia and euinsulinaemia or hyperglycaemia and hyperinsulinaemia, and this study examined the relative roles of insulin-like growth factor 1 (IGF-I), insulin-like growth factor binding protein 5 (IGFBP-5) in this system.
Cells were treated for 5 days with 5.5 mM glucose and 1 nM insulin, or 25 mM glucose and 10 nM insulin. Subsequently, migration studies examined rates of scrape-wound closure in cell monolayers and organotypic skin models over 48 hours in cells in these conditions in the presence or absence of 100 ng/ml IGF-I, 4 µg/ml IGFBP-5 and 100 µM Gap27. Immunocytochemistry with confocal microscopy was used to examine the effect of IGF-I and IGFBP-5 on Cx43 and Cx26 distribution in organotypic models.
Addition of IGF-I increased scrape-wound closure in both euglycaemia/euinsulinaemia and hyperglycaemia/hyperinsulinaemia in 2D and 3D cultures (P < 0.001). Further, the presence of IGFBP-5 counteracted this effect (P < 0.05). Gap27 increased wound closure in addition to that seen with IGF-I, and overrode the inhibitory effects of IGFBP-5 (P < 0.001). Under euglycaemia/euinsulinaemia Cx43 and Cx26 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 treatment increased Cx43-ser368 levels. Organotypic models treated with hyperglycaemia/hyperinsulinaemia showed an upregulation of Cx26 expression, with Cx43 still present in the basal layers, and an upregulation of Cx43-ser368. This seemed to be further enhanced following Gap27 treatment. Treatment with IGF-I did not alter these observations.
Gap27 blocked CMC and increased cell migration rates in both euglycaemia/euinsulinaemia and hyperglycaemia/hyperinsulinaemia. IGF-1 increased migration of Gap27-treated cells further, while IGFBP-5 retarded them. Gap27 overcame the inhibited migration due to IGFBP-5. The connexin expression pattern observed in organotypic models suggests that IGF-I does not influence connexin expression status in epidermal tissue. 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.