Regulation of Connexin 43 Expression in Human Gingival Fibroblasts

Objectives: Previous studies have shown that downregulation of connexin 43 (Cx43) in skin wounds promotes wound closure and granulation tissue formation. We have shown previously that Cx43 abundance is rapidly downregulated in human gingival wounds, which heal faster with less scarring as compared to skin wounds. Additionally, blocking of Cx43 function by mimetic peptides in gingival fibroblasts regulates the expression of genes that may promote fast and scarless wound healing. Thus, a reduced abundance of Cx43 in fibroblasts may facilitate fast gingival wound healing, although the mechanisms involved in its downregulation are unknown. Thus, our aim was to find out the mechanisms involved in Cx43 downregulation in gingival fibroblasts. We hypothesized that inflammatory cytokines/growth factors released in early wounds and/or disruption of cell-cell contacts by wounding downregulates Cx43 abundance in gingival fibroblasts.
Methods: Cultured human gingival fibroblasts (GFBLs) from healthy individuals (n=4) were treated with various wound-healing associated cytokines, growth factors (TNF-α; IL-1α; IL-1β; IL-2; IL-6; IL-17; PGE₂; IFN-g; EGF; HB-EGF; bFGF; IGF-1; TGF-β1 and TGF-β3) and 10% serum (present in early wounds), and expression of Cx43 was assessed by qRT- PCR. Effect of disruption of cell-cell contacts by scratch-wounding on Cx43 expression and localization was also studied over-time by qRT-PCR and immunostaining, respectively.
Results: Treatment of GFBLs with cytokines, growth factors and serum, and physical wounding, did not have a significant effect on Cx43 expression at mRNA level. However, wounding caused a robust downregulation of Cx43 gap-junction and hemichannel plaques at the leading edge of GFBLs at the wound margins.
Conclusions: The findings suggest that the expression of Cx43 mRNA is stable and not significantly affected by the wound environment (cytokines, growth factors, serum, or disruption of cell-cell contacts). However, disruption of cell contacts by wounding modulates Cx43 gap-junction and hemichannel subcellular abundance and localization.