Expression and Function of Connexin 43 in Human Gingival Wound Healing and Fibroblasts

Connexins (Cxs) are a family of transmembrane proteins that form hemichannels and gap junctions (GJs) on the cell membranes, and transfer small signaling molecules between the cytoplasm and extracellular space and between connecting cells, respectively. Among Cxs, suppressing Cx43 expression or function promotes skin wound closure and granulation tissue formation, and may alleviate scarring, but the mechanisms are not well understood. Oral mucosal gingiva is characterized by faster wound closure and scarless wound healing outcome as compared to skin wounds. Therefore, we hypothesized that Cx43 function is down regulated during human gingival wound healing, which in fibroblasts promotes expression of genes conducive for fast and scarless wound healing. Cultured gingival fibroblasts expressed Cx43 as their major connexin. Immunostaining of unwounded human gingiva showed that Cx43 was abundantly present in the epithelium, and in connective tissue formed large Cx43 plaques in fibroblasts. At the early stages of wound healing, Cx43 was strongly down regulated in wound epithelial cells and fibroblasts, returning to the level of normal tissue by day 60 post-wounding. Blocking of Cx43 function by Cx43 mimetic peptide Gap27 suppressed GJ-mediated dye transfer, promoted migration, and caused significant changes in the expression of wound healing-associated genes in gingival fibroblasts. In particular, out of 54 genes analyzed, several MMPs and TGF-beta 1, involved in regulation of inflammation and extracellular matrix (ECM) turnover, and VEGF-A, involved in angiogenesis, were significantly upregulated while pro-fibrotic ECM molecules, including Collagen type I, and cell contractility-related molecules were significantly down regulated. These responses involved MAPK, GSK3 alpha/beta and TGF-beta signaling pathways, and AP1 and SP1 transcription factors. Thus, suppressed function of Cx43 in fibroblasts promotes their migration, and regulates expression of wound healing-associated genes via AP1, SP1, MAPK, GSK3 alpha/beta and TGF-beta signaling pathways, and may promote fast and scarless wound healing in human gingiva.