Expression and function of connexin 43 in human gingival wound healing and fibroblasts

Abstract

Connexins (C×s) 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 C×s, suppressing C×43 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 C×43 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 C×43 as their major connexin. Immunostaining of unwounded human gingiva showed that C×43 was abundantly present in the epithelium, and in connective tissue formed large C×43 plaques in fibroblasts. At the early stages of wound healing, C×43 was strongly down regulated in wound epithelial cells and fibroblasts, returning to the level of normal tissue by day 60 post-wounding. Blocking of C×43 function by C×43 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-β1, 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, GSK3α/β and TGF-β signaling pathways, and AP1 and SP1 transcription factors. Thus, suppressed function of C×43 in fibroblasts promotes their migration, and regulates expression of wound healing-associated genes via AP1, SP1, MAPK, GSK3α/β and TGF-β signaling pathways, and may promote fast and scarless wound healing in human gingiva.

Figure 1. Gingival fibroblasts express C×43 as their major connexin protein.

(A) Results show real-time PCR analysis of major connexins previously described in fibroblasts (C×32, C×40, C×43 and C×45) in cultured human gingival fibroblasts from four different individuals (GFBL-HN, GFBL-CM, GFBL-OL and GFBL-DC). All cell lines expressed C×43 as their major connexin, with moderate levels of C×45, low level of C×32, and no expression of C×40. Range of Ct-values obtained from real-time PCR is indicated below each gene name. (B) Similar findings were found when the same connexins were analyzed in cell lysates using Western blotting. (C) Immunostaining of a representative confluent cell culture (GFBL-DC) for C×43 and C×45. Gingival fibroblasts contained numerous C×43-positive plaques, while much fewer similar structures positive for C×45 were noted. In general, connexin-positive plaques were localized at cell-cell contact areas, possibly representing GJs (arrows), and other areas not associated with cell-cell contacts (arrowheads).

Figure 2. C×43 is down regulated in gingival fibroblasts during wound healing.

Representative immunostainings of C×43 (red) and vimentin (green; a mesenchymal cell marker) in unwounded human oral mucosal connective tissue (attached gingiva) (A and B), and in gingival granulation and wound connective tissue 3 (C and D), 7 (E and F), 14 (G and H), 28 (I and J) and 60 days (K and L) post-wounding. (A and B) In the unwounded gingival connective tissue, abundant C×43 immunoreactivity was present as punctate staining, likely representing C×43 plaques, in vimentin-positive fibroblast-like cells throughout the tissue. (C and D) At day 3 post-wounding, C×43 was down regulated in fibroblasts at wound edge as compared to unwounded tissue (A and B). First fibroblasts that had migrated into the wound area showed no immunoreactivity for C×43 (D). (E–H) At day 7 (E and F) and 14 (G and H) post-wounding, very few C×43 positive structures were noted in fibroblasts in the highly cellular granulation and connective tissue. (I and J) At day 28 after wounding, abundance of C×43-positive plaques in connective tissue cells in the newly formed connective tissue at the wound area was increased as compared to earlier time points. However, size of these plaques was clearly smaller than in the unwounded tissue (A and B). (K and L) At day 60 after wounding, structure of the connective tissue formed at the wound area was closely similar to unwounded tissue. Size and number of C×43-positive plaques in fibroblast-like cells in the regenerated wound area was similar to the unwounded tissue (A and B). Data shown represents minimum of three sections stained in parallel samples from two to three individual donors at each time point. CT: connective tissue; W Edge: wound edge; W Area: wound area; GT: granulation tissue; WCT: wound connective tissue. Nuclear staining (blue) was performed using DAPI.

Figure 3. Gap27 and MFA suppress GJ-mediated dye transfer in gingival fibroblasts.

(A–F) Confluent GFBL-DC cultures maintained in DMEM were scrape-loaded with Lucifer Yellow (0.5%; green) in the presence of control peptide (A and B; 150 μM), Gap27 (C; 150 μM), vehicle (dH₂O; D and E), or MFA (50 μM; F), and dye transfer was followed for 5 min. Treatment of cells with Gap27 (C) or MFA (F) markedly reduced dye transfer as compared to control samples treated with the control peptide (A and B) or vehicle (D and E). Results show representative images from minimum of three repeated experiments. For the experiments, cells were pretreated with Gap27 and control peptide or MFA and vehicle for 24 h or 1 h before the experiments, respectively. Magnification bars: 50 μm.

Figure 4. Effect of Gap27-mediated blocking of C×43 function on gene expression in parallel gingival fibroblast lines.

Confluent cultures of gingival fibroblasts from three different individuals (GFBL-HN, GFBL-CM and GFBL-DC) were treated with Gap27 or control peptide (150 μM) for 24 h, and expression of a set of genes involved in wound healing was analyzed by real-time PCR. Results represent mean +/− SEM of mRNA expression relative to control peptide-treated cells from triplicate samples in one experiment (*p<0.05, **p<0.01, ***p<0.001; Student’s t-test). Horizontal line indicates relative mRNA expression for the control-peptide treated samples. EDA-FN: Extra Domain A-Fibronectin; EDB-FN: Extra Domain B-Fibronectin; TN-C: Tenascin-C; CTGF: Connective Tissue Growth factor (CCN2); α-SMA: α-Smooth Muscle Actin; VEGF-A: Vascular Endothelial Growth Factor-A.

Figure 5. Blocking of C×43 by Gap27 resulted in significantly increased secretion of active MMP-1 and MMP-10, and pro-MMP-3 by gingival fibroblasts.

Confluent cultures of gingival fibroblasts (GFBL-DC) were treated with Gap27 or control peptide (150 μM) for 24 h, and abundance of MMP-1 (A–D), MMP-3 (E–G), and MMP-10 (H–J) in the conditioned medium and cell layer was analyzed by Western blotting. (B, D, G and J) Quantitation of MMP levels in Western blots shows mean +/− SEM from three independent experiments (*p<0.05, ***p<0.001; Student’s t-test). Sample loading for cell layer fraction was normalized for β-Tubulin levels. Identity of active and pro-forms of the enzymes was confirmed by pretreatment of a set of samples with or without APMA to activate latent enzymes prior to Western blotting (data not shown).

Figure 6. Blocking of C×43 function by Gap27 promotes secretion of VEGF-A, and suppresses DCN levels in gingival fibroblast cultures.

Confluent cultures of gingival fibroblasts (GFBL-DC) were treated with Gap27 or control peptide (150 μM) for 24 h, and Vascular Endothelial Growth Factor-A (VEGF-A) and Decorin (DCN) levels were analyzed in the conditioned medium by Western Blotting. Representative results from three independent experiments are shown.

Figure 7. Gap27 treatment increases C×43 protein abundance significantly.

(A) Confluent cultures of gingival fibroblasts (GFBL-DC) were treated with Gap27 or control peptide (150 μM) for 24 h, and abundance of C×43 was analyzed by Western blotting. Gap27 treatment did not affect the relative intensities of three bands corresponding to differently phosphorylated forms of C×43 (P0: pS368; P1:pS279/282 and pS255; P2: pS262). (B) Quantitation of C×43 levels in Western blots shows mean +/− SEM from three independent experiments (**p<0.01; Student’s t-test). Sample loading was normalized for β-Tubulin levels.

Figure 8. Western blotting analysis of key signaling pathways modulated by Gap27 in gingival fibroblasts.

Confluent cultures of gingival fibroblasts (GFBL-DC) were treated with Gap27 or control peptide (150 μM) for 1, 2, 6, and 24 h. Cell lysates were analyzed for protein levels of total SMAD3 and phosphorylated SMAD3 (p-SMAD3) (A), total p38 and phosphorylated p38 (p-p38) (C), total ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) (E), total GSK3α/β and phosphorylated GSK3α/β (p-GSK3α/β) (G), and total β-Catenin, phosphorylated β-Catenin (p-β-Catenin) and non-p-β-Catenin (I). (B, D, F, H and J) Quantitation of the phosphorylated or non-phosphorylated signaling molecules relative to their total levels at time 0 (control samples), and at 1, 2, 6 and 24 h after Gap27 treatment. Sample loading was normalized for β-Tubulin levels. Results from one experiment are shown.