Role of connexin 43 in regulating wound healing-related gene expression in human skin and gingival fibroblasts

Wound healing in human oral mucosal gingiva is faster and results in significantly reduced scar formation as compared to similar skin wounds. Fibroblasts are the most abundant group of connective tissue cells that play a key role in wound healing and scar formation. It is possible that differential healing outcomes in skin and gingiva may relate to the distinct phenotypic features of fibroblasts residing in these tissues. In fibroblasts, cells-to-cell communication occurs partly through connexin (Cx) hemichannels (HCs) and gap junctions (GJs). Findings from previous studies have shown that functional blockage of connexin 43 (Cx43), the most ubiquitous Cx in skin (SFBLs) and gingival fibroblasts (GFBLs), accelerates wound closure in skin and may alleviate scarring, but the mechanisms are poorly understood and may involve modulation of Cx43 function in fibroblasts. In the present dissertation, we show that (1) Cx43 was the most abundant Cx present in cultured human SFBLs and GFBLs. (2) Its abundance was potently downregulated at the early stage of human gingival wound healing. (3) Cx43 assembled into GJ and HC plaques in skin and gingival epithelium and connective tissue fibroblasts, although its distribution into GJs or HCs was markedly different in these two tissues. (4) Cx43 mainly assembled into HCs in GFBLs while in SFBLs only a few HCs were present in vivo and in vitro. (5) Using an in vivo-like 3D culture model and Cx43 mimetic peptides to block its function, we showed that the GJ, HC, and channel-independent functions of Cx43 distinctly upregulate anti-fibrotic and downregulate profibrotic wound healing-related genes in GFBLs and SFBLs. (6) In GFBLs this response was mainly mediated by activation of ERK1/2 pathway via Cx43 HC blockage. Thus, Cx43 assembly into GJs and HCs and its function are distinct in SFBLs and GFBLs, which may contribute to the different wound healing outcomes in these tissues. Furthermore, specific blockage of Cx43 HC functions may provide a novel target to promote wound healing and alleviate scar formation.Dentistry, Faculty ofGraduat

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

<div><p>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.</p></div

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

<p>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.</p

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.

<p>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).</p

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

<p>(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.</p

Blocking of C×43 function with Gap27 treatment modulates significantly expression of genes involved in TGF-β signaling and encoding VEGF-A and CXCL12/SDF-1α in gingival fibroblasts.

<p>Results show real-time PCR analysis of relative mRNA expression in confluent GFBL-DC cultures treated with Gap27 (150 μM) relative to control peptide-treated samples for 24 h. Results represent mean +/− SEM from minimum of three repeated experiments (*p<0.05, **p<0.01, ***p<0.001; Student’s t-test). Genes that are bolded show ≥1.5-fold up or down regulation relative to control peptide treated samples. Genes with negligible expression (Ct>30) were not analyzed further. TGF-βR1: TGF-β Receptor 1; TGF-βR2: TGF-β Receptor 2; EGR1: Early Growth Response 1; EGR2: Early Growth Response 2; EGR3: Early Growth Response 3; NAB1: NGFI-A Binding Protein-1; NAB2: NGFI-A Binding Protein-2; VEGF-A: Vascular Endothelial Growth Factor-A; FGF-2: Fibroblast Growth Factor-2; IL1β Interleukin-1β; IL Interleukin-10; TNF-α: Tumor Necrosis Factor-α.</p><p>Blocking of C×43 function with Gap27 treatment modulates significantly expression of genes involved in TGF-β signaling and encoding VEGF-A and CXCL12/SDF-1α in gingival fibroblasts.</p

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

<p>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.</p

Blocking of C×43 function with Gap27 treatment modulates significantly expression of extracellular matrix proteins and cell contractility and myofibroblast-associated genes in gingival fibroblasts.

<p>Results show real-time PCR analysis of relative mRNA expression in confluent GFBL-DC cultures treated with Gap27 (150 μM) relative to control peptide-treated samples for 24 h. Results represent mean +/− SEM from minimum of three repeated experiments (*p<0.05, ***p<0.001; Student’s t-test). Genes that are bolded show ≥1.5-fold up or down regulation relative to control peptide treated samples. EDA-FN: Extra Domain A-Fibronectin; EDB-FN: Extra Domain B-Fibronectin; TN-C: Tenascin-C; BGN: Biglycan; DCN: Decorin; FMOD: Fibromodulin; LUM: Lumican; α-SMA: α-Smooth Muscle Actin; NMMIIA: Non-Muscle Myosin IIA; NMMIIB: Non-Muscle Myosin IIB.</p><p>Blocking of C×43 function with Gap27 treatment modulates significantly expression of extracellular matrix proteins and cell contractility and myofibroblast-associated genes in gingival fibroblasts.</p

Blocking of C×43 function with Gap27 treatment modulates significantly expression of genes involved in protein degradation during wound healing in gingival fibroblasts.

<p>Results show real-time PCR analysis of relative mRNA expression in confluent GFBL-DC cultures treated with Gap27 (150 μM) relative to control peptide-treated samples for 24 h. Results represent mean +/− SEM from minimum of three repeated experiments (**p<0.01, ***p<0.001; Student’s t-test). Genes that are bolded show ≥1.5-fold up or down regulation relative to control peptide treated samples. Genes with negligible expression (Ct>30) were not analyzed further. MMP: Matrix Metalloproteinase; TIMP: Tissue Inhibitor of Metalloproteinase; CTSK: Cathepsin K.</p><p>Blocking of C×43 function with Gap27 treatment modulates significantly expression of genes involved in protein degradation during wound healing in gingival fibroblasts.</p