Guoqiao Jiang

Absence of αvβ6 Integrin Is Linked to Initiation and Progression of Periodontal Disease

Integrin alphavbeta6 is generally not expressed in adult epithelia but is induced in wound healing, cancer, and certain fibrotic disorders. Despite this generalized absence, we observed that alphavbeta6 integrin is constitutively expressed in the healthy junctional epithelium linking the gingiva to tooth enamel. Moreover, expression of alphavbeta6 integrin was down-regulated in human periodontal disease, a common medical condition causing tooth loss and also contributing to the development of cardiovascular diseases by increasing the total systemic inflammatory burden. Remarkably, integrin beta6 knockout mice developed classic signs of spontaneous, chronic periodontal disease with characteristic inflammation, epithelial down-growth, pocket formation, and bone loss around the teeth. Integrin alphavbeta6 acts as a major activator of transforming growth factor-beta1 (TGF-beta1), a key anti-inflammatory regulator in the immune system. Co-expression of TGF-beta1 and alphavbeta6 integrin was observed in the healthy junctional epithelium. Moreover, an antibody that blocks alphavbeta6 integrin-mediated activation of TGF-beta1 initiated inflammatory periodontal disease in a rat model of gingival inflammation. Thus, alphavbeta6 integrin is constitutively expressed in the epithelium sealing the gingiva to the tooth and plays a central role in protection against inflammatory periodontal disease through activation of TGF-beta1.

Expression of Endo180 is spatially and temporally regulated during wound healing

Aims:  Interactions of cells with the extracellular matrix are important for normal wound healing and may play a role in scar formation. Remarkably, wound healing in human gingiva does not result in scar formation and serves as a model for wound regeneration. Endo180 (CD280) is a cell surface receptor that has novel functions to regulate cell migration and bind and internalize collagens that are key processes in wound healing. The aim of this study was to examine the expression of Endo180 during gingival wound regeneration.

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

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.

Critical role for αvβ6 integrin in enamel biomineralization

Summary Tooth enamel has the highest degree of biomineralization of all vertebrate hard tissues. During the secretory stage of enamel formation, ameloblasts deposit an extracellular matrix that is in direct contact with the ameloblast plasma membrane. Although it is known that integrins mediate cell–matrix adhesion and regulate cell signaling in most cell types, the receptors that regulate ameloblast adhesion and matrix production are not well characterized. We hypothesized that &agr;v&bgr;6 integrin is expressed in ameloblasts where it regulates biomineralization of enamel. Human and mouse ameloblasts were found to express both &bgr;6 integrin mRNA and protein. The maxillary incisors of Itgb6−/− mice lacked yellow pigment and their mandibular incisors appeared chalky and rounded. Molars of Itgb6−/− mice showed signs of reduced mineralization and severe attrition. The mineral-to-protein ratio in the incisors was significantly reduced in Itgb6−/− enamel, mimicking hypomineralized amelogenesis imperfecta. Interestingly, amelogenin-rich extracellular matrix abnormally accumulated between the ameloblast layer of Itgb6−/− mouse incisors and the forming enamel surface, and also between ameloblasts. This accumulation was related to increased synthesis of amelogenin, rather than to reduced removal of the matrix proteins. This was confirmed in cultured ameloblast-like cells, in which &agr;v&bgr;6 integrin was not an endocytosis receptor for amelogenins, although it participated in cell adhesion on this matrix indirectly via endogenously produced matrix proteins. In summary, integrin &agr;v&bgr;6 is expressed by ameloblasts and it plays a crucial role in regulating amelogenin deposition and/or turnover and subsequent enamel biomineralization.

Localization and potential function of kindlin-1 in periodontal tissues

Kindlin-1 is an intracellular focal adhesion protein that regulates the actin cytoskeleton. Patients suffering from Kindler syndrome have a homologous mutation of the kindlin-1 gene and develop skin blisters, periodontal disease, and intestinal complications because of deficient adhesion of the basal epithelial cells. We investigated kindlin-1 localization in periodontal tissue and its functions in cultured keratinocytes and showed that kindlin-1 co-localizes with migfilin and paxillin in the basal epithelial cells of oral mucosa and in cultured keratinocytes. The kindlin-1-deficient oral mucosal tissue from a patient with Kindler syndrome showed a complete lack of paxillin and reduced migfilin immunostaining in the basal keratinocytes. Co-immunoprecipitation showed that migfilin directly interacted with kindlin-1. RNA interference-induced kindlin-1 deficiency in keratinocytes led to an altered distribution of migfilin-containing focal adhesions, reduced cell spreading, decreased cell proliferation, and decelerated cell migration. Disruption of microtubules in the kindlin-1-deficient cells further reduced cell spreading, suggesting that microtubules can partially compensate for kindlin-1 deficiency. Kindlin-1 supported mature cell-extracellular matrix adhesions of keratinocytes, as downregulation of kindlin-1 expression significantly reduced the cell-adhesion strength. In summary, kindlin-1 interacts with migfilin and plays a crucial role in actin-dependent keratinocyte cell adhesion essential for epidermal and periodontal health.

Connexin 43 Hemichannels Regulate the Expression of Wound Healing-Associated Genes in Human Gingival Fibroblasts

Connexin 43 (Cx43) is the most ubiquitous connexin in various cells, and presents as hemichannels (HCs) and gap junctions (GJs) on the cell membrane. We have recently shown that Cx43 abundance was strongly reduced in fibroblasts of human gingival wounds, and blocking Cx43 function in cultured human gingival fibroblasts (GFBLs) strongly regulated the expression of wound healing-related genes. However, it is not known whether these responses involved Cx43 HCs or GJs. Here we show that Cx43 assembled into distinct GJ and HC plaques in GFBLs both in vivo and in vitro. Specific blockage of Cx43 HC function by TAT-Gap19, a Cx43 mimetic peptide, significantly upregulated the expression of several MMPs, TGF-β signaling molecules, Tenascin-C, and VEGF-A, while pro-fibrotic molecules, including several extracellular matrix proteins and myofibroblast and cell contractility-related molecules, were significantly downregulated. These changes were linked with TAT-Gap19-induced suppression of ATP signaling and activation of the ERK1/2 signaling pathway. Collectively, our data suggest that reduced Cx43 HC function could promote fast and scarless gingival wound healing. Thus, selective suppression of Cx43 HCs may provide a novel target to modulate wound healing.

Elevated CD26 Expression by Skin Fibroblasts Distinguishes a Profibrotic Phenotype Involved in Scar Formation Compared to Gingival Fibroblasts

Compared to skin, wound healing in oral mucosa is faster and produces less scarring, but the mechanisms involved are incompletely understood. Studies in mice have linked high expression of CD26 to a profibrotic fibroblast phenotype, but this has not been tested in models more relevant for humans. We hypothesized that CD26 is highly expressed by human skin fibroblasts (SFBLs), and this associates with a profibrotic phenotype distinct from gingival fibroblasts (GFBLs). We compared CD26 expression in human gingiva and skin and in gingival and hypertrophic-like scar-forming skin wound healing in a pig model, and used three-dimensional cultures of human GFBLs and SFBLs. In both humans and pigs, nonwounded skin contained abundantly CD26-positive fibroblasts, whereas in gingiva they were rare. During skin wound healing, CD26-positive cells accumulated over time and persisted in forming hypertrophic-like scars, whereas few CD26-positive cells were present in the regenerated gingival wounds. Cultured human SFBLs displayed significantly higher levels of CD26 than GFBLs. This was associated with an increased expression of profibrotic genes and transforming growth factor-β signaling in SFBLs. The profibrotic phenotype of SFBLs partially depended on expression of CD26, but was independent of its catalytic activity. Thus, a CD26-positive fibroblast population that is abundant in human skin but not in gingiva may drive the profibrotic response leading to excessive scarring.

Connexin 43 regulates the expression of wound healing-related genes in human gingival and skin fibroblasts

ABSTRACT Fibroblasts are the most abundant connective tissue cells and play an important role in wound healing. It is possible that faster and scarless wound healing in oral mucosal gingiva relative to skin may relate to the distinct phenotype of the fibroblasts residing in these tissues. Connexin 43 (Cx43) is the most ubiquitous Cx in skin (SFBLs) and gingival fibroblasts (GFBLs), and assembles into hemichannels (HCs) and gap junctions (GJs) on the cell membrane. We hypothesized that SFBLs and GFBLs display distinct expression or function of Cx43, and that this may partly underlie the different wound healing outcomes in skin and gingiva. Here we show that Cx43 distinctly formed Cx43 GJs and HCs in human skin and gingiva in vivo. However, in SFBLs, in contrast to GFBLs, only a small proportion of total Cx43 assembled into HC plaques. Using an in vivo‐like 3D culture model, we further show that the GJ, HC, and channel‐independent functions of Cx43 distinctly regulated wound healing‐related gene expression in GFBLs and SFBLs. Therefore, the distinct wound healing outcomes in skin and gingiva may partly relate to the inherently different assembly and function of Cx43 in the resident fibroblasts. HIGHLIGHTSCx43 distinctly forms gap junctions and hemichannels (HCs) in human skin and gingiva.Compared to human gingival fibroblasts, skin fibroblasts contained few Cx43 HCs.Cx43 distinctly regulates gene expression in human gingival and skin fibroblasts.

Regulation of connexin 43 expression in human gingival fibroblasts

Aims: Abundance of connexin 43 (Cx43), a transmembrane protein that forms hemichannels (HCs) and gap junctions (GJs), is dynamically regulated in human gingival fibroblasts (GFBLs) during wound healing. This may be important for fast and scarless gingival wound healing as Cx43 is involved in key cell functions important during this process. Our aim was to uncover the factors that regulate Cx43 expression and abundance in GFBLs. We hypothesized that cytokines and growth factors released during wound healing coordinately regulate Cx43 abundance in GFBLs. Results: TGF‐&bgr;1, ‐&bgr;2, ‐&bgr;3, PGE2 and IL‐1&bgr; significantly upregulated, while TNF‐&agr; and IFN‐&ggr; downregulated Cx43 in cultured GFBLs. TGF‐&bgr;1, ‐&bgr;2, ‐&bgr;3, IL‐1&bgr; and IFN‐&ggr; modulated Cx43 abundance at both mRNA and protein levels, while TNF‐&agr; and PGE2 regulated only Cx43 protein abundance, suggesting involvement of distinct transcriptional/post‐transcriptional and translational/post‐translational mechanisms, respectively. TGF‐&bgr;1‐induced upregulation of Cx43 was mediated by TGF&bgr;RI (ALK5) and SMAD2/3 signaling, and this was potently suppressed by PGE2, IL‐1&bgr;, TNF‐&agr; and IFN‐&ggr; that inhibited SMAD2/3 phosphorylation. Conclusion: Regulation of Cx43 abundance in GFBLs involves transcriptional/post‐transcriptional and translational/post‐translational mechanisms that are distinctly modulated by an interplay between TGF‐&bgr; isoforms and PGE2, IL‐1&bgr;, TNF‐&agr; and IFN‐&ggr;. HighlightsWound healing‐associated cytokines modulate connexin 43 abundance in human gingival fibroblasts.Interplay between these cytokines distinctly determines connexin 43 abundance via transcriptional/post‐transcriptional and translational/post‐translational mechanisms.TGF‐&bgr;‐induced upregulation of connexin 43 is mediated by SMAD2/3 pathway.TNF‐&agr;, IL‐1&bgr;, IFN‐&ggr; and PGE2 inhibit TGF‐&bgr;‐induced connexin 43 upregulation by suppressing SMAD2/3 phosphorylation.

Suppression of αvβ6 Integrin Expression by Polymicrobial Oral Biofilms in Gingival Epithelial Cells

Periodontal diseases manifest by the formation of deep pockets between the gingiva and teeth where multispecies bacterial biofilms flourish, causing inflammation and bone loss. Epithelial cell receptor αvβ6 integrin that regulates inflammation by activating the anti-inflammatory cytokine transforming growth factor-β1, is highly expressed in healthy junctional epithelium that connects the gingiva to the tooth enamel. However, its expression is attenuated in human periodontal disease. Moreover, Itgb6−/− mice display increased periodontal inflammation compared to wild-type mice. We hypothesized that bacterial biofilms present in the periodontal pockets suppress αvβ6 integrin levels in periodontal disease and that this change aggravates inflammation. To this end, we generated three-week-old multi-species oral biofilms in vitro and treated cultured gingival epithelial cells (GECs) with their extracts. The biofilm extracts caused suppression of β6 integrin expression and upregulation of pro-inflammatory cytokines, including interleukin-1β and -6. Furthermore, GECs with β6 integrin siRNA knockdown showed increased interleukin-1β expression, indicating that αvβ6 integrin-deficiency is associated with pro-inflammatory cytokine responsiveness. FSL-1, a synthetic bacterial lipopeptide, also suppressed β6 integrin expression in GECs. Therefore, biofilm components, including lipopeptides, may downregulate αvβ6 integrin expression in the pocket epithelium and thus promote epithelial cell-driven pro-inflammatory response in periodontal disease.