Irsogladine maleate regulates epithelial barrier function in tumor necrosis factor-α-stimulated human gingival epithelial cells

Fujita T, Yumoto H, Shiba H, Ouhara K, Miyagawa T, Nagahara T, Matsuda S, Kawaguchi H, Matsuo T, Murakami S, Kurihara H. Irsogladine maleate regulates epithelial barrier function in tumor necrosis factor‐α‐stimulated human gingival epithelial cells. J Periodont Res 2012; 47: 55–61. © 2011 John Wiley & Sons A/S Background and Objective: As epithelial cells function as a mechanical barrier, the permeability of the gingival epithelial cell layer indicates a defensive capability against invasion by periodontal pathogens. We have reported the expression of claudin‐1 and E‐cadherin, key regulators of permeability, in the gingival junctional epithelium. Irsogladine maleate (IM) is a medication for gastric ulcers and also regulates Aggregatibacter actinomycetemcomitans‐stimuated chemokine secretion and E‐cadherin expression in gingival epithelium. In this study, we have further investigated the effects of IM on the barrier functions of gingival epithelial cells under inflammatory conditions. Material and methods: We examined the permeability, and the expression of claudin‐1 and E‐cadherin, in human gingival epithelial cells (HGECs) stimulated with tumor necrosis factor (TNF)‐α, with or without IM. Results: TNF‐α increased the permeability of HGECs, and IM abolished the increase. TNF‐α reduced the expression of E‐cadherin in HGECs, and IM reversed the reduction. In addition, immunofluorescence staining showed that TNF‐α disrupted claudin‐1 expression in HGECs, and IM reversed this effect. Conclusion: The results suggest that IM reverses the TNF‐α‐induced disruption of the gingival epithelial barrier by regulating E‐cadherin and claudin‐1.     

Interleukin-1β regulates odontogenic ameloblast-associated protein gene transcription in human gingival epithelial cells

Odontology - Junction epithelium (JE) is located apical to the bottom of the gingival sulcus and binds enamel to hemidesmosomes to protect the periodontal tissue from bacterial infection. Function...     

The induction expression of human β-defensins in gingival epithelial cells and fibroblasts

ObjectiveThe gingival epithelial cells and fibroblasts can produce antimicrobial peptides when stimulated by inflammatory cytokines. The purpose of the present study was to test whether gingival keratinocytes and gingival fibroblasts respond differently to inflammatory cytokine activation. This will enable us to understand the chronic inflammatory response in the process of periodontal disease.DesignGingival keratinocytes and fibroblasts were isolated and treated with different concentrations of IL-1β and quantitative real-time PCR was performed to evaluate the induced expressions of hBD-1, hBD-2 and hBD-3. The induced response was compared between the gingival epithelial cells and fibroblasts. The inhibitors of p38 protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) were applied to explore the molecular mechanism during the induction of hBDs in both cells.ResultsThe results showed that the hBDs expressions were found to be induced by different concentrations of IL-1β, but with several differences between gingival epithelial cells and fibroblasts. The hBDs mRNA expression in gingival fibroblasts was more sensitive compared with keratinocytes to different concentrations of IL-1β. The hBD-1 and hBD-3 expressions in these two cells were down-regulated by IL-1β and hBD-2 expression was up-regulated. The inflammatory cytokine IL-1β had dual effect on hBDs expression.ConclusionsThe gingival epithelial cells and fibroblasts respond differently to the inflammatory cytokine IL-1β which indicated different roles played by the two cells in the host defense. The dual effect of IL-1β on hBDs expression may contribute to the defensins down-regulation in periodontal disease.     

MiR-200b suppresses TNF-α-induced AMTN production in human gingival epithelial cells

Odontology - Amelotin (AMTN) is an enamel protein that is localized in junctional epithelium (JE) of gingiva and suggested to be involved in the attachment between JE and tooth enamel. MicroRNA is...     

Azithromycin recovers reductions in barrier function in human gingival epithelial cells stimulated with tumor necrosis factor-α

ObjectiveThe gingival epithelium plays an important role in protecting against the invasion of periodontal pathogens, and the permeability of gingival epithelial cells has been implicated in the initiation of periodontitis. Azithromycin (AZM) has been used in the treatment of chronic inflammatory airway diseases because it regulates cell–cell contact in airway epithelial cells. Therefore, AZM may also regulate barrier function in gingival epithelial cells. In the present study, we examined the effects of AZM on the permeability of human gingival epithelial cells (HGEC) under inflammatory conditions in vitro.Materials and methodsHGEC were stimulated by tumor necrosis factor-α (TNF-α) in the presence of AZM or p38 MAP kinase and ERK inhibitors. Permeability was assessed based on transepithelial electrical resistance (TER). The expression of E-cadherin, phosphorylated p38 MAP kinase, and ERK was analyzed by Western blotting.ResultsTNF-α decreased TER in HGEC, and AZM and the p38 MAP kinase and ERK inhibitors recovered this decrease. AZM inhibited the phosphorylation of ERK and p38 MAP kinase in TNF-α-stimulated HGEC. Furthermore, AZM recovered the decrease in E-cadherin expression in HGEC stimulated with TNF-α. Conclusions: These results suggested that AZM regulated gingival epithelial permeability through p38 MAP kinase and ERK signaling, and may contribute to suppress the inflammation in gingival tissue.     

Anodized-hydrothermally treated titanium with a nanotopographic surface structure regulates integrin-α6β4 and laminin-5 gene expression in adherent murine gingival epithelial cells

PurposePeri-implant epithelium associated with the structure of the internal basal lamina is in contact with a transmucosal portion of the endosseous implant surface. This contact is important to protect the many complex factors required for the long-term stability and maintenance of the implant. This study investigated the effect of initial adhesion of gingival epithelial cells to anodized-hydrothermally treated commercially pure titanium with nanotopographic structure (SA-treated c.p.Ti). Changes in cell morphology and gene expression of integrin-α6β4 and laminin-5 were assessed.MethodsMurine immortalized gingival epithelial (GE1) cells were cultured for 1–3 days on c.p.Ti, anodic oxide (AO) c.p.Ti, and SA-treated c.p.Ti disks. Cell morphology was analyzed using scanning electron microscopy (SEM). Cell proliferation was analyzed using the WST-1 assay. Integrin-α6β4 and laminin-5 (α3, β3, γ2) mRNA levels were measured using real-time quantitative RT-PCR.ResultsThe GE1 cells appeared flattened with extensions on all disks by SEM analysis. Filopodium-like extensions were bound closely to the nanotopographic structure surface of SA-treated c.p.Ti especially at day 3 of culture. GE1 cell proliferation as well as the expression of integrin-α6β4 and laminin-5 (α3, β3, γ2) mRNAs was significantly higher on SA-treated c.p.Ti than on c.p.Ti and AO c.p.Ti disks after 3 days (P < 0.05).ConclusionsGingival epithelial cells initially attach to a transmucosal portion of SA-treated c.p.Ti implant material and subsequently express the integrin-α6β4 adhesion molecule and the laminin-5 extracellular matrix molecule. This cell behavior may play a key role in maintaining the peri-implant oral mucosal tissue barrier.     

β-Phenethyl isothiocyanate induces death receptor 5 to induce apoptosis in human oral cancer cells via p38

Oral Diseases (2012) 18 , 513–519 Objectives: β‐Phenylethyl isothiocyanate (PEITC) has been demonstrated to fight many types of cancers through various molecular pathways. In this study, we focused on its effect on the induction of apoptosis to inhibit cell growth and molecular mechanism in oral cancer. Materials and methods: 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(2,4‐disulfophenyl)‐2‐(4 sulfophenyl)‐2H‐tetrazolium (MTS) assay was used to examine cell viability. The apoptotic effect was investigated using 4′‐6‐Diamidino‐2‐phenylindole (DAPI) staining or Western blotting. Inhibitors were used to determine the molecular target and mechanism of PEITC‐mediated apoptosis. Results: β‐Phenylethyl isothiocyanate inhibited the growth of HN22 human oral cancer cells and induced caspase‐dependent apoptosis in HN22 cells as evidenced by nuclear fragmentation and the activation of caspase 3. It increased cleaved caspase 8, truncated BID, and death receptor 5 (DR5) through the activation of p38 MAPK. This result was confirmed by blockage of PEITC‐induced cleavages of Poly(ADP‐ribose) Polymerase, caspase‐3, caspase‐8, and DR5 by p38 MAPK inhibitor, SB203580. We also found that PEITC activated p38 and augmented DR5 to induce apoptosis in other human oral cancer cells. Conclusions: These results suggest that DR5 is a potential molecular target for PEITC‐induced apoptosis in oral cancer via p38 MAPK.     

PERIOSTIN regulates MMP-2 expression via the αvβ3 integrin/ERK pathway in human periodontal ligament cells

ObjectiveDuring orthodontic tooth movement, activation of the vascular system in the compressed periodontal ligament (PDL), which becomes hypoxic, is essential for periodontal tissue remodelling. PERIOSTIN, an extracellular matrix protein, is expressed in PDL and its concentration is increased on the compressive side during orthodontic tooth movement. PERIOSTIN promotes angiogenesis through upregulation of matrix metalloproteinase (MMP)-2, which has been shown to be expressed via αvβ3 integrin/extracellular signal-related kinase (ERK) signalling pathway and vascular endothelial growth factor (VEGF). Therefore, we hypothesized that hypoxia-induced PERIOSTIN promotes MMP-2 expression via αvβ3 integrin/ERK signalling and VEGF in PDL cells.MethodsHuman PDL cells were cultured in condition medium containing desferrioxamine (DFO) to mimic hypoxia. The total RNA, cell lysates or supernatant were collected, and MMP2 and VEGF expression, PERIOSTIN expression and ERK phosphorylation, and MMP-2 activity were analysed by real-time RT-PCR, western blot analysis, and zymography, respectively. A recombinant human PERIOSTIN or PERIOSTIN siRNA was applied to the cells, then the total RNA was extracted to measure MMP-2 and VEGF expression. The cells were treated with αvβ3 integrin-blocking antibody or ERK inhibitor followed by PERIOSTIN stimulation. MMP-2 expression was measured by real-time RT-PCR.ResultsPERIOSTIN was upregulated in a time-dependent manner in human PDL cells treated with DFO, a chemical hypoxia mimic. MMP-2 and VEGF expression, and MMP-2 activity were increased by DFO or PERIOSTIN treatment, and decreased by PERIOSTIN silencing. PERIOSTIN treatment also induced ERK phosphorylation, and PERIOSTIN-induced MMP-2 was reduced by αvβ3 integrin-blocking antibody or ERK inhibitor.ConclusionThese data suggest that PERIOSTIN upregulates MMP-2 expression via the αvβ3 integrin/ERK signalling pathway and VEGF expression in human PDL cells.     

IL-1 and TNF-α regulation of aryl hydrocarbon receptor (AhR) expression in HSY human salivary cells

ObjectivesRecent findings demonstrate that nuclear receptor – aryl hydrocarbon receptor (AhR) may play an important role in the pathogenesis of Sjögren's syndrome (SS) via involvement in Epstein–Barr virus reactivation. In that study a reporter system was used. Therefore, it was decided to define AhR expression in human salivary cell line (HSY) and its functional regulators.DesignThe expression and functional regulation of AhR was studied in HSY cells. The cells were incubated with dioxin (TCDD) – AhR model inducer, IL-1 and TNF-α. qRT-PCR was applied to assess the expression of AHR, AHRR (AhR repressor), ARNT (AhR nuclear translocator) as well as AhR dependent genes: CYP1A1 and CYP1B1. Enzymatic activity of CYP1A1 and CYP1B1 was evaluated using luciferin-labelled CYPs substrate.ResultsIn general, dioxin did not significantly influence the expression of AHR and ARNT, but reduced AHRR level. AhR dependent gene expression, i.e. CYP1A1 and CYP1B1 increased gradually with TCDD incubation time. TNF-α significantly induced AHR along with CYP1A1 and CYP1B1 expression. IL-1β did not affect AHR expression, and had minimal effects on CYP1 mRNA levels. Exposure of HSY cells to TCDD resulted in time-dependent induction of CYP1A1 and CYP1B1 enzymatic activity.ConclusionsThis study documents functional expression of AhR in HSY as well as induction of AhR and its dependent genes by TNF-α.     

Is collagen breakdown during periodontitis linked to inflammatory cells and expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human gingival tissue?

BACKGROUND: Evidence of the role of matrix metalloproteinases (MMPs) produced by resident and inflammatory cells in periodontal destruction is now well established. The purpose of this study was to quantify, in healthy and diseased upper gingival connective tissue, the area fraction (AA%) occupied by collagen fibers, the cell number belonging to inflammatory cell subsets, and the amounts of MMPs and TIMPs (tissue inhibitors of MMPs) in order to investigate the possible correlations, if any, between such molecules, collagen loss, and inflammatory cell subsets. METHODS: Gingival tissue specimens from 6 healthy controls (C) and 6 patients with severe periodontitis (P) were divided into 2 groups. The first group of specimens was frozen and used for the staining of collagen fibers by sirius red F3Ba and for immunohistochemistry with antibodies against CD8, CD4, CD22, CD68, and TIA-1 molecules. The second group was used for organ culture, zymography, Western blotting, and dot blotting. Morphometric and automated image analysis was performed for the evaluation of the area fraction occupied by collagen fibers, the number of inflammatory cell subsets and for enzymatic activities developed by MMPs, and the amounts of TIMPs expressed during periodontal disease. RESULTS: In group P, the area fraction of collagen fibers (33 +/- 10%) was significantly decreased (P < 0.0002) when compared to group C (60 +/- 7%), and was correlated with the number of all inflammatory cells and amounts of MMPs and TIMPs. In group P, there were significant increases of CD8+, CD22+, CD68+, and TIA-1+ cells, as well as increases in the amounts of MMP-1, MMP-2, MMP-3, MMP-9, and the active form of MMP-9. The active form of MMP-9 and the amount of TIMP-1 were positively correlated with the number of CD22+, CD68+, and TIA-1+ cells. CONCLUSIONS: The present study showed an imbalance between MMPs and TIMPs associated with the pathologic breakdown of the extracellular matrix during periodontitis. The active form of MMP-9 could be a marker for the clinical severity of periodontal disease.     

Maresin 1 regulates autophagy and inflammation in human periodontal ligament cells through glycogen synthase kinase–3β/β-catenin pathway under inflammatory conditions

ObjectiveAccumulating lines of evidence suggest that maresin 1 (MaR-1) exerts anti-inflammatory effects in many cell types and plays beneficial roles in inflammatory disease, such as peritonitis and colitis. Moreover, it has been demonstrated that MaR-1 play protective roles against localized aggressive periodontitis. However, the function and mechanism of MaR-1 in human periodontal ligament cells (PDL) cells from periodontitis are poorly understood. The present study aimed to clarify the effects and molecular mechanism of MaR-1 in PDL cell survival and inflammation.MethodsPDL cells were isolated from the middle third of the root surface of premolars from four healthy humans; MTT assay and cell death detection ELISA assay were used to detect cell survival and apoptosis; Inflammatory cytokines level was measured by ELISA assay; RT-PCR and western blot was used to measure the mRNA and protein expression in this study.ResultsHere we found that MaR-1 treatment markedly promotes survival and inhibits apoptosis in PDL cell treated by LPS. MaR-1 treatment strikingly suppressed the production of LPS-induced pro-inflammatory cytokines IL-6, IL-8, TNF-α and IL-1β. MaR-1 also promotes autophagy by increasing the ratio of LC3II/LC3I, the level of beclin-1 and reduced the expression of p62 in LPS treated PDL cells, which is beneficial to cell survival. Moreover, the results showed that MaR-1-mediated autophagy is dependent on the glycogen synthase kinase–3β(GSK-3β)/β-catenin signal pathway. The inhibitor of autophagy 3-MA and the inhibitor of the GSK-3β/β-catenin signal pathway LiCL both reverse the effects of MaR-1 on LPS-treated PDL cell survival and inflammation.ConclusionMaR-1 promotes cell survival and alleviates cell inflammation by activating GSK-3β/β-catenin-dependent autophagy. These results provide new insights into the mechanism of chronic periodontitis.     

Effect of hydrogen sulphide on the expression of osteoprotegerin and receptor activator of NF-κB ligand in human periodontal ligament cells induced by tension-force stimulation

ObjectiveHydrogen sulphide (H₂S) is an endogenous gaseous signalling molecule, the generation rate of which is affected by mechanical force in cells. Recently, it was reported that mechanical force plays an important role in some pathways such as osteoprotegerin (OPG) and the receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) in human periodontal ligament cells (hPDLCs). Here, we investigated whether H₂S played a regulatory role within the periodontal remodelling process by addressing the expression level of OPG/RANKL in hPDLCs.DesignhPDLCs were first applied with tension force for 0–120 min to select the optimal time for force application. These cells were treated with H₂S for 24 h followed by stimulation with tension-force application. Cell proliferation and apoptosis were assessed by the methyl thiazolyl tetrazolium (MTT) assay and flow cytometry analysis. For OPG, RANKL and cystathionine-γ-lyase (CSE), real-time polymerase chain reaction (PCR) was used to analyse the messenger RNA (mRNA) expression; enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of OPG and soluble RANKL (sRANKL).ResultsTension force promoted the mRNA expression of CSE and the optimal application time was 60 min. The expression of OPG was increased in a concentration-dependent manner by H₂S treatment. Importantly, the relative OPG/RANKL expression ratio was significantly increased upon induction by H₂S, an effect that was enhanced by tension-force application.ConclusionsH₂S could promote osteogenic differentiation by regulating the relative OPG/RANKL expression ratio of hPDLCs, which is enhanced by tension force. These findings may be valuable for understanding the mechanism of H₂S in the periodontal remodelling, especially in the process of tooth movement.