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.     

Targeting TNF-α suppresses the production of MMP-9 in human salivary gland cells

Objective

Tumour necrosis factor-α (TNF-α) is a pleiotropic cytokine that plays an essential role in inflammation and apoptosis. Our previous study suggested that TNF-α-induced activation of matrix metalloproteinase-9 (MMP-9) resulted in the destruction of acinar tissue in the salivary glands of patients with Sjögren's syndrome (SS) via disruption of the acinar cell-basement membrane. Recently, a wide array of biological agents has been designed to inhibit TNF, including etanercept and adalimumab.In this study, we demonstrate the suppressive effect of anti-TNF agents on TNF-α-induced MMP-9 production in NS-AV-AC, an immortalized human salivary gland acinar cell line.

Materials and methods

NS-AV-AC cells were treated with etanercept or adalimumab after TNF-α treatment. MMP-9 production and enzymatic activity were, respectively, visualized by real-time PCR and ELISA assay, and evaluated by gelatin zymography, and apoptosis was evaluated by DNA fragmentation assay.

Results

TNF-α induced the production of MMP-9 in NS-SV-AC cells. However, this production was greatly inhibited by treatment with etanercept or adalimumab. In addition, TNF-α-induced DNA fragmentation was prevented by treatment with etanercept or adalimumab.

Conclusions

These results may indicate that anti-TNF agents would have therapeutic efficacy for preventing destruction of the acinar structure in the salivary glands of patients with SS.     

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.     

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.     

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...     

Estrogen regulates DNA synthesis in human gingival epithelial cells displaying strong estrogen receptor β immunoreactivity

Nebel D, Bratthall G, Ekblad E, Norderyd O, Nilsson B‐O. Estrogen regulates DNA synthesis in human gingival epithelial cells displaying strong estrogen receptor β immunoreactivity. J Periodont Res 2011; 46: 622–628. © 2011 John Wiley & Sons A/S Background and Objective: Estrogen acts via estrogen receptor (ER) α and β. The expression pattern of ERs and their importance in gingival tissues are not fully understood. In this study, we investigate gingival ER expression and effects of estrogen on gingival epithelial cell proliferation. Material and Methods: Gingival biopsies were obtained from both healthy and diseased sites in three male and three female subjects. Expression of ERα and β was determined by immunohistochemistry. Effects of 17β‐estradiol (E₂) on cell proliferation, monitored by measuring DNA synthesis, were studied in cultured human gingival epithelial HGEPp.05 cells. Results: Estrogen receptor β, but not ERα, immunoreactivity was demonstrated in nuclei of epithelial cells in all layers of the gingival epithelium, but also in cells of the lamina propria. No differences were observed between male and female subjects. The same pattern, i.e. high ERβ expression but no ERα expression, was observed in both healthy and diseased sites within each individual. No differences in the intensity of the ERβ immunoreactive signal and the number of ERβ‐positive nuclei were observed between healthy and diseased gingiva. Treatment with a physiological concentration of E₂ (10 nm ) had no effect on DNA synthesis in ERβ‐ and ERα‐expressing HGEPp.05 cells. In contrast, E₂ at high concentrations (500 nm and 10 μm ) reduced DNA synthesis by 60–70%. Conclusion: Human gingival epithelial cells display strong ERβ but low ERα immunoreactivity both in vivo and in culture. Estrogen attenuates gingival epithelial cell DNA synthesis at high but not low concentrations, suggesting a concentration‐dependent mechanism.     

Involvement of angiotensin II type 1 receptors in interleukin-1β-induced interleukin-6 production in human gingival fibroblasts

The renin-angiotensin system is thought to be involved in inflammatory processes such as periodontitis. However, its precise role is still unclear. Therefore, in the present study the expression of the angiotensin II type 1 receptor (AT1R) was investigated in inflamed human gingival tissue, and the possible involvement of the AT1R in interleukin-1β (IL-1β)-induced interleukin-6 (IL-6) production by cultured human gingival fibroblasts (HGFs) was also studied. Immunohistochemical staining revealed that inflammatory cells and fibroblast-like cells were positive for the AT1R. However, in healthy gingival tissue, AT1R staining was very weak. The levels of AT1R mRNA and AT1R protein increased in HGFs after stimulation with IL-1β. The levels of IL-1β-induced IL6 mRNA and IL-6 protein were significantly reduced in AT1R gene-silenced HGFs compared with control HGFs. The data suggest that the AT1R may be involved in the regulation of gingival inflammation by modulating IL-1β-induced IL-6 production in HGFs.     

Statins regulate interleukin-1β-induced RANKL and osteoprotegerin production by human gingival fibroblasts

Stein SH, Dean IN, Rawal SY, Tipton DA. Statins regulate interleukin‐1β ‐ induced RANKL and osteoprotegerin production by human gingival fibroblasts. J Periodont Res 2011; 46: 483–490. © 2011 John Wiley & Sons A/S Background and Objective: Three‐hydroxy‐3‐methyl‐glutaryl‐CoA (HMG‐CoA) reductase competitive inhibitors, or ‘statins’, are widely used for lowering cholesterol and thereby reducing the risk of a heart attack. Recent data suggest that statins influence metabolic bone activity by their actions on three molecules: RANKL; RANK; and osteoprotegerin (OPG), the soluble decoy receptor for RANKL. The purpose of this study was to evaluate OPG and RANKL production in resting and interleukin‐1β (IL‐1β)‐activated human gingival fibroblasts (HGFs), and to determine the effect of statins on their production. Material and Methods: Fibroblasts were pre‐incubated with atorvastatin or simvastatin for 24 h in serum‐free medium, and then incubated with IL‐1β for 6 d. The concentration of OPG or RANKL in culture supernatants was measured by specific ELISA. Data were analyzed using analysis of variance and Scheffe’s F procedure for post hoc comparison. Results: IL‐1β (1 × 10^?8 m ) stimulated a significant increase in the production of OPG on days 1, 3 and 6. There was a trend towards an increase in RANKL production as a result of stimulation with IL‐1β. Both statins, at multiple concentrations, significantly increased the constitutive RANKL/OPG ratio. Only atorvastatin at the highest concentration (5 × 10^?6 m ) significantly increased the IL‐1β‐stimulated RANKL/OPG ratio. Conclusion: IL‐1β significantly increased OPG production by HGFs. The statins differed minimally in their effects on OPG and RANKL production by resting and IL‐1β‐activated HGFs. Both statins increased constitutive RANKL/OPG ratios, but generally not IL‐1β‐stimulated ratios. Thus, statins may influence the production of RANKL and OPG by HGFs to favor bone catabolism, under noninflammatory conditions.     

Fibronectin supports TNF-α-induced osteopontin expression through β1 integrin and ERK in HN-22 cells

The extracellular matrix (ECM), in collaboration with intracellular signal, plays a critical role in the modulation of cellular behavior and function. Herein, we investigated the influence of fibronectin (FN) and tumor necrosis factor-alpha (TNF-α) on OPN expression in HN-22, a human head and neck squamous cell carcinoma (HNSCC) cell line. The data showed that TNF-α significantly increased OPN expression only in the FN-coated condition. Application of function-blocking antibody directed against β1 integrin abolished this OPN induction. Moreover, TNF-α when added together with activating β1 integrin antibody is sufficient to induced OPN expression. The combination effect of FN and TNF-α was significantly deteriorated by a MEK inhibitor, but not NF-κB inhibitor. We further demonstrated that the phosphorylation of ERK1/2 was strongly enhanced by TNF-α and FN compared to the application of either one alone. Synergistic effect on ERK1/2 phosphorylation was also detected by TNF-α and activating β1 integrin antibody, whereas inhibitory antibody to β1 integrin attenuated FN and TNF-α-induced phosphorylation of ERK1/2. Our results indicate that FN coordinates TNF-α-mediated OPN induction via β1 integrin-dependent signaling mechanism that activates ERK. The results suggest the critical role of tumor micro-environment signaling networks on the regulation of cytokine expression profiles during tumor progression.     

Regulation of antimicrobial peptide expression in human gingival keratinocytes by interleukin-1α

In the oral cavity, mucosal keratinocytes resist bacterial infection, in part, by producing broad-spectrum antimicrobial peptides (AMPs) including defensin, adrenomedullin and calprotectin. Epidermal keratinocyte expression of many AMPs increases in response to interleukin-1α (IL-1α). IL-1α is produced by epidermal keratinocytes and regulates cell differentiation. To better understand innate immunity in the oral cavity, we sought to determine how IL-1α might regulate expression of AMPs by human gingival keratinocytes (HGKs) using DNA microarray and Western blot analyses. HGKs from three subjects expressed eleven AMPs, including S100A7, S100A8, S100A9, S100A12, secretory leucocyte protease inhibitor, lipocalin 2 (LCN2), cystatin C and β-defensin 2. Of the expressed AMPs, S100A7, S100A12 and LCN2 were up-regulated by IL-1α (inducible AMPs); the other AMPs were considered to be constitutive. Human gingival keratinocytes, therefore, express constitutive and IL-1α-inducible AMPs to provide a rapid and robust innate response to microbial infection.     

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.     

ALK5 inhibition blocks TGFß-induced CCN2 expression in gingival fibroblasts

Connective tissue growth factor (CCN2/CTGF) is not normally expressed in gingival fibroblasts, but is induced by the potent profibrotic cytokine TGFβ and is overexpressed in gingival fibrosis. Since CCN2 is a marker and mediator of fibrosis, targeting CCN2 expression in gingival fibroblasts may provide new insights into the future development of novel therapeutic opportunities to treat oral fibrosis. Herein we used real-time polymerase chain-reaction, Western blot, and indirect immunofluorescence analysis to evaluate whether SB-431542, a specific pharmacological inhibitor of TGFβ type I receptor (ALK5), blocks the ability of TGFβ to induce CCN2 mRNA and protein expression in human gingival fibroblasts. Our results indicate that CCN2 mRNA and protein are induced by TGFβ in gingival fibroblasts in a SB-431542-sensitive fashion. These results suggest that blocking ALK5 may be useful in blocking the profibrotic effects of TGFβ in gingival fibroblasts.     

Involvement of HMGB1 and RAGE in IL-1β-induced gingival inflammation

ObjectiveExtracellularly released high mobility group box 1 (HMGB1) protein behaves as a cytokine, promotes inflammation and participates in the pathogenesis of several disorders in peripheral organs. The role of HMGB1 and receptor for advanced glycation end products (RAGE) expressed in gingival inflammatory tissues was explored.MethodsReal time PCR was applied to assay HMGB1 and RAGE mRNA expression in gingival epithelial and fibroblast cells induced by interleukin-1β (IL-1β). A highly selective inhibitor of inducible nitric oxide (iNOS) was employed. ELISA was done for measurement of HMGB1 concentrations in cell culture media of gingival epithelial and fibroblast cells. Immunohistochemistry was performed to analyse the expression and sub-cellular localization of HMGB1, together with RAGE, in specimens obtained from patients with chronic inflammation.ResultsA time-dependent response of HMGB1 and RAGE expression in gingival cells to IL-1β induction was observed. IL-1β promotes HMGB1 production in human gingival epithelial cells in a nitric oxide-dependent manner. HMGB1 and RAGE appeared highly expressed in gingival inflammatory tissues.ConclusionThese results demonstrate that HMGB1 and RAGE are abundantly expressed in gingiva and promptly released during gingival inflammation. We suggest a role for HMGB1/RAGE/iNOS signalling on inflamed gingival epithelial cells.     

Rho plays a key role in TGF-β1-induced proliferation and cytoskeleton rearrangement of human periodontal ligament cells

Human periodontal ligament cells (hPDLCs) form specialised connective tissues that influence the lifespan of the tooth. Periodontal disease is a chronic infectious disease of the periodontal supporting tissues caused by a variety of factors, particularly the loss of hPDLCs. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine known to play an important role in periodontal disease, but little is known about the effects of TGF-β1 on human PDL cells. To determine how TGF-β1 mediates the changes in hPDLCs, we characterised the effects of TGF-β1 treatment on hPDLCs. We then elucidated the signalling pathway that mediates these effects. Serum-starved hPDLCs were incubated with 10 ng/mL TGF-β1, and their proliferation was examined using the Cell Counting Kit-8, while their morphological changes were examined by phase-contrast microscopy. F-actin reorganisation was visualised by phalloidin staining and confocal microscopy. Protein expression was analysed by western blotting. We found that TGF-β1 treatment induced proliferation and cytoskeletal reorganisation, decreased Rho-GDIa protein expression, activated ROCK protein expression, and increased the phosphorylation of LIM kinase and cofilin. Proliferation and cytoskeletal rearrangement were suppressed by pre-treatment with the ROCK inhibitor Y-27632; additionally, expression of ROCK protein and phosphorylation of LIM kinase and cofilin were decreased by Y-27632, while Rho-GDIa knockdown by targeted siRNA transfection causes opposite effects. Therefore, we propose that TGF-β1 induces proliferation and cytoskeletal rearrangement in hPDLCs via Rho GTPase-dependent pathways that modulate ROCK, LIM kinase, and cofilin activity.     

MicroRNA-375 sensitizes tumour necrosis factor-alpha (TNF-α)-induced apoptosis in head and neck squamous cell carcinoma in vitro

We investigated the effect of microRNA-375 (miR-375) on tumour necrosis factor-alpha (TNF-α)-induced cell death in head and neck squamous cell carcinoma, and further explored the potential molecular mechanism underlying this phenomenon. Cal27 cells were transfected with miR-375 mimic and subsequently treated with or without TNF-α (10 ng/ml). An additional group of cells were treated with TNF-α alone. The resulting morphological changes were observed, and the percentage of sub-G1 cells was measured. The protein expression and cleavage of caspase 3, caspase 8, and poly(ADP ribose) polymerase (PARP) were determined through Western blotting. The results showed a significant increase in cell death in the combination group, but not in the groups treated with miR-375 mimic, TNF-α alone, or control. The data obtained from sub-G1 cells supported the notion that miR-375 increases the accumulation of sub-G1. In the combination group, the degradation of caspase 3, caspase 8, and PARP was observed and the cleavage of these enzymes was detected. The pan-caspase inhibitor, Z-VAD, inhibited the apoptosis of Cal27 cells treated with a combination of miR-375 mimic and TNF-α. In addition, the apoptosis inhibitory proteins, cFLIP-L and cIAP1, were down-regulated in a time-dependent manner. Taken together, these data suggest that miR-375 sensitizes TNF-α-induced apoptosis, and the reduction in the expression of the apoptosis inhibitory proteins cFLIP-L and cIAP2 plays an important role in this sensitization.