Role of the nucleotide-binding oligomerization domain-containing protein 1 pathway in the development of periodontitis.

OBJECTIVES: During innate immune defense, host pattern recognition receptors, including toll-like receptors and nucleotide-binding oligomerization domain-like receptors (NLRs), can activate downstream pathways by recognizing pathogen-associated molecular patterns produced by microorganisms, triggering immune responses. NOD1, an important cell membrane protein in the NLR-like receptor protein family, exerts anti-infective effects through γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) recognition. Oral epithelial cells resist bacterial invasion through iE-DAP-induced interleukin (IL)-8 production, recruiting neutrophils to sites of inflammation in response to bacterial threats to periodontal tissues. To date, the regulatory mechanisms of iE-DAP in gingival epithelial cells (GECs) are poorly understood. This study was conducted to investigate the role of the NOD1 pathway in the development of periodontitis by examining the effect of iE-DAP on IL-8 production in Ca9-22 cells. METHODS: IL-8 production by iE-DAP-stimulated-Ca9-22 cells was assessed using an enzyme-linked immunosorbent assay. Phosphorylation levels of intracellular signaling molecules were evaluated using western blot analyses. RESULTS: iE-DAP induced NOD1 receptor expression in Ca9-22 cells. Additionally, iE-DAP induced expression of pro-IL-1ß protein without extracellular secretion. Our results suggest that iE-DAP regulates IL-8 production by activating p38 mitogen-activated protein kinase (MAPK) and ERK1/2 signaling pathways. iE-DAP also promoted nuclear factor kappa-B p65 phosphorylation, facilitating its nuclear translocation. Notably, p38 MAPK and ERK1/2 inhibitors suppressed iE-DAP-stimulated IL-8 production, suggesting that JNK is not involved in this mechanism. CONCLUSIONS: Our results indicate that p38 MAPK and ERK1/2, but not JNK, are involved in innate immune responses in GECs.

P38α contributes to TNF-α-induced IL-8 production in human gingival cells.

Tumor necrosis factor-alpha (TNF-α) is a major inflammatory cytokine that induces interleukin (IL)-8 production. Although some studies have reported the involvement of the p38 MAPK signaling pathway in TNF-α-induced IL-8 production, its specific regulatory mechanisms in gingival epithelial cells (GECs) are still poorly understood. In the present study, Ca9-22 cells were used as representative GECs to investigate the effect of p38 signaling on TNF-α-induced IL-8 production. We found that TNF-α enhanced IL-8 production in Ca9-22 cells by activating the p38 signaling pathway, and one of its isoforms, p38α, played a key role. P38α deletion markedly inhibited TNF-α-induced IL-8 expression in Ca9-22 cells, while p38α gene rescue could reverse this effect. Further studies revealed that TNF-α-induced IL-8 production was markedly reduced when the threonine 180 and tyrosine 182 p38α phosphorylation sites were targeted for mutagenesis to alanine and phenylalanine, respectively, suggesting their critical role in the process. In conclusion, p38α plays an important role in TNF-α-induced IL-8 production, providing a potential therapeutic target to prevent and treat periodontal disease.

Effects of a co-stimulation with S-PRG filler eluate and muramyl dipeptide (MDP) on matrix metalloproteinase-1 production by human dental pulp fibroblast-like cells.

The present study investigated the effects of a co-stimulation with surface reaction-type pre-reacted glass-ionomer (S-PRG) filler eluate and muramyl dipeptide (MDP) on matrix metalloproteinase (MMP)-1 production by human dental pulp fibroblast-like cells (hDPFs). S-PRG filler eluate contains 6 ions (F, Na, Al, B, Sr, and Si) released from S-PRG filler. Each S-PRG filler eluate and MDP stimulation enhanced MMP-1 production by hDPFs. The co-stimulation with S-PRG filler eluate and MDP enhanced MMP-1 production more than the MDP stimulation alone. A similar stimulation induced the phosphorylation of ERK 1/2. The increased secretion of MMP-1 and enhanced phosphorylation of ERK 1/2 by the co-stimulation with S-PRG filler eluate and MDP were suppressed by the selective and potent CaSR antagonist NPS 2143. Since strontium binds to CaSR, these results suggest that the enhanced production of MMP-1 by the co-stimulation with S-PRG filler eluate and MDP was due to the effects of strontium.

Effects of S-PRG filler eluate on MMP-1 and MMP-3 secretion by human gingival fibroblasts.

The aim of this study was to investigate the effects of surface reaction-type pre-reacted glass-ionomer (S-PRG) filler eluate on Matrix metalloproteinase (MMP)-1 and MMP-3 secretion by human gingival fibroblasts (HGF). The S-PRG filler eluate contains 6 ions (F, Na, Al, B, Sr and Si) released from the S-PRG filler. The S-PRG filler eluate stimulation induced a slight secretion of MMP-1 and MMP-3 by HGF. It also enhanced the phosphorylation of p38 and ERK. The increase in MMP-1 and MMP-3 secretion by the inflammatory cytokine TNF-α was suppressed by the S-PRG filler eluate. TNF-α-induced increases in the phosphorylation of ERK were slightly enhanced by S-PRG filler eluate. These findings may prompt the development of new therapeutic agents for oral inflammation with materials composed of S-PRG filler eluate.

The Effect of Transforming Growth Factor Beta 1 on the Mineralization of Human Cementoblasts.

INTRODUCTION: Transforming growth factor beta 1 (TGF-ß1) plays an important role in bone mineralization and has been reported to promote osteoblast proliferation and differentiation. However, there is no report about the effects of TGF-ß1 on human cementoblasts. The purpose of this study was to clarify the effect of TGF-ß1 on the proliferation and differentiation of the human cementoblast cell line (HCEM) in vitro. METHODS: HCEM cells were stimulated with TGF-ß1 at concentrations of 0.05, 0.5, 5, and 10 ng/mL. A proliferation assay was performed from 24-72 hours. The effect of TGF-ß1 on mineralization was analyzed by quantifying the area stained with alizarin red on days 7 and 14. Real-time polymerase chain reaction was used to assess the effect of TGF-ß1 on the mineralization-related genes alkaline phosphatase, bone sialoprotein, and type I collagen on days 3, 7, and 14. RESULTS: TGF-ß1 did not affect cell proliferation. TGF-ß1 together with the mineralization medium (consisting of ascorbic acid, dexamethasone, and ß-glycerophosphate) increased the alizarin red-stained area on days 7 and 14. Real-time polymerase chain reaction revealed that alkaline phosphatase messenger RNA expression was increased in TGF-ß1-stimulated HCEM cells in mineralization medium on days 3 and 7, whereas bone sialoprotein and type I collagen messenger RNA expression was increased on day 7. CONCLUSIONS: Although TGF-ß1 does not affect cell proliferation, it does promote cell differentiation and mineralization of HCEM cells.

Optimal dilutions of S-PRG filler eluate for experiments on human gingival fibroblasts in vitro.

The present study attempted to identify the optimal dilution at which at which the effects of surface reaction-type pre-reacted glass-ionomer (S-PRG) filler eluate on human gingival fibroblasts (HGF) may be safely examined in vitro. S-PRG filler is a material that releases six ions and exerts strong caries-suppressing effects. We prepared S-PRG filler eluate in which S-PRG filler and α-MEM were mixed as a medium for HGF. This eluate contains six ions that are released from S-PRG filler. All cells died in proliferation experiments on HGF using S-PRG filler eluate, which demonstrated that unless S-PRG filler eluate was diluted, the ion concentration was strongly cytotoxic. S-PRG filler eluate diluted by 1/100 or more with the addition of 2% or more of FBS was safe for use. We herein successfully established the optimal dilution of S-PRG filler eluate at which HGF may be safely examined in vitro.

Human Vγ9Vδ2 T cells show potent antitumor activity against zoledronate-sensitized OSCC cell lines.

PURPOSE: Vγ9Vδ2 T cells exhibit potent antitumor effects against multiple types of tumors in preclinical models. In the present study, we examined whether human Vγ9Vδ2 T cells can be effective against oral squamous cell carcinoma (OSCC) cell lines in vitro because the interaction between OSCC and Vγ9Vδ2 T cells has not been explored previously. METHODS: Eight OSCC cell lines were analyzed for their expression of ligands that potentially activate Vγ9Vδ2 T cells. Vγ9Vδ2 T cells were expanded in vitro from peripheral blood mononuclear cells (PBMCs) using zoledronate and IL-2. Expanded Vγ9Vδ2 T cells were tested for IFNγ production and cytotoxicity in response to zoledronate-treated OSCC cell lines. Flow cytometry was used to obtain and analyze data. RESULTS: All OSCC cell lines expressed CD277. The cell lines also expressed at least one type of NKG2D ligand. Zoledronate-treated OSCC cell lines induced IFNγ expression in Vγ9Vδ2 T cells. We thus found that Vγ9Vδ2 T cells efficiently kill zoledronate-sensitized OSCC cell lines. CONCLUSIONS: We found that zoledronate-treated OSCC cell lines are effectively killed by Vγ9Vδ2 T cells. Our results indicate that developing Vγ9Vδ2 T cell-based immunotherapy will be promising in treating patients with OSCC.

Cytokine-mediated activation of human ex vivo-expanded Vγ9Vδ2 T cells.

Vγ9Vδ2 T cells, the major subset of the human peripheral blood γδ T-cell, respond to microbial infection and stressed cells through the recognition of phosphoantigens. In contrast to the growing knowledge of antigen-mediated activation mechanisms, the antigen-independent and cytokine-mediated activation mechanisms of Vγ9Vδ2 T cells are poorly understood. Here, we show that interleukin (IL) -12 and IL-18 synergize to activate human ex vivo-expanded Vγ9Vδ2 T cells. Vγ9Vδ2 T cells treated with IL-12 and IL-18 enhanced effector functions, including the expression of IFN-γ and granzyme B, and cytotoxicity. These enhanced effector responses following IL-12 and IL-18 treatment were associated with homotypic aggregation, enhanced expression of ICAM-1 and decreased expression of the B- and T-lymphocyte attenuator (BTLA), a co-inhibitory receptor. IL-12 and IL-18 also induced the antigen-independent proliferation of Vγ9Vδ2 T cells. Increased expression of IκBζ, IL-12Rß2 and IL-18Rα following IL-12 and IL-18 stimulation resulted in sustained activation of STAT4 and NF-κB. The enhanced production of IFN-γ and cytotoxic activity are critical for cancer immunotherapy using Vγ9Vδ2 T cells. Thus, the combined treatment of ex vivo-expanded Vγ9Vδ2 T cells with IL-12 and IL-18 may serve as a new strategy for the therapeutic activation of these cells.

RNA interference-mediated knockdown of Smad1 inhibits receptor activator of nuclear factor κB ligand expression induced by BMP-2 in primary osteoblasts.

OBJECTIVE: BMP-2 induces osteoblast differentiation and activates osteoclast formation. Here, we investigated the role of Smad1, a molecule that signals downstream of BMP-2, in mediating the effects of BMP-2 on osteoclast differentiation induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in osteoblasts. DESIGN: The effects of 1,25(OH)2D3 and BMP-2 in osteoclasts were examined using polymerase chain reaction and Western blotting to measure changes in target gene and protein expression. Immunostaining was carried out to investigate the localization of the vitamin D receptor (VDR) in the nucleus in response to BMP-2. RESULTS: Stimulation with both 1,25(OH)2D3 and BMP-2 resulted in significantly greater osteoclast formation and receptor activator of nuclear factor κB ligand (RANKL) mRNA expression compared to stimulation with 1,25(OH)2D3 alone. In addition, expression of the VDR protein was increased, enhancing the activity of 1,25(OH)2D3. Interestingly, knockdown of Smad1 resulted in reduced osteoclast formation, RANKL mRNA expression, and VDR protein expression compared with control cells. Costimulation with 1,25(OH)2D3 and BMP-2 enhanced VDR localization in the nucleus. CONCLUSIONS: We found that BMP-2 induced Smad1 activation, thereby influencing the localization of VDR in the nucleus in the presence of 1,25(OH)2D3 and resulting in increased RANKL mRNA expression. These effects ultimately resulted in enhanced osteoclast differentiation.

Enamel matrix derivative protein enhances production of matrixmetalloproteinase-2 by osteoblasts.

BACKGROUND: Matrix metalloproteinases (MMPs) degrade the extracellular matrix (ECM) and regulate remodeling and regeneration of bone. Enamel matrix derivative (EMD) protein has been used clinically for periodontal regeneration, although its molecular mechanisms are not clear. We evaluated the role of matrix metalloproteinases (MMPs) in regulating EMD-dependent degradation of gelatin on oeoblast-like cell line MG63. METHODS: MG-63 cells (osteoblast cell line) were incubated with 100 µg/ml EMD protein in the presence or absence of MMP-2 tissue inhibitor for 20 h followed by incubation on DQ-gelatin-coated plates for 4 h. MG-63 cells (1 × 10(6)) were preincubated with SB203580 for 30 min at 37°C and were then placed in 100 µg/ml EMD protein for 24 h. Conditioned media were collected and detected by Western blot analysis. RESULTS: EMD protein enhanced cell-mediated degradation of gelatin, which was inhibited by the MMP inhibitor TIMP-2. Furthermore, MMP-2 was produced by MG63 cells in response to EMD protein in a P38 MAPK-dependent manner. In addition, blocking of p38 MAPK activation by SB203580 significantly inhibited generation of the active form of MMP-2. CONCLUSION: P38 MAPK pathway promotes expression MMP-2 in EMD activated osteoblasts, which in turn stimulates periodontal regeneration by degrading matrix proteins in periodontal connective tissue.

Enamel matrix derivative protein stimulated wound healing via phosphoinositide 3-kinase.

BACKGROUND: Enamel matrix derivative (EMD) protein has been clinically used for periodontal regeneration, but the molecular mechanisms are not clear. Previous studies suggested that the activation of phosphoinositide 3-kinase (PI 3-kinase) plays a key role in facilitating cell migration. Given that the migration of osteoblasts is one of the key steps in the wound-healing processes, we hypothesized that EMD protein would stimulate osteoblast migration by activating PI 3-kinase. In this study, we tested this hypothesis using MG-63 cells as model systems to evaluate mechanisms of migration by stimulation with EMD protein. METHODS: Confluent MG-63 cells were mechanically scratched using a sterilized 1-mm pipette tip that removed the cells within a circular area. The wells were incubated for 24 hours in various stimulation conditions (25, 50, or 100 microg/ml EMD protein) with or without the PI 3-kinase inhibitor wortmannin (1, 10, and 100 nM) or LY294002 (1, 10, and 100 microM). Migrated cells in the wound section were counted by randomly selecting three areas from one well. The activation of PI 3-kinase by EMD protein was evaluated by the phosphorylation of Akt using Western blot analysis. RESULTS: Although EMD protein did not affect proliferation, it enhanced migration into wounds on MG-63 cells. We showed that EMD protein enhanced the phosphorylation of Akt in a dose-dependent manner. We demonstrated that the PI 3-kinase inhibitors wortmannin and LY294002 blocked migration into wounds and the phosphorylation of Akt enhanced by EMD protein in MG-63 cells. CONCLUSION: These results demonstrated that the activation of PI 3-kinase plays a key role in the EMD protein-stimulated migration of osteoblasts.

Impaired TCR signaling through dysfunction of lipid rafts in sphingomyelin synthase 1 (SMS1)-knockdown T cells.

During T cell activation, TCRs cluster at the center of the T cell-antigen-presenting cell interface forming the central supramolecular activation cluster. Although it has been suggested that sphingolipid- and cholesterol-rich microdomains, termed lipid rafts, form platforms for the regulation and transduction of TCR signals, an actual role for membrane sphingomyelin (SM), a key component of lipid rafts, has not been reported. After cloning a gene responsible for SM synthesis, sphingomyelin synthase (SMS) 1, we established a SM-knockdown cell line (Jurkat-SMS1/kd) by transfection of SMS1-short-interfering RNA into Jurkat T cells, which is deficient in membrane expression of SM. Upon CD3 stimulation, expression of CD69 (the earliest leukocyte activation antigen), activation-induced cell adhesion and proliferation as well as TCR clustering was severely impaired in Jurkat-SMS1/kd cells. CD3-induced tyrosine phosphorylation and association of linker for activation of T cell with ZAP-70 and Grb2 and phosphorylation of protein kinase C (PKC) were also severely impaired in Jurkat-SMS1/kd cells. Finally, translocation of TCR, ZAP-70 and PKC into lipid rafts was markedly decreased in Jurkat-SMS1/kd cells. These findings indicate that membrane SM is crucial for TCR signal transduction, leading to full T cell activation through lipid raft function.

[Osteoporosis and oral biology].

Estrogen deficiency in post-menopausal osteoporosis not only causes decreased bone mass in mandibular bone, but also affect cartilaginous ossification in mandibular condyle. Although the mechanisms of action are not entirely clear, estrogen is thought to promote the programmed cell death of osteoclasts and hence reduce their period of activity. Treatment with estrogen or some agents prevent bone loss in alveolar bone through blocking production of cytokines in osteoblasts and promoting osteoclast apoptosis. It's necessary to address basic principles and current concepts in bone remodeling, mediators of bone resorption and their clinical relevance.

Matrix metalloproteinase-1 produced by human CXCL12-stimulated natural killer cells.

Natural killer (NK) cells play a key role in inflammation and tumor regression through their ability to migrate into tissues. CXCL12 is a chemokine that promotes lymphocyte invasion and migration into tissues; however, the mechanism for this process remains incompletely understood. In this study, we show that CXCL12 significantly enhanced CD16(+)CD56(+) human peripheral NK-cell invasion into type I collagen by the catalytic activity of matrix metalloproteinase-1 (MMP-1). Confocal immunofluorescence and co-immunoprecipitation studies suggest that MMP-1 colocalized with alpha(2)beta(1) integrin on CXCL-12-stimulated NK-cell surface. The binding of pro-MMP-1 with alpha(2)beta(1) integrin required activation of G(i)-coupled pathway. However, the production of MMP-1 from CXCL12-stimulated NK cells was mediated by p38 and mitogen-activated or extracellular signal-regulation protein kinase kinase 1/2 in a manner independent of the G(i)-coupled pathway. These results suggest that CXCL12/CXCR4 interaction transduces the two signaling pathways to promote NK-cell invasion, which stimulates pericellular degradation of extracellular matrix proteins by membrane-associated MMP-1. The mechanisms would thus play a role in facilitating lymphocyte trafficking and accumulation in tissues during physiological and pathological processes.

[Metabolism of alveolar bone].

The alveolar bone is a special tissue originating from the dental sac. This bone is constantly subjected to mechanical stress such as occlusal pressure and it responds to chronic inflammatory stimuli. Furthermore, the alveolar bone proper contains Sharpey's fibers (collagen) having a very short biological metabolic half-life of one day. In this manner, because the local environment of the alveolar bone is markedly different from that of other bones, the metabolic mechanism of the alveolar bone, and the alveolar bone proper in particular, may be different from that of other bones.

Control of TCR-mediated activation of beta 1 integrins by the ZAP-70 tyrosine kinase interdomain B region and the linker for activation of T cells adapter protein.

One of the earliest functional responses of T lymphocytes to extracellular signals that activate the Ag-specific CD3/TCR complex is a rapid, but reversible, increase in the functional activity of integrin adhesion receptors. Previous studies have implicated the tyrosine kinase zeta-associated protein of 70 kDa (ZAP-70) and the lipid kinase phosphatidylinositol 3-kinase, in the activation of beta(1) integrins by the CD3/TCR complex. In this report, we use human ZAP-70-deficient Jurkat T cells to demonstrate that the kinase activity of ZAP-70 is required for CD3/TCR-mediated increases in beta(1) integrin-mediated adhesion and activation of phosphatidylinositol 3-kinase. A tyrosine to phenylalanine substitution at position 315 in the interdomain B of ZAP-70 inhibits these responses, whereas a similar substitution at position 292 enhances these downstream signals. These mutations in the ZAP-70 interdomain B region also specifically affect CD3/TCR-mediated tyrosine phosphorylation of residues 171 and 191 in the cytoplasmic domain of the linker for activation of T cells (LAT) adapter protein. CD3/TCR signaling to beta(1) integrins is defective in LAT-deficient Jurkat T cells, and can be restored with expression of wild-type LAT. Mutant LAT constructs with tyrosine to phenylalanine substitutions at position 171 and/or position 191 do not restore CD3/TCR-mediated activation of beta(1) integrins in LAT-deficient T cells. Thus, these studies demonstrate that the interdomain B region of ZAP-70 regulates beta(1) integrin activation by the CD3/TCR via control of tyrosine phosphorylation of tyrosine residues 171 and 191 in the LAT cytoplasmic domain.