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1.
J Oral Biosci ; 65(2): 206-210, 2023 06.
Article in English | MEDLINE | ID: mdl-36963631

ABSTRACT

OBJECTIVES: Candidalysin is a peptide toxin produced by Candida albicans that causes damage to epithelial cells by destabilizing the plasma membrane. This study aimed to evaluate heparin's ability to neutralize candidalysin and protect epithelial cells from lysis. METHODS: The study was conducted using a human oral epithelial cell line and synthetic candidalysin. Cell damage was assessed by measuring lactate dehydrogenase release. Enzyme-linked immunosorbent assay and immunoblotting were used to determine cytokine concentrations and assess activation of intracellular signaling molecules and transcription factors, respectively. Flow cytometry was used to measure cell-bound candidalysin. RESULTS: Heparin diminished the cell-lytic activity of candidalysin and subsequent epithelial responses. Additionally, heparin inhibited the interaction between candidalysin and epithelial cells. Furthermore, polyacrylic acid, a synthetic polymer, mimicked the neutralizing effects of candidalysin. CONCLUSION: Our findings suggest that negatively charged polymers could be a potential therapeutic option for preventing the damage caused by candidalysin. Further research is needed to explore the effectiveness of other anionic polymers and their potential clinical applications.


Subject(s)
Fungal Proteins , Heparin , Humans , Heparin/pharmacology , Heparin/metabolism , Fungal Proteins/metabolism , Candida albicans/metabolism , Epithelial Cells/metabolism
2.
Dent Mater J ; 41(4): 624-632, 2022 Jul 30.
Article in English | MEDLINE | ID: mdl-35584937

ABSTRACT

Recently, the potential of ß-cyclodextrin-thread acid-degradable polyrotaxane (AdPRX) has been emphasized as a therapeutic agent for cholesterol-related metabolic disorders. In this study, we investigated whether carboxymethyl carbamate-modified AdPRX (CMC-AdPRX) can be used for adsorption to calcium phosphate to treat bone diseases. We first synthesized CMC-AdPRX and used it to coat the calcium phosphate plate. RAW264.7 cells were then differentiated into osteoclasts via a receptor activator of nuclear factor-κB ligand, and the number of osteoclasts and the area of absorption lacunae were determined. The number of tartrate-resistant acid phosphatase-positive multinucleated cells was reduced on the CMC-AdPRX-coated plate. The area of the absorption lacunae was smaller with CMC-AdPRX than with AdPRX, which was not carboxy-modified. Our results suggest that CMC-AdPRX can adsorb to calcium phosphate and act on differentiated osteoclasts to suppress their functional expression.


Subject(s)
Bone Resorption , Rotaxanes , beta-Cyclodextrins , Acid Phosphatase/metabolism , Animals , Calcium Phosphates/pharmacology , Cell Differentiation , Isoenzymes/metabolism , Mice , Osteoclasts/metabolism , RANK Ligand/metabolism , RAW 264.7 Cells , Rotaxanes/pharmacology , Tartrate-Resistant Acid Phosphatase/metabolism , beta-Cyclodextrins/pharmacology
3.
Int Immunol ; 33(3): 161-170, 2021 03 01.
Article in English | MEDLINE | ID: mdl-33038250

ABSTRACT

Candida albicans is a commensal fungus that predominantly resides on mucosal surfaces and can cause lethal systemic infection when the host defense is compromised. Candidalysin is a cytolytic peptide toxin produced by C. albicans hyphae that is essential for mucosal tissue damage and is believed to contribute to the establishment of systemic infection and mortality. Candidalysin is also required for the epithelial innate response in which proinflammatory cytokines and chemokines are produced and neutrophil recruitment is initiated. It was recently reported that epidermal growth factor receptor (EGFR) was essential for the candidalysin-triggered epithelial response. The present study identified IL-1α as another component of candidalysin-mediated initial epithelial activation. We found that human oral epithelial cells released IL-1α rapidly after candidalysin exposure. Blockade of IL-1α/IL-1 receptor (IL-1R) signaling in candidalysin-exposed cells resulted in decreased phosphorylation of IκBα, decreased induction of IκBζ and decreased production of granulocyte-macrophage colony-stimulating factor and IL-8. Expression of c-Fos, which is induced downstream of EGFR signaling in candidalysin-treated cells, is less affected by IL-1R blockade. Inversely, blockade of EGFR signaling does not affect candidalysin-mediated phosphorylation of IκBα and induction of IκBζ, suggesting that independent signaling pathways contribute to the induction of NF-κB and c-Fos downstream of the candidalysin pore formation site. Consistently, antibody inhibition of both EGFR and IL-1R enhanced the suppressive effect of cytokine production in candidalysin-treated cells. Thus, we identified the immediate release of IL-1α and its synergistic role with EGFR ligands on the initial activation of oral epithelial cells in response to candidalysin.


Subject(s)
Epithelial Cells/immunology , Fungal Proteins/toxicity , Interleukin-1alpha/metabolism , Mouth Mucosa/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Candida albicans/immunology , Cell Line , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , I-kappa B Proteins/metabolism , Immunity, Innate/immunology , Interleukin-1alpha/antagonists & inhibitors , Interleukin-8/metabolism , Mouth Mucosa/cytology , Phosphorylation , Receptors, Interleukin-1/antagonists & inhibitors , Receptors, Interleukin-1/metabolism , Signal Transduction/physiology
4.
J Biochem ; 168(1): 33-40, 2020 Jul 01.
Article in English | MEDLINE | ID: mdl-32065632

ABSTRACT

The RNA helicase, DDX17 is a member of the DEAD-box protein family. DDX17 has two isoforms: p72 and p82. The p82 isoform has additional amino acid sequences called intrinsically disordered regions (IDRs), which are related to the formation of membraneless organelles (MLOs). Here, we reveal that p72 is mostly localized to the nucleoplasm, while p82 is localized to the nucleoplasm and nucleoli. Additionally, p82 exhibited slower intranuclear mobility than p72. Furthermore, the enzymatic mutants of both p72 and p82 accumulate into the stress granules. The enzymatic mutant of p82 abolishes nucleolar localization of p82. Our findings suggest the importance of IDRs and enzymatic activity of DEAD-box proteins in the intracellular distribution and formation of MLOs.


Subject(s)
Cell Nucleolus/metabolism , Cell Nucleus/metabolism , DEAD-box RNA Helicases/metabolism , Organelles/metabolism , RNA/metabolism , Uterine Cervical Neoplasms/pathology , Female , HeLa Cells , Humans , Protein Isoforms , Uterine Cervical Neoplasms/metabolism
5.
Biomed Res ; 40(5): 189-196, 2019.
Article in English | MEDLINE | ID: mdl-31597904

ABSTRACT

Sphingomyelin is a major lipid of the plasma membrane and is enriched in microdomains of the plasma membrane that are critical for signal transduction. However, the function of sphingomyelin in the cell membrane of osteoblasts has not been clarified. Therefore, we examined how sphingomyelin synthase 2 (SMS2) affects osteoclast differentiation by osteoblasts. We knocked down the expression of SMS2 with siRNA targeting the Sgms2 gene in mouse primary osteoblasts. The effects of SMS2 knockdown in osteoblasts were examined using polymerase chain reaction and western blotting. The knockdown of SMS2 suppressed the formation of TRAP-positive multinucleated cells by co-culture of osteoblasts and bone marrow cells compared to the control. We found that receptor activator of nuclear factor κB ligand (RANKL) mRNA expression was significantly reduced by 1,25(OH)2D3 stimulation in SMS2 siRNA osteoblasts. The knockdown of SMS2 repressed the expression of retinoid-X-receptor-α (RXRα) regardless of 1,25(OH)2D3 stimulation. TRAP-positive multinucleated cell formation was significantly reduced by RXRα siRNA in osteoblasts in a co-culture system. These results suggest that SMS2 regulates osteoclast differentiation by inducing RANKL expression via RXRα.


Subject(s)
Gene Expression Regulation , Osteoblasts/metabolism , Osteogenesis/genetics , RANK Ligand/genetics , Transferases (Other Substituted Phosphate Groups)/genetics , Animals , Cell Differentiation/genetics , Cells, Cultured , Gene Knockdown Techniques , Gene Silencing , Mice , Osteoclasts/metabolism , RNA Interference , RNA, Small Interfering/genetics , Retinoid X Receptor alpha/genetics , Retinoid X Receptor alpha/metabolism
6.
Virus Res ; 263: 179-183, 2019 04 02.
Article in English | MEDLINE | ID: mdl-30769121

ABSTRACT

Borna disease virus (BoDV) is a nonsegmented, negative-strand RNA virus that uniquely replicates and establishes persistent infection in cell nucleus. Recent studies have demonstrated the presence of actin in the nucleus and its role in intranuclear phenomena such as transcription and DNA repair. Although nuclear actin is involved in the life cycle of some intranuclear DNA viruses, the interaction between BoDV and nuclear actin has not been reported. In this study, we show that the inhibition of the nucleocytoplasmic transport of actin affects the replication of BoDV in the nucleus. The knockdown of a nuclear export factor of actin, exportin 6, results in the induction of structural aberration in intranuclear viral factories of BoDV. Furthermore, the inhibition of the nuclear export of actin promotes accumulation of viral matrix protein in the cytoplasm and periphery of the infected cells. These results suggest that the dynamics of actin affect the replication of BoDV by disturbing the structure of viral factories in the nucleus.


Subject(s)
Actins/metabolism , Borna disease virus/physiology , Cell Nucleus/virology , Host-Pathogen Interactions , Virus Replication , Active Transport, Cell Nucleus , Animals , Cell Line , Humans , Karyopherins/metabolism
7.
J BUON ; 23(7): 132-138, 2018 12.
Article in English | MEDLINE | ID: mdl-30722122

ABSTRACT

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.


Subject(s)
Apoptosis/drug effects , Carcinoma, Squamous Cell/pathology , Leukocytes, Mononuclear/pathology , Mouth Neoplasms/pathology , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Zoledronic Acid/pharmacology , Bone Density Conservation Agents/pharmacology , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/metabolism , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Mouth Neoplasms/drug therapy , Mouth Neoplasms/metabolism , Receptors, Antigen, T-Cell, gamma-delta/immunology , Tumor Cells, Cultured
8.
Oncotarget ; 8(28): 45928-45942, 2017 Jul 11.
Article in English | MEDLINE | ID: mdl-28521284

ABSTRACT

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.


Subject(s)
Cytokines/metabolism , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Receptors, Antigen, T-Cell, gamma-delta/metabolism , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/physiology , Adaptor Proteins, Signal Transducing , Cell Proliferation/drug effects , Cells, Cultured , Humans , I-kappa B Proteins/genetics , Nuclear Proteins/genetics
9.
Arch Oral Biol ; 60(9): 1319-26, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26123746

ABSTRACT

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.


Subject(s)
Bone Morphogenetic Protein 2/pharmacology , RNA Interference , Smad1 Protein/physiology , Animals , Blotting, Western , Cell Differentiation/drug effects , Cells, Cultured , Ligands , Mice , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoclasts/cytology , Osteoclasts/drug effects , Osteogenesis/drug effects , Polymerase Chain Reaction , RNA, Messenger/metabolism , Receptor Activator of Nuclear Factor-kappa B , Receptors, Calcitriol/metabolism , Signal Transduction
10.
BMC Oral Health ; 14: 85, 2014 Jul 10.
Article in English | MEDLINE | ID: mdl-25011999

ABSTRACT

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.


Subject(s)
Dental Enamel Proteins/pharmacology , Matrix Metalloproteinase 2/drug effects , Osteoblasts/enzymology , Cell Line , Culture Media, Conditioned , Enzyme Inhibitors/pharmacology , Gelatin/metabolism , Humans , Imidazoles/pharmacology , MAP Kinase Signaling System/drug effects , Matrix Metalloproteinase Inhibitors/pharmacology , Osteoblasts/drug effects , Pyridines/pharmacology , Temperature , Time Factors , Tissue Inhibitor of Metalloproteinase-2/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors
11.
J Immunol ; 191(7): 3681-93, 2013 Oct 01.
Article in English | MEDLINE | ID: mdl-23997225

ABSTRACT

A hallmark of T cell activation in vitro and in vivo is the clustering of T cells with each other via interaction of the LFA-1 integrin with ICAM-1. The functional significance of these homotypic aggregates in regulating T cell function remains unknown. We used an APC-free in vitro activation system to demonstrate that stimulation of purified naive CD8 T cells results in enhanced expression of ICAM-1 on T cells that is sustained by the inflammatory cytokine IL-12 and associated with robust T cell aggregates. ICAM-1-deficient CD8 T cells proliferate normally but demonstrate a striking failure to aggregate. Interestingly, loss of ICAM-1 expression results in elevated levels of IFN-γ and granzyme B, as well as enhanced cytotoxicity. Similar results were obtained when anti-LFA-1 Ab was used to block the clustering of wild-type T cells. ICAM-1 ligation is not required for IFN-γ regulation, as clustering of ICAM-1-deficient CD8 T cells with wild-type T cells reduces IFN-γ expression. Analysis using a fluorescent reporter that monitors TCR signal strength indicates that T cell clustering limits T cell exposure to Ag during activation. Furthermore, T cell clustering promotes the upregulation of the CTLA-4 inhibitory receptor and the downregulation of eomesodermin, which controls effector molecule expression. Activation of ICAM-1-deficient CD8 T cells in vivo results in an enhanced percentage of KLRG-1(+) T cells indicative of short-lived effectors. These results suggest that T cell clustering represents a mechanism that allows continued proliferation but regulates T cell effector function and differentiation.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Cell Differentiation/immunology , Intercellular Adhesion Molecule-1/metabolism , Lymphocyte Activation/immunology , Animals , Antigens/immunology , CD8-Positive T-Lymphocytes/cytology , CTLA-4 Antigen/genetics , CTLA-4 Antigen/metabolism , Cell Communication/immunology , Cell Differentiation/genetics , Dendritic Cells/immunology , Dendritic Cells/metabolism , Gene Expression Regulation , Intercellular Adhesion Molecule-1/genetics , Interferon-gamma/biosynthesis , Interleukin-12/pharmacology , Lymphocyte Activation/drug effects , Lymphocyte Activation/genetics , Mice , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism
12.
J Periodontol ; 80(10): 1631-7, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19792853

ABSTRACT

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.


Subject(s)
Dental Enamel Proteins/pharmacology , Osteoblasts/drug effects , Phosphatidylinositol 3-Kinases/drug effects , Androstadienes/pharmacology , Animals , Blotting, Western , Cell Count , Cell Line , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Chromones/pharmacology , Dose-Response Relationship, Drug , Enzyme Activation , Enzyme Inhibitors/pharmacology , Humans , Morpholines/pharmacology , Osteoblasts/enzymology , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/drug effects , Wortmannin , Wound Healing/drug effects
13.
Clin Calcium ; 16(1): 117-21, 2006 Jan.
Article in Japanese | MEDLINE | ID: mdl-16397361

ABSTRACT

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.


Subject(s)
Alveolar Process/metabolism , Bone and Bones/metabolism , Collagen/metabolism , Dental Enamel Proteins/pharmacology , Humans , Inflammation/metabolism , Periodontal Diseases/metabolism , Stress, Mechanical
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