Seiji Goda

CX3C-Chemokine, Fractalkine-Enhanced Adhesion of THP-1 Cells to Endothelial Cells Through Integrin-Dependent and -Independent Mechanisms

Leukocyte adhesion and trafficking at the endothelium requires both cellular adhesion molecules and chemotactic factors. A newly identified CX3C chemokine, fractalkine, expressed on activated endothelial cells, plays an important role in leukocyte adhesion and migration. We examined the functional effects of fractalkine on β1 and β2 integrin-mediated adhesion using a macrophage-like cell line, THP-1 cells. In this study, we report that THP-1 cells express mRNA encoding a receptor for fractalkine, CX3CR1, determined by Northern blotting. Scatchard analysis using fractalkine-SEAP (secreted form of placental alkaline phosphatase) chimeric proteins revealed that THP-1 cells express a single class of CX3CR1 with a dissociation constant of 30 pM and a mean expression of 440 sites per cell. THP-1 cells efficiently adhered, in a fractalkine-dependent manner, to full-length of fractalkine immobilized onto plastic and to the membrane-bound form of fractalkine expressed on ECV304 cells or TNF-α-activated HUVECs. Moreover, soluble-fractalkine enhanced adhesion of THP-1 cells to fibronectin and ICAM-1 in a dose-dependent manner. Pertussis toxin, an inhibitor of Gi, inhibited the fractalkine-mediated enhancement of THP-1 cell adhesion to fibronectin and ICAM-1. Finally, we found that soluble-fractalkine also enhanced adhesion of freshly separated monocytes to fibronectin and ICAM-1. These results indicate that fractalkine may induce firm adhesion between monocytes and endothelial cells not only through an intrinsic adhesion function itself, but also through activation of integrin avidity for their ligands.

Fractalkine-Mediated Endothelial Cell Injury by NK Cells

Endothelial cells (ECs) are primary targets of immunological attack, and their injury can lead to vasculopathy and organ dysfunction in vascular leak syndrome and in rejection of allografts or xenografts. A newly identified CX3C-chemokine, fractalkine, expressed on activated ECs plays an important role in leukocyte adhesion and migration. In this study we examined the functional roles of fractalkine on NK cell activity and NK cell-mediated endothelial cell injury. Freshly separated NK cells expressed the fractalkine receptor (CX3CR1) determined by FACS analysis and efficiently adhered to immobilized full-length fractalkine, but not to the truncated forms of the chemokine domain or mucin domain, suggesting that fractalkine functions as an adhesion molecule on the interaction between NK cells and ECs. Soluble fractalkine enhanced NK cell cytolytic activity against K562 target cells in a dose- and time-dependent manner. This enhancement correlated well with increased granular exocytosis from NK cells, which was completely inhibited by the G protein inhibitor, pertussis toxin. Transfection of fractalkine cDNA into ECV304 cells or HUVECs resulted in increased adhesion of NK cells and susceptibility to NK cell-mediated cytolysis compared with control transfection. Moreover, both enhanced adhesion and susceptibility of fractalkine-transfected cells were markedly suppressed by soluble fractalkine or anti-CX3CR1 Ab. Our results suggest that fractalkine plays an important role not only in the binding of NK cells to endothelial cells, but also in NK cell-mediated endothelium damage, which may result in vascular injury.

Fractalkine, a CX 3 C-chemokine, functions predominantly as an adhesion molecule in monocytic cell line THP-1

A newly identified CX3C‐chemokine, fractalkine, expressed on activated endothelial cells plays an important role in leucocyte adhesion and migration. Co‐immobilized fractalkine with fibronectin or intercellular adhesion molecule‐1 enhanced adhesion of THP‐1 cells, which express the fractalkine receptor (CX3CR1), compared with that observed for each alone. That adherence was fractalkine‐dependent and was confirmed in blocking studies. However, soluble fractalkine induced little chemotaxis in THP‐1 cells in comparison to monocyte chemotactic protein‐1 (MCP‐1), which induced a strong chemotactic response. Moreover, the membrane form of fractalkine expressed on ECV304 cells reduced MCP‐1 mediated chemotaxis of THP‐1 cells. These results indicate that fractalkine may function as an adhesion molecule between monocytes and endothelial cells rather than as a chemotactic factor.

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.

The effect of matrix metalloproteinase-9 on the differentiation into osteoclast cells on RAW264 cells

Abstract Matrix metalloproteinases (MMPs) break down the extracellular matrix (ECM) and are important proteins for bone remodeling. Here, to clarify relationships between MMPs and mechanisms of osteoclast differentiation, we used MMP inhibitor, GM6001, to investigate osteoclast differentiation engage to receptor activator of nuclear factor κB ligand (RANKL)/receptor activator of nuclear factor κB (RANK) stimulation using the RAW264 osteoclast precursor cell line. Also, since differentiation of RAW264 due to RANKL/RANK stimulation is dependent on p38 mitogen-activated protein kinase (p38 MAPK), we investigated expression of MMP-9 and osteocyte differentiation of RAW264 due to RANKL stimulation using SB203580, a p38 MAPK inhibitor. After confirming that GM6001 did not significantly affect cellular proliferation of the cell line, RAW264 was cultured with GM6001 and RANKL. GM6001 suppressed RANKL stimulation-induced tartrate-resistant acid phosphatase (TRAP)-positive polynuclear osteoclasts in a dose-dependent manner, and RANKL increased expression of MMP-9 in a dose-dependent manner. SB203580 also suppressed differentiation into TRAP-positive multinucleated osteocytes in a dose-dependent manner and blocked expression of MMP-9. These findings suggest that differentiation of osteoclasts by RANKL stimulation involves expression of MMP-9, which in turn involves p38 MAPK.

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.

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-12Rβ2 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.

Corrigendum to “Effects of gallotannin on osteoclastogenesis and the p38 MAP kinase pathway” [Orthod. Waves 75 (2016) 105–113]

Yukiko Hosoyama D.D.S. , Eisuke Domae D.D.S., Ph.D. *, Seiji Goda D.D.S., Ph.D. , Naoyuki Matsumoto D.D.S., Ph.D. d Graduate School of Dentistry, Department of Orthodontics, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan Department of Biochemistry, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan Department of Oral Science, Graduate School of Dentistry, Kanagawa Dental University, Yokosuka, Kanagawa 2388580, Japan Department of Orthodontics, Osaka Dental University, 8-1 Kuzuhahanazonocho, Hirakata, Osaka 573-1121, Japan

Effects of gallotannin on osteoclastogenesis and the p38 MAP kinase pathway

Abstract Purpose Osteoclasts are multinucleated giant cells that specialize in bone resorption and work together with bone-forming osteoblasts to maintain bone homeostasis. However, excessive osteoclast activation accounts for bone diseases, such as osteoporosis and periodontitis. In previous studies, natural small-molecule compounds have been shown to regulate osteoclastogenesis and osteoclast functions. Here we demonstrate that gallotannin, a hydrolyzable plant tannin, suppresses osteoclast differentiation. Methods We first used an ex vivo bone marrow culture system containing both osteoclast precursors and surrounding cells, thereby resembling physiological conditions, to evaluate the suppressive effect of gallotannin. We also used a RANKL-induced osteoclastogenesis assay containing only osteoclast precursors to confirm the suppressive effect of gallotannin in the absence of effects from other cells. Results The suppressive effect of gallotannin was associated with the reduced RANKL-mediated induction of NFATc1, a critical transcription factor involved in osteoclast differentiation. We further confirmed that gallotannin reduced the p38 MAPK pathway activation, which is mediated by M-CSF and RANKL. This pathway suppression might underlie the suppression of NFATc1 production and subsequent reduction in osteoclast differentiation. Conclusion Our data indicate that the natural small-molecule compound gallotannin might be useful as a novel anti-bone resorptive agent.

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

BackgroundMatrix 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.MethodsMG-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 × 106) 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.ResultsEMD 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.ConclusionP38 MAPK pathway promotes expression MMP-2 in EMD activated osteoblasts, which in turn stimulates periodontal regeneration by degrading matrix proteins in periodontal connective tissue.