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1.
Int J Mol Sci ; 23(6)2022 Mar 09.
Article in English | MEDLINE | ID: mdl-35328359

ABSTRACT

Effects of the antiosteoblastogenesis factor Semaphorin 4D (Sema4D), expressed by thrombin-activated platelets (TPs), on osteoblastogenesis, as well as osteoclastogenesis, were investigated in vitro. Intact platelets released both Sema4D and IGF-1. However, in response to stimulation with thrombin, platelets upregulated the release of Sema4D, but not IGF-1. Anti-Sema4D-neutralizing monoclonal antibody (mAb) upregulated TP-mediated osteoblastogenesis in MC3T3-E1 osteoblast precursors. MC3T3-E1 cells exposed to TPs induced phosphorylation of Akt and ERK further upregulated by the addition of anti-sema4D-mAb, suggesting the suppressive effects of TP-expressing Sema4D on osteoblastogenesis. On the other hand, TPs promoted RANKL-mediated osteoclastogenesis in the primary culture of bone-marrow-derived mononuclear cells (BMMCs). Among the known three receptors of Sema4D, including Plexin B1, Plexin B2 and CD72, little Plexin B2 was detected, and no Plexin B1 was detected, but a high level of CD72 mRNA was detected in RANKL-stimulated BMMCs by qPCR. Both anti-Sema4D-mAb and anti-CD72-mAb suppressed RANKL-induced osteoclast formation and bone resorptive activity, suggesting that Sema4D released by TPs promotes osteoclastogenesis via ligation to a CD72 receptor. This study demonstrated that Sema4D released by TPs suppresses osteogenic activity and promotes osteoclastogenesis, suggesting the novel property of platelets in bone-remodeling processes.


Subject(s)
Osteogenesis , Semaphorins , Antigens, CD , Blood Platelets , Nerve Tissue Proteins/genetics , Receptors, Cell Surface/genetics , Semaphorins/genetics , Semaphorins/pharmacology , Thrombin/pharmacology
2.
Free Radic Biol Med ; 97: 330-341, 2016 08.
Article in English | MEDLINE | ID: mdl-27343691

ABSTRACT

Osteoclastogenesis was induced by RANKL stimulation in mouse monocytes to examine the possible bactericidal function of osteoclast precursors (OCp) and mature osteoclasts (OCm) relative to their production of NO and ROS. Tartrate-resistant acid phosphatase (TRAP)-positive OCp, but few or no OCm, phagocytized and killed Escherichia coli in association with the production of reactive oxygen species (ROS) and nitric oxide (NO). Phagocytosis of E. coli and production of ROS and NO were significantly lower in TRAP+ OCp derived from Toll-like receptor (TLR)-4 KO mice than that derived from wild-type (WT) or TLR2-KO mice. Interestingly, after phagocytosis, TRAP+ OCp derived from wild-type and TLR2-KO mice did not differentiate into OCm, even with continuous exposure to RANKL. In contrast, E. coli-phagocytized TRAP+ OCp from TLR4-KO mice could differentiate into OCm. Importantly, neither NO nor ROS produced by TRAP+ OCp appeared to be engaged in phagocytosis-induced suppression of osteoclastogenesis. These results suggested that TLR4 signaling not only induces ROS and NO production to kill phagocytized bacteria, but also interrupts OCm differentiation. Thus, it can be concluded that TRAP+ OCp, but not OCm, can mediate bactericidal activity via phagocytosis accompanied by the production of ROS and NO via TLR4-associated reprograming toward phagocytic cell type.


Subject(s)
Nitric Oxide/physiology , Osteoclasts/metabolism , Reactive Oxygen Species/metabolism , Tartrate-Resistant Acid Phosphatase/physiology , Toll-Like Receptor 4/physiology , Animals , Escherichia coli/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Microbial Viability , Osteoclasts/microbiology , Phagocytosis , RANK Ligand/physiology , RAW 264.7 Cells
3.
J Periodontol ; 85(6): e198-204, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24228808

ABSTRACT

BACKGROUND: MicroRNAs (miRNAs) are short, non-coding RNAs that are involved in post-transcriptional regulation of gene expression. Differential miRNA expression in innate and acquired immunity has been shown to regulate immune cell development and function. miRNA expression has been demonstrated to affect pathophysiology of inflammatory diseases, such as rheumatoid arthritis and lupus. As such, this study explores the role of miRNA in the context of pathophysiology of destructive periodontitis. Specifically, this investigation profiles the differentially expressed miRNA of Porphyromonas gingivalis (Pg)-stimulated human gingival epithelial cells (HGECs). METHODS: The specific miRNAs differentially expressed in Pg-stimulated OBA-9, immortalized HGECs, were analyzed using microarray. Real-time polymerase chain reaction (PCR) and Western blotting were performed to confirm the level of miRNA expression and determine target production of miRNA in OBA-9. The production of interleukin (IL)-8 was measured to determine the bioactivity of target protein regulated by miRNA. RESULTS: miR-584, which targets lactoferrin receptor (LfR), was 3.39-fold upregulated by Pg stimulation. This upregulation of miR-584 was confirmed by real-time PCR. Pg stimulation resulted in the suppression of LfR at mRNA and protein levels. The transfection of the miR inhibitor for miR-584 in OBA-9 recovered Pg-induced suppression of LfR. The addition of human lactoferrin (hLf) had a suppressive effect on IL-8 production in Pg-stimulated OBA-9. However, hLf also decreased IL-8 production strongly in Pg-stimulated OBA-9 in the presence of the miR inhibitor for miR-584. CONCLUSION: These findings suggest that the upregulation of miR-584 by Pg in OBA-9 inhibits the anti-inflammatory effects of hLf via the suppression of LfR.


Subject(s)
Gingiva/cytology , Interleukin-8/genetics , Lactoferrin/genetics , MicroRNAs/genetics , Porphyromonas gingivalis/immunology , Receptors, Cell Surface/genetics , Adult , Aggregatibacter actinomycetemcomitans/immunology , Cell Line, Transformed , Cells, Cultured , Coculture Techniques , Epithelial Cells/immunology , Epithelial Cells/microbiology , Female , Gene Expression Regulation, Bacterial/genetics , Gene Knockdown Techniques , Gingiva/immunology , Gingiva/microbiology , Humans , Inflammation Mediators/analysis , Inflammation Mediators/immunology , Interleukin-8/analysis , Lactoferrin/analysis , MicroRNAs/antagonists & inhibitors , MicroRNAs/immunology , Receptors, Cell Surface/analysis , Up-Regulation , Young Adult
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