Eiko Sakai

Molecular analysis of RANKL-independent cell fusion of osteoclast-like cells induced by TNF-α, lipopolysaccharide, or peptidoglycan

Focusing on the final step of osteoclastogenesis, we studied cell fusion from tartrate‐resistant acid phosphatase (TRAP)‐positive mononuclear cells into multinuclear cells. TRAP‐positive mononuclear cells before generation of multinuclear cells by cell fusion were differentiated from RAW264.7 cells by treatment with receptor activator of nuclear factor kappa B ligand (RANKL), and then the cells were treated with lipopolysaccharide (LPS), followed by culturing for further 12 h. LPS‐induced cell fusion even in the absence of RANKL. Similarly, tumor necrosis factor (TNF)‐α and peptidoglycan (PGN) induced cell fusion, but M‐CSF did not. The cell fusion induced by RANKL, TNF‐α, and LPS was specifically blocked by osteoprotegerin (OPG), anti‐TNF‐α antibody, and polymyxin B, respectively. LPS‐ and PGN‐induced cell fusion was partly inhibited by anti‐TNF‐α antibody but not by OPG. When TRAP‐positive mononuclear cells fused to yield multinuclear cells, phosphorylation of Akt, Src, extracellular signal‐regulated kinase (ERK), p38MAPK (p38), and c‐Jun NH2‐terminal kinase (JNK) was observed. The specific chemical inhibitors LY294002 (PI3K), PP2 (Src), U0126 (MAPK‐ERK kinase (MEK)/ERK), and SP600125 (JNK) effectively suppressed cell fusion, although SB203580 (p38) did not. mRNA of nuclear factor of activated T‐cells c1 (NFATc1) and dendritic cell‐specific transmembrane protein (DC‐STAMP) during the cell fusion was quantified, however, there was no obvious difference among the TRAP‐positive mononuclear cells treated with or without M‐CSF, RANKL, TNF‐α, LPS, or PGN. Collectively, RANKL, TNF‐α, LPS, and PGN induced cell fusion of osteoclasts through their own receptors. Subsequent activation of signaling pathways involving PI3K, Src, ERK, and JNK molecules was required for the cell fusion. Although DC‐STAMP is considered to be a requisite for cell fusion of osteoclasts, cell fusion‐inducing factors other than DC‐STAMP might be necessary for the cell fusion. J. Cell. Biochem. 101: 122–134, 2007.

The major structural components of two cell surface filaments of Porphyromonas gingivalis are matured through lipoprotein precursors

Bacterial cell surface filaments play significant roles in adherence to and invasion of host cells. They are generated by the chaperone/usher pathway system (class I fimbriae), the type II secretion system (type IV pili) and the nucleation‐dependent polymerization system (Curli filaments) that are categorized by their modes of expression and assembly. In this study, we found that the periodontal pathogen Porphyromonas gingivalis expressed the major structural components of two cell surface filaments (fimbrilin and the 75 kDa protein) that had extremely long prosequences in their primary gene products. N‐terminal amino acid sequencing of the prosequences, treatment of P. gingivalis cells with globomycin, an inhibitor for lipoprotein‐specific signal peptidase, amino acid substitution of the cysteine residue of the prosequence of fimbrilin and [3H]‐palmitic acid labelling implied that fimbrilin and the 75 kDa protein were matured through their lipoprotein precursor forms. Accumulation of precursor forms of fimbrilin and the 75 kDa protein on the cell surface of the gingipain‐null mutant revealed that Arg‐gingipain processed these precursors on the surface to yield their mature forms, which subsequently assembled into the filamentous structures, suggesting that the transport and assembly of the major component proteins appear to be novel.

Porphyromonas gingivalis-induced platelet aggregation in plasma depends on Hgp44 adhesin but not Rgp proteinase

Evidence from recent epidemiological studies suggests a link between periodontal infections and increased risk of atherosclerosis and related cardiovascular and cerebrovascular events in human subjects. One of the major pathogens of periodontitis, Porphyromonas gingivalis, has the ability to aggregate human platelets in platelet‐rich plasma (PRP). Mechanism of P. gingivalis‐induced platelet aggregation in PRP was investigated. Proteinase inhibitors toward Arg‐gingipain (Rgp) and Lys‐gingipain (Kgp) did not suppress P. gingivalis‐induced platelet aggregation in PRP, whereas the Rgp inhibitor markedly inhibited P. gingivalis‐induced platelet aggregation using  washed  platelets.  Mutant  analysis  revealed  that P. gingivalis‐induced platelet aggregation in PRP depended on Rgp‐, Kgp‐ and haemagglutinin A (HagA)‐encoding genes that intragenically coded for adhesins such as Hgp44. Hgp44 adhesin on the bacterial cell surface, which was processed by Rgp and Kgp proteinases, was essential for P. gingivalis‐induced platelet aggregation in PRP. P. gingivalis cell‐reactive IgG in plasma, and FcγRIIa receptor and to a lesser extent GPIbα receptor on platelets were found to be a prerequisite for P. gingivalis‐induced platelet aggregation in PRP. These results reveal a novel mechanism of platelet aggregation by P. gingivalis.

Force-induced Rapid Changes in Cell Fate at Midpalatal Suture Cartilage of Growing Rats

The application of expansional force induces replacement of the cartilaginous tissue with bone at the midpalatal suture of growing rats. We examined the early cellular events evoked by force by analyzing the expression of proliferating cell nuclear antigen (PCNA), an operational marker of cell proliferation, and of several bone matrix proteins. A rectangular orthodontic appliance was set between the right and left upper molars of four-week-old rats, with 50 g of initial expansional force. Two days after application of the force, the pre-existing cartilage was separated laterally. Mesenchymal cells with stretched shapes were arranged parallel to the expansional force and filled the center of the suture. Only a few of these stretched cells exhibited nuclear accumulation of PCNA. In contrast, many polygonal mesenchymal cells distributed along the inner lateral side of the cartilaginous tissue exhibited strong immunoreactivity for PCNA. Localization of alkaline phosphatase activity overlapped into this proliferating cell zone. Nascent extracellular matrix under the proliferating cells was positive for osteocalcin, indicating commencement of active bone formation. These findings indicated that, among mesenchymal cells subjected to expansional forces, only cells located on the inner side of the cartilaginous tissue proliferate and differentiate into osteoblasts. In agreement with rapid bone growth progression, apoptosis was also observed in the zone of proliferating cells, as measured by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assays.

Suppression of RANKL‐dependent heme oxygenase‐1 is required for high mobility group box 1 release and osteoclastogenesis

The differentiation of osteoclasts is regulated by several essential cytokines, such as receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony‐stimulating factor. Recently, high mobility group box 1 (HMGB1), a chromatin protein, also has been identified as one of these osteoclast differentiation cytokines. However, the molecular mechanisms that control HMGB1 release from osteoclast precursor cells are not known. Here, we report that RANKL‐induced suppression of heme oxygenase‐1 (HO‐1), a heme‐degrading enzyme, promotes HMGB1 release during osteoclastogenesis. In contrast, induction of HO‐1 with hemin or curcumin in bone marrow‐derived macrophages or RAW‐D murine osteoclast precursor cells inhibited osteoclastogenesis and suppressed HMGB1 release. Since an inhibitor for p38 mitogen‐activated protein kinase (MAPK) prevented the RANKL‐mediated HO‐1 suppression and extracellular release of HMGB1, these effects were p38 MAPK‐dependent. Moreover, suppression of HO‐1 in RAW‐D cells by RNA interference promoted the activation of caspase‐3 and HMGB1 release, whereas overexpression of HO‐1 inhibited caspase‐3 activation as well as HMGB1 release. Furthermore, these effects were regulated by redox conditions since antioxidant N‐acetylcysteine abolished the HO‐1/HMGB1/caspase‐3 axis. These results suggest that RANKL‐dependent HO‐1 suppression leads to caspase‐3 activation and HMGB1 release during osteoclastogenesis. J. Cell. Biochem. 113: 486–498, 2012.

Engineering Bone Formation from Human Dental Pulp- and Periodontal Ligament-Derived Cells

A robust method for inducing bone formation from cultured dental mesenchymal cells has not been established. In this study, a method for generating bone tissue in vivo from cultured human dental pulp- and periodontal ligament-derived cells (DPCs and PDLCs, respectively) was designed using exogenous bone morphogenetic protein 2 (BMP2). DPCs and PDLCs showed enhanced alkaline phosphatase (ALP) activity and calcified nodule formation in medium containing dexamethasone, β-glycerophosphate, and ascorbic acid (osteogenic medium). However, the addition of recombinant human bone morphogenetic protein 2 (rhBMP2) to osteogenic medium remarkably increased ALP activity and in vitro calcification above the increases observed with osteogenic medium alone. rhBMP2 also significantly upregulated the expression of osteocalcin, osteopontin, and dentin matrix protein 1 mRNA in both cell types cultured in osteogenic medium. Finally, we detected prominent bone-like tissue formation in vivo when cells had been exposed to rhBMP2 in osteogenic medium. In contrast, treatments with osteogenic medium or rhBMP2 alone could not induce abundant mineralized tissue formation. We propose here that treatment with rhBMP2 in osteogenic medium can make dental mesenchymal tissues a highly useful source of cells for bone tissue engineering. In addition, both DPCs and PDLCs showed similar and remarkable osteo-inducibility.

Construction of recombinant hemagglutinin derived from the gingipain-encoding gene of Porphyromonas gingivalis, identification of its target protein on erythrocytes, and inhibition of hemagglutination by an interdomain regional peptide

Porphyromonas gingivalis, an anaerobic gram-negative bacterium associated with chronic periodontitis, can agglutinate human erythrocytes. In general, hemagglutination can be considered the ability to adhere to host cells; however, P. gingivalis-mediated hemagglutination has special significance because heme markedly accelerates growth of this bacterium. Although a number of studies have indicated that a major hemagglutinin of P. gingivalis is intragenically encoded by rgpA, kgp, and hagA, direct evidence has not been obtained. We demonstrated in this study that recombinant HGP44(720-1081), a fully processed HGP44 domain protein, had hemagglutinating activity but that an unprocessed form, HGP44(720-1138), did not. A peptide corresponding to residues 1083 to 1102, which was included in HGP44(720-1138) but not in HGP44(720-1081), could bind HGP44(720-1081) in a dose-dependent manner and effectively inhibited HGP44(720-1081)-mediated hemagglutination, indicating that the interdomain regional amino acid sequence may function as an intramolecular suppressor of hemagglutinating activity. Analyses by solid-phase binding and chemical cross-linking suggested that HGP44 interacted with glycophorin A on the erythrocyte membrane. Glycophorin A and, more effectively, asialoglycophorin, which were added exogenously, inhibited HGP44(720-1081)-mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and a decrease in HGP44(720-1081)-mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44(720-1081) could bind to asialoglycophorin with a dissociation constant of 3.0 x 10(-7) M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A.

Expression and localization of MGP in rat tooth cementum

Tooth cementum, a calcified hard tissue covering the root surfaces, is an important component connecting the teeth to the collagenous fibres of the periodontal ligament. Although the overall composition of cementum may closely resemble that of bone, each part has not been fully characterized. Here, the localization of the matrix gamma-carboxyglutamic acid (Gla) protein (MGP), one of the major Gla-containing proteins in the body, in cementum was investigated using immunohistochemistry and in situ hybridization. (1) Strong MGP antigenicity was observed in the acellular cementum, but was only moderate in the cellular cementum; (2) polygonal periodontal ligament cells facing the acellular cementum and the uncalcified cellular cementum expressed MGP mRNA, indicating that these cells produced MGP and deposited it on the cementum; (3) MGP accumulated at the junction between the uncalcified and calcified cellular cementum; and (4) the distribution pattern of MGP antigenicity resembled that of osteopontin. As one function of MGP could be as a negative regulator for mineral apposition, the expression of MGP in the cells adjacent to the cementum may be important to prevent hyperapposition of minerals.

Rab27A Regulates Transport of Cell Surface Receptors Modulating Multinucleation and Lysosome-Related Organelles in Osteoclasts

Rab27A regulates transport of lysosome-related organelles (LROs) and release of secretory granules in various types of cells. Here, we identified up-regulation of Rab27A during differentiation of osteoclasts (OCLs) from bone-marrow macrophages (BMMs), by DNA microarray analysis. Rab27A deficiency in OCLs, using small interfering RNA (siRNA) knockdown in RAW-D cell line or BMMs derived from ashen mice, which display genetic defects in Rab27A expression, induced multinucleated and giant cells. Upon stimulation with macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL), essential cytokines for OCL differentiation, phosphorylation levels of extracellular signal-regulated kinase (Erk), proto-oncogene tyrosine-protein kinase (Src), and p-38 were slightly enhanced in ashen BMMs than in wild-type BMMs. The cell surface level of c-fms, an M-CSF receptor, was slightly higher in ashen BMMs than in wild-type BMMs, and down-regulation of RANK, a RANKL receptor, was delayed. In addition to receptors, OCLs derived from ashen mice exhibited aberrant actin ring formation, abnormal subcellular localization of lysosome-associated membrane protein (LAMP2) and cathepsin K (CTSK), and marked reduction in resorbing activity. Thus, these findings suggest that Rab27A regulates normal transport of cell surface receptors modulating multinucleation and LROs in OCLs.

The Hemoglobin Receptor Protein of Porphyromonas gingivalis Inhibits Receptor Activator NF-κB Ligand-Induced Osteoclastogenesis from Bone Marrow Macrophages

ABSTRACT Extracellular proteinaceous factors of Porphyromonas gingivalis, a periodontal pathogen, that influence receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis from bone marrow macrophages were investigated. The culture supernatant of P. gingivalis had the ability to inhibit RANKL-induced in vitro osteoclastogenesis. A major protein of the culture supernatant, hemoglobin receptor protein (HbR), suppressed RANKL-induced osteoclastogenesis in a dose-dependent fashion. HbR markedly inhibited RANKL-induced osteoclastogenesis when present in the culture for the first 24 h after addition of RANKL, whereas no significant inhibition was observed when HbR was added after 24 h or later, implying that HbR might interfere with only the initial stage of RANKL-mediated differentiation. HbR tightly bound to bone marrow macrophages and had the ability to induce phosphorylation of ERK, p38, NF-κB, and Akt. RANKL-induced phosphorylation of ERK, p38, and NF-κB was not suppressed by HbR, but that of Akt was markedly suppressed. HbR inhibited RANKL-mediated induction of c-Fos and NFATc1. HbR could induce beta interferon (IFN-β) from bone marrow macrophages, but the induction level of IFN-β might not be sufficient to suppress RANKL-mediated osteoclastogenesis, implying presence of an IFN-β-independent pathway in HbR-mediated inhibition of osteoclastogenesis. Since rapid and extensive destruction of the alveolar bone causes tooth loss, resulting in loss of the gingival crevice that is an anatomical niche for periodontal pathogens such as P. gingivalis, the suppressive effect of HbR on osteoclastogenesis may help the microorganism exist long in the niche.