The dental resin monomers HEMA and TEGDMA have inhibitory effects on osteoclast differentiation with low cytotoxicity.

The dental resin monomers 2-hydroxyethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) are released from the resin matrix due to unpolymerized monomers; once released, they influence various biological functions and the viability of cells in the oral environment. Although HEMA and TEGDMA have various effects on cells, including inflammation, inhibition of cell proliferation or differentiation, and apoptosis, the effects of these monomers on osteoclasts remain unknown. In this study, we investigated the effects of HEMA and TEGDMA on osteoclast differentiation of bone marrow-derived macrophages or murine monocytic cell line RAW-D. Both HEMA and TEGDMA inhibited osteoclast formation and their bone-resorbing activity at non-cytotoxic concentrations. Moreover, HEMA and TEGDMA decreased the expression of nuclear factor of activated T cells cytoplasmic-1 (NFATc1), a master regulator of osteoclast differentiation, and of osteoclast markers that are transcriptionally regulated by NFATc1, including Src and cathepsin K. Regarding their effects on signaling pathways involved in osteoclast differentiation, HEMA impaired the phosphorylation of extracellular signal-regulated kinase and Jun N-terminal kinase, whereas TEGDMA attenuated the phosphorylation of Akt and Jun N-terminal kinase. Thus, HEMA and TEGDMA inhibit osteoclast differentiation through different signaling pathways. This is the first report on the effects of the monomers HEMA and TEGDMA on osteoclasts. Copyright © 2017 John Wiley & Sons, Ltd.

Outer membrane vesicles of Porphyromonas gingivalis attenuate insulin sensitivity by delivering gingipains to the liver.

Outer membrane vesicles (OMVs) are nanosized particles derived from the outer membrane of gram-negative bacteria. Oral bacterium Porphyromonas gingivalis (Pg) is known to be a major pathogen of periodontitis that contributes to the progression of periodontal disease by releasing OMVs. The effect of Pg OMVs on systemic diseases is still unknown. To verify whether Pg OMVs affect the progress of diabetes mellitus, we analyzed the cargo proteins of vesicles and evaluated their effect on hepatic glucose metabolism. Here, we show that Pg OMVs were equipped with Pg-derived proteases gingipains and translocated to the liver in mice. In these mice, the hepatic glycogen synthesis in response to insulin was decreased, and thus high blood glucose levels were maintained. Pg OMVs also attenuated the insulin-induced Akt/glycogen synthase kinase-3 ß (GSK-3ß) signaling in a gingipain-dependent fashion in hepatic HepG2 cells. These results suggest that the delivery of gingipains mediated by Pg OMV elicits changes in glucose metabolisms in the liver and contributes to the progression of diabetes mellitus.

Determination of active site of lysine-specific cysteine proteinase (Lys-gingipain) by use of a Porphyromonas gingivalis plasmid system.

Porphyromonas gingivalis, a major etiological bacterium of periodontal diseases, produces a unique lysine-specific cysteine proteinase (Lys-gingipain, Kgp) implicated in the virulence of this organism. Our observations show the expression of a catalytically active recombinant Kgp in a P. gingivalis Kgp-null mutant and the restoration of its functions by the use of a shuttle plasmid vector stable in P. gingivalis. The Kgp-expressing mutant exhibited a similar catalytic activity to that of the wild-type strain. This mutant also restored the ability to form black-pigmented colonies on blood agar plates and to generate a 19-kDa haemoglobin receptor protein responsible for haemoglobin binding. In order to establish the importance of the active-site Cys residue and elucidate its role in bacterial black pigmentation we constructed three Kgp mutants with changed potential active-site Cys residues. The cells expressing a single mutation (C476A) showed the high Kgp activity and the black pigmentation. In contrast, the cells expressing the single mutant (C477A) and the double mutant (C476A/C477A) exhibited neither Kgp activity nor black pigmentation. These results indicate that the 477th Cys residue is essential for both the Kgp activity and the black pigmentation of P. gingivalis.

Cathepsin E-deficient mice show increased susceptibility to bacterial infection associated with the decreased expression of multiple cell surface Toll-like receptors.

Cathepsin E, an intracellular aspartic proteinase, is predominantly localized in the endosomal compartments of immune system cells. In the present study, we investigated the role of cathepsin E in immune defense systems against bacterial infection. Cathepsin E-deficient (CatE(-/-)) mice showed dramatically increased susceptibility to infection with both the Gram-positive bacterium Staphyrococcus aureus, and the Gram-negative bacterium Porphyromonas gingivalis when compared with syngeneic wild-type mice, most likely due to impaired regulation of bacterial elimination. Peritoneal macrophages from CatE(-/-) mice showed significantly impaired tumor necrosis factor-alpha and IL-6 production in response to S. aureus and decreased bactericidal activities toward this bacterium. Moreover, the cell surface levels of Toll-like receptor-2 (TLR2) and TLR4, which recognize specific components of Gram-positive and -negative bacteria, respectively, were decreased in CatE(-/-) macrophages, despite no significant difference in the total cellular expression levels of these receptors between the wild-type and CatE(-/-) macrophages, implying trafficking defects in these surface receptors in the latter. These results indicate an essential role of cathepsin E in immune defense against invading microorganisms, most probably due to regulation of the cell surface expression of TLR family members required for innate immune responses.

Suzuki-Miyaura coupling catalyzed by polymer-incarcerated palladium, a highly active, recoverable, and reusable Pd catalyst.

[reaction: see text] Suzuki-Miyaura coupling using a highly efficient and reusable polymer-incarcerated palladium (PI Pd) is described. Various coupling reactions proceeded smoothly using PI Pd with phosphine ligands, and the catalyst could be recovered by simple filtration and reused several times without loss of activity.

Dentin phosphophoryn promotes cellular migration of human dental pulp cells.

Dentin phosphophoryn (DPP) is a dentin sialophosphoprotein gene product that has an RGD motif and repeat sequences of aspartic acid and phosphoserine. To date, the function of DPP in the early stage of reparative dentin formation still remains unclear. The objective of this study was to evaluate the effects of DPP on pulp cell migration and proliferation. DPP promoted cell migration in a concentration-dependent manner, thus increasing it by about 3-fold at 1000 ng/mL compared with the control, but it had no effect on cell proliferation. Dephosphorylated DPP also promoted cell migration, similarly to DPP. However, cell migration was significantly suppressed by the addition of alphavbeta3 integrin antibody to the medium. Furthermore, porcine DPP-derived RGD peptide, but not its mutant RAD peptide, significantly promoted cell migration. These results indicated that the RGD motif of DPP plays an important role in the migration of human dental pulp cells.

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.

Gingipains inactivate a cell surface ligand on Porphyromonas gingivalis that induces TLR2-and TLR4-independent signaling.

Arginine-specific gingipain and lysine-specific gingipain are two major cysteine proteinases produced by Porphyromonas gingivalis. To clarify the role of gingipains in the interaction between P. gingivalis and the innate immune system, CHO reporter cells expressing TLR2 or TLR4 were stimulated with wildtype or gingipain-deficient P. gingivalis cells and activation of nuclear factor-kappaB in these cells was examined. While CHO/CD14 cells and 7.19 cells, an MD-2-defective mutant derived from CHO/CD14 cells, failed to respond to wild-type P. gingivalis, they responded to gingipain-deficient P. gingivalis. On the other hand, CHO/CD14/TLR2 cells responded to both wild-type and gingipain-deficient P. gingivalis. These results suggested that gingipains have no effects on TLR2-dependent signaling from P. gingivalis but have inhibitory effects on TLR2-and TLR4-independent signaling in CHO cells. Indeed, the activity of gingipain-deficient P. gingivalis to induce the activation of 7.19 cells was diminished after treatment of the bacterial cells with gingipains. We next partially purified bacterial cell components activating 7.19 cells from gingipain-deficient P. gingivalis. The activity of the partially purified components was diminished by treatment with heat or gingipains. It is also noteworthy that anti-CD14 mAb inhibited the activation of 7.19 cells induced by the partially purified components. These results indicated that the components of P. gingivalis that were able to induce TLR2-and TLR4-independent signaling were inactivated by gingipains before being recognized by CD14. The inactivation of the components would be helpful for P. gingivalis to escape from the innate immune system.

Identification of a new membrane-associated protein that influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis.

The dual membrane envelopes of Gram-negative bacteria provide two barriers of unlike nature that regulate the transport of molecules into and out of organisms. Organisms have developed several systems for transport across the inner and outer membranes. The Gram-negative periodontopathogenic bacterium Porphyromonas gingivalis produces proteinase and adhesin complexes, gingipains/adhesins, on the cell surface and in the extracellular milieu as one of the major virulence factors. Gingipains and/or adhesins are encoded by kgp, rgpA, rgpB, and hagA on the chromosome. In this study, we isolated a P. gingivalis mutant (porT), which showed very weak activities of gingipains in the cell lysates and culture supernatants. Subcellular fractionation and immunoblot analysis demonstrated that precursor forms of gingipains and adhesins were accumulated in the periplasmic space of the porT mutant cells. Peptide mass fingerprinting and N-terminal amino acid sequencing of the precursor proteins and the kgp'-'rgpB chimera gene product in the porT mutant indicated that these proteins lacked the signal peptide regions, consistent with their accumulation in the periplasm. The PorT protein seemed to be membrane-associated and exposed to the periplasmic space, as revealed by subcellular fractionation and immunoblot analysis using anti-PorT antiserum. These results suggest that the membrane-associated protein PorT is essential for transport of the kgp, rgpA, rgpB, and hagA gene products across the outer membrane from the periplasm to the cell surface, where they are processed and matured.

The regulation of bone resorption in tooth formation and eruption processes in mouse alveolar crest devoid of cathepsin k.

Osteoclastic bone resorption has recently been implicated in the tooth formation and eruption in alveolar bone. Cathepsin K (CK) is a cysteine proteinase expressed predominantly in osteoclasts and is believed to play a critical role in degradation of bone matrix proteins. Here we present evidence that the alveolar bone resorption is essential for the tooth formation and that eruption proceeds normally in CK-deficient (CK-/-) mice. Radiographic and histological analyses revealed that the alveolar bone from these animals had no significant abnormalities during the tooth development between 5 and 28 days after birth. The tooth crown was normally erupted through the alveolar bone layer at 28 days after birth. The number of tartrate-resistant acid phosphatase-positive multinuclear cells in the alveolar bone around the tooth germ was apparently increased in 5-day-old CK-/- mice compared with age-matched littermates. More important, however, the immunohistochemical localization of matrix metalloproteinase-9 (MMP-9) was clearly increased in the CK-/- osteoclasts. In contrast, no significant difference in the immunoreactivity for cathepsin D was observed between the CK-/- osteoclasts and the wild-type ones. These results indicate that CK-/- osteoclasts are fully differentiated and are capable of degrading the organic phase of alveolar bone during the tooth formation and eruption, which may result from the compensatory action by MMP-9 increasingly expressed in the osteoclasts.

Roles of Arg- and Lys-gingipains in coaggregation of Porphyromonas gingivalis: identification of its responsible molecules in translation products of rgpA, kgp, and hagA genes.

Arg- (Rgp) and Lys-gingipains (Kgp) are two individual cysteine proteinases produced by Porphyromonas gingivalis , an oral anaerobic bacterium, and are implicated as major virulence factors in a wide range of pathologies of adult periodontitis. Coaggregation of this bacterium with other oral bacteria is an initial and critical step in infectious processes, yet the factors and mechanisms responsible for this process remain elusive. Here we show that the initial translation products of the rgpA , kgp and hemagglutinin hagA genes are responsible for coaggregation of P. gingivalis and that the proteolytic activity of Rgp and Kgp is indispensable in this process. The rgpA rgpB kgp- and rgpA kgp hagA -deficient triple mutants exhibited no coaggregation activity with Actinomyces viscosus , whereas the kgp -null and rgpA rgpB -deficient double mutants significantly retained this activity. Consistently, the combined action of Rgp- and Kgp-specific inhibitors strongly inhibited the coaggregation activity of the bacterium, although single use of Rgp- or Kgp-specific inhibitor significantly retained this activity. We also demonstrate that the 47- and 43-kDa proteins produced from the translation products of the rgpA , kgp , and hagA genes by proteolytic activity of both Rgp and Kgp are responsible for the coaggregation of P. gingivalis.

Laminin alpha2 is essential for odontoblast differentiation regulating dentin sialoprotein expression.

Laminin alpha2 is subunit of laminin-2 (alpha2beta1gamma1), which is a major component of the muscle basement membrane. Although the laminin alpha2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin alpha2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin alpha2 is expressed in dental mesenchymal cells, especially in odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wild-type and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin alpha2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.