Noriaki Kamio

Angiotensin II induces the production of MMP-3 and MMP-13 through the MAPK signaling pathways via the AT1 receptor in osteoblasts

Angiotensin II (Ang II) plays an important role in the maintenance of bone mass and integrity by activation of the mitogen-activated protein kinases (MAPKs) and by modulation of balance between resorption by osteoclasts and formation by osteoblasts. However, the role of Ang II in the turnover of extracellular matrix (ECM) in osteoid by osteoblasts remains unclear. Therefore, we examined the effect of Ang II on the expression of matrix metalloproteinases (MMPs), plasminogen activators (PAs), and their inhibitors [i.e., tissue inhibitors of metalloproteinases (TIMPs) and PA inhibitor-1 (PAI-1)] using osteoblastic ROS17/2.8 cells. Treatment with Ang II strikingly increased the expressions of MMP-3 and -13 and promoted cell proliferation associated with reduced alkaline phosphatase activity as well as enhanced phosphorylated expression of extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, and stress-activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) in ROS17/2.8 cells. However, Ang II had no effect on the expression of MMP-2, -9, -14, urokinase-type PA, tissue-type PA, TIMP-1, -2, -3, and PAI-1 in cells. Losartan (AT1 receptor blocker) blocked Ang II-induced expression of MMP-3 and -13, whereas PD123319 (AT2 receptor blocker) did not completely block these responses. Losartan also blocked the Ang II-induced phosphorylation of ERK1/2, p38 MAPK, and SAPK/JNK. MAPK kinase 1/2 inhibitor PD98059 and JNK inhibitor SP600125 suppressed Ang II-induced expression of MMP-3 and -13. These results suggested that Ang II stimulated the degradation process that occurs during ECM turnover in osteoid by increasing the production of MMP-3 and -13 through MAPK signaling pathways via the AT1 receptor in osteoblasts. Furthermore, our findings suggest that Ang II does not influence the plasminogen/plasmin pathway in osteoblasts.

Vaspin Attenuates RANKL-Induced Osteoclast Formation in RAW264.7 Cells

Visceral adipose tissue-derived serine protease inhibitor (vaspin), an adipokine that was recently identified in a rat model of type 2 diabetes, has been suggested to have an insulin-sensitizing effect. In this study, we investigated whether vaspin inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis using two types of osteoclast precursors: RAW264.7 cells and bone marrow cells (BMCs). Vaspin inhibited RANKL-induced osteoclastogenesis in RAW264.7 cells and BMCs. Interestingly, vaspin also inhibited the RANKL-induced expression of nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells and BMCs. Furthermore, it inhibited the RANKL-induced upregulation of matrix metalloproteinase-9 and cathepsin K in RAW264.7 cells. Thus, we suggest that vaspin downregulates osteoclastogenesis in part by inhibiting expression of the transcription factor NFATc1.

High butyric acid amounts induce oxidative stress, alter calcium homeostasis, and cause neurite retraction in nerve growth factor-treated PC12 cells

Butyric acid (BA) is a common secondary metabolite by-product produced by oral pathogenic bacteria and is detected in high amounts in the gingival tissue of patients with periodontal disease. Previous works have demonstrated that BA can cause oxidative stress in various cell types; however, this was never explored using neuronal cells. Here, we exposed nerve growth factor (NGF)-treated PC12 cells to varying BA concentrations (0.5, 1.0, 5.0xa0mM). We measured total heme, H2O2, catalase, and calcium levels through biochemical assays and visualized the neurite outgrowth after BA treatment. Similarly, we determined the effects of other common periodontal short-chain fatty acids (SCFAs) on neurite outgrowth for comparison. We found that high (1.0 and 5.0xa0mM) BA concentrations induced oxidative stress and altered calcium homeostasis, whereas low (0.5xa0mM) BA concentration had no significant effect. Moreover, compared to other SCFAs, we established that only BA was able to induce neurite retraction.

Role of the histone H3 lysine 9 methyltransferase Suv39 h1 in maintaining Epsteinn–Barr virus latency in B95–8 cells

The ability of Epstein–Barr Virus (EBV) to establish latent infection is associated with infectious mononucleosis and a number of malignancies. In EBV, the product of the BZLF1 gene (ZEBRA) acts as a master regulator of the transition from latency to the lytic replication cycle in latently infected cells. EBV latency is primarily maintained by hypoacetylation of histone proteins in the BZLF1 promoter by histone deacetylases. Although histone methylation is involved in the organization of chromatin domains and has a central epigenetic role in gene expression, its role in maintaining EBV latency is not well understood. Here we present evidence that the histone H3 lysine 9 (H3K9) methyltransferase suppressor of variegation 3–9 homolog 1 (Suv39 h1) transcriptionally represses BZLF1 in B95–8 cells by promoting repressive trimethylation at H3K9 (H3K9me3). Suv39 h1 significantly inhibited basal expression and ZEBRA‐induced BZLF1 gene expression in B95–8 B cells. However, mutant Suv39 h1 lacks the SET domain responsible for catalytic activity of histone methyl transferase and thus had no such effect. BZLF1 transcription was augmented when Suv39 h1 expression was knocked down by siRNA in B95–8 cells, but not in Akata or Raji cells. In addition, treatment with a specific Suv39 h1 inhibitor, chaetocin, significantly enhanced BZLF1 transcription. Furthermore, chromatin immunoprecipitation assays revealed the presence of Suv39 h1 and H3K9me3 on nucleosome histones near the BZLF1 promoter. Taken together, these results suggest that Suv39 h1–H3K9me3 epigenetic repression is involved in BZLF1 transcriptional silencing, providing a molecular basis for understanding the mechanism by which EBV latency is maintained.

Neuraminidase-producing oral mitis group streptococci potentially contribute to influenza viral infection and reduction in antiviral efficacy of zanamivir

Influenza is a serious respiratory disease among immunocompromised individuals, such as the elderly, and its prevention is an urgent social issue. Influenza viruses rely on neuraminidase (NA) activity to release progeny viruses from infected cells and spreading the infection. NA is, therefore, an important target of anti-influenza drugs. A causal relationship between bacteria and influenza virus infection has not yet been established, however, a positive correlation between them has been reported. Thus, in this study, we examined the biological effects of oral mitis group streptococci, which are predominant constituents of human oral florae, on the release of influenza viruses. Among them, Streptococcusoralis ATCC 10557 and Streptococcus mitis ATCC 6249 were found to exhibit NA activity and their culture supernatants promoted the release of influenza virus and cell-to-cell spread of the infection. In addition, culture supernatants of these NA-producing oral bacteria increased viral M1 protein expression levels and cellular ERK activation. These effects were not observed with culture supernatants of Streptococcus sanguinis ATCC 10556 which lacks the ability to produce NA. Although the NA inhibitor zanamivir suppressed the release of progeny viruses from the infected cells, the viral release was restored upon the addition of culture supernatants of NA-producing S. oralis ATCC 10557 or S. mitis ATCC 6249. These findings suggest that an increase in the number of NA-producing oral bacteria could elevate the risk of and exacerbate the influenza infection, hampering the efficacy of viral NA inhibitor drugs.

Varying butyric acid amounts induce different stress- and cell death-related signals in nerve growth factor-treated PC12 cells: implications in neuropathic pain absence during periodontal disease progression

Neuropathic pain is absent from the early stages of periodontal disease possibly due to neurite retraction. Butyric acid (BA) is a periodontopathic metabolite that activates several stress-related signals and, likewise, induce neurite retraction. Neuronal cell death is associated to neurite retraction which would suggest that BA-induced neurite retraction is ascribable to neuronal cell death. However, the underlying mechanism of BA-related cell death signaling remains unknown. In this study, we exposed NGF-treated PC12 cells to varying BA concentrations [0 (control), 0.5, 1.0, 5.0xa0mM] and determined selected stress-related (H2O2, glutathione reductase, calcium (Ca2+), plasma membrane Ca2+ ATPase (PMCA), and GADD153/CHOPS) and cell death-associated (extrinsic: FasL, TNF-α, TWEAK, and TRAIL; intrinsic: cytochrome C (CytC), NF-kB, CASP8, CASP9, CASP10, and CASP3) signals. Similarly, we confirmed cell death execution by chromatin condensation. Our results showed that low (0.5xa0mM) and high (1.0 and 5.0xa0mM) BA levels differ in stress and cell death signaling. Moreover, at periodontal disease-level BA concentration (5xa0mM), we observed that only FasL amounts were affected and occurred concurrently with chromatin condensation insinuating that cells have fully committed to neurodegeneration. Thus, we believe that both stress and cell death signaling in NGF-treated PC12 cells are affected differently depending on BA concentration. In a periodontal disease scenario, we hypothesize that during the early stages, low BA amounts accumulate resulting to both stress- and cell death-related signals that favor neurite non-proliferation, whereas, during the later stages, high BA amounts accumulate resulting to both stress- and cell death-related signals that favor neurodegeneration. More importantly, we propose that neuropathic pain absence at any stage of periodontal disease progression is ascribable to BA accumulation regardless of amount.

Structural Significance of the β1K396 Residue Found in the Porphyromonas gingivalis Sialidase β-Propeller Domain: A Computational Study with Implications for Novel Therapeutics Against Periodontal Disease

Porphyromonas gingivalis sialidase activity is associated with virulence and initiated by sialic acid (SA) binding to the β-propeller domain (BPD). Sialidase BPD is structurally conserved in various bacterial species and the protein binding interfaces have the tendency to form salt bridges, whereas uncommitted charged residues may affect binding and protein structure. However, it is not clear whether the sialidase BPD of varying strains of the same bacterial species differ, particularly with regards to salt bridge formation. Here, we determined the P. gingivalis ATCC 33277 and W50 sialidase homology models and sialidase activities, while the putative salt bridge residues found in the sialidase BPDs were compared. We established that both ATCC 33277 and W50 have different sialidase homology models and activities, whereas, the BPD (β1-6) is structurally conserved with most salt bridge-forming residues following a common orientation. Moreover, β2D444-β6K338 distance measurement in ATCC 33277 (5.99 Å) and W50 (3.09 Å) differ, while β1K396A substitution alters the β2D444-β6K338 distance measurements in ATCC 33277 (3.09 Å) and W50 (3.01 Å) consequentially affecting each model. P. gingivalis plays a major role in periodontitis induction and its virulence is greatly influenced by the sialidase enzyme wherein the sialidase BPD is highly conserved. Our results suggest that alterations in the salt bridge formation within the BPD interface may affect the P. gingivalis sialidase structure. This would imply that disrupting the salt bridge formation within the P. gingivalis sialidase BPD could serve as a potential therapeutic strategy for the treatment of P. gingivalis-related periodontitis.

Cynaropicrin from Cynara scolymus L. suppresses Porphyromonas gingivalis LPS-induced production of inflammatory cytokines in human gingival fibroblasts and RANKL-induced osteoclast differentiation in RAW264.7 cells

Periodontal diseases are a major public health problem affecting over half of the adult population worldwide. Lipopolysaccharide (LPS) produced by the periodontopathic bacterium Porphyromonas gingivalis induces the expression of inflammatory cytokines that promote inflammatory bone destruction. Mounting evidence supports that periodontal diseases are involved in the onset and progression of several systemic diseases, such as aspiration pneumonia and diabetes. Although treatment of periodontal diseases by removing the periodontopathic bacteria by brushing is a standard practice, it has limitations and is not effective in all cases. Therefore, a new method to replace or complement brushing is needed for the treatment of periodontal diseases. In this study, we investigated the anti-inflammatory effects of an extract from Cynara scolymus L. and its pharmacologically effective compound cynaropicrin, a sesquiterpene lactone, on human gingival fibroblasts (HGFs) stimulated by LPS and the potential anti-osteoclastogenic effects on RAW264.7 cells induced by receptor activator of NF-κB ligand (RANKL). We found that cynaropicrin inhibited IL-8 and IL-6 mRNA and protein synthesis in LPS-stimulated HGFs in a dose-dependent manner. P. gingivalis LPS-induced degradation of IκBα and phosphorylation of NF-κB p65 were also suppressed by cynaropicrin, as was LPS-stimulated NF-κB transactivation. Thus, cynaropicrin’s inhibition of P. gingivalis LPS-induced IL-8 and IL-6 expression may be due to the inhibition of the NF-κB pathway. Furthermore, we showed that cynaropicrin dramatically reduced RANKL-induced osteoclast differentiation. These results suggest that cynaropicrin may be useful for preventing periodontal diseases and could prove valuable in the development of more effective preventative approaches for periodontal diseases.

MiR-200b attenuates IL-6 production through IKKβ and ZEB1 in human gingival fibroblasts

ObjectiveMicroRNAs (miRNAs) play important roles in biological processes such as cell differentiation, development, infection, immune response, inflammation and tumorigenesis. We previously reported that the expression of miR-200b was significantly increased in inflamed gingiva compared with non-inflamed gingiva. To elucidate the roles of miR-200b in the inflamed gingiva, we havexa0analyzed the effects of miR-200b on the expression of IL-6 in human gingival fibroblasts (HGF).Materials and methodsTotal RNA and protein were extracted from HGF after stimulation by interleukin-1β (IL-1β; 1xa0ng/ml) or tumor necrosis factor-α (TNF-α; 10xa0ng/ml) and transfected with miR-200b expression plasmid or miR-200b inhibitor. IL-6, IL-1β, inhibitor of nuclear factorxa0kappa-B kinaseβ (IKKβ), Zinc-finger E-box-binding homeobox 1 (ZEB1) and E-cadherin mRNA and protein levels were analyzed by real-time PCR and Western blot.ResultsIL-1β and TNF-α increased IL-6 mRNA and protein levels, and they were significantly suppressed by miR-200b overexpression, whereas they were further increased by miR-200b inhibitor in HGF. IKKβ and ZEB1 which are target genes of miR-200b negatively regulate E-cadherin. MiR-200b suppressed the expression of IKKβ and ZEB1 and increased E-cadherin mRNA and protein levels in HGF.ConclusionsThese results suggest that miR-200b attenuates inflammatory response via IKKβ and ZEB1 in periodontal tissue.

Ions released from a S-PRG filler induces oxidative stress in Candida albicans inhibiting its growth and pathogenicity

Candida albicans causes opportunistic fungal infections usually hidden among more dominant bacteria and does not exhibit high pathogenicity in vivo. Among the elderly, due to reduced host resistance to pathogens attributable to immunoscenesence, oral candidiasis is more likely to develop often leading to systemic candidiasis. Surface pre-reacted glass ionomer filler (S-PRG filler) is an ion-releasing functional bioactive glass that can release and recharge six ions which in turn strengthens tooth structure, inhibits demineralization arising from dental caries, and suppresses dental plaque accumulation. However, its effects on C. albicans have never been elucidated. Here, we evaluated the effects of ion released from S-PRG filler on C. albicans. Results show that extraction liquids containing released ions (ELIS) decreased the amount of hydrogen peroxide and catalase activity in C. albicans. Moreover, ELIS presence was found to affect C. albicans: (1) suppression of fungal growth and biofilm formation, (2) prevent adherence to denture base resin, (3) inhibit dimorphism conversion, and (4) hinder the capability to produce secreted aspartyl proteinase. Taken together, our findings suggest that ELIS induces oxidative stress in C. albicans and suppresses its growth and pathogenicity. In this regard, we propose that ELIS has the potential to be clinically used to help prevent the onset and inhibition of oral candidiasis among the elderly population.