Effect of 5-aminolevulinic acid-mediated photodynamic therapy against Fusobacterium nucleatum in periodontitis prevention.

Periodontitis, a chronic infectious disease leading to gingival atrophy and potential tooth loss through alveolar bone resorption, is closely linked to the oral microbiome. Fusobacterium nucleatum, known to facilitate late-stage bacterial colonization in the oral microbiome, plays a crucial role in the onset of periodontitis. Controlling F. nucleatum abundance is vital for preventing and treating periodontal disease. Photodynamic therapy combined with 5-aminolevulinic acid (ALA-PDT) has been reported to be bactericidal against Pseudomonas aeruginosa and Staphylococcus aureus. We aimed to investigate the bactericidal potential of ALA-PDT against F. nucleatum, which was evaluated by examining the impact of varying 5-ALA concentrations, culture time, and light intensity. After ALA-PDT treatment, DNA was extracted from interdental plaque samples collected from 10 volunteers and sequenced using the Illumina MiSeq platform. To further elucidate the bactericidal mechanism of ALA-PDT, porphyrins were extracted from F. nucleatum following cultivation with 5-ALA and subsequently analyzed using fluorescence spectra. ALA-PDT showed a significant bactericidal effect against F. nucleatum. Its bactericidal activity demonstrated a positive correlation with culture time and light intensity. Microbiota analysis revealed no significant alteration in α-diversity within the ALA-PDT group, although there was a noteworthy reduction in the proportion of the genus Fusobacterium. Furthermore, fluorescence spectral analysis indicated that F. nucleatum produced an excitable photosensitive substance following the addition of 5-ALA. Overall, if further studies confirm these results, this combined therapy could be an effective strategy for reducing the prevalence of periodontitis.

Transposon insertion in Rothia dentocariosa.

OBJECTIVES: Rothia spp. are emerging as significant bacteria associated with oral health, with Rothia dentocariosa being one of the most prevalent species. However, there is a lack of studies examining these properties at the genetic level. This study aimed to establish a genetic modification platform for R. dentocariosa. METHODS: Rothia spp. were isolated from saliva samples collected from healthy volunteers. Subsequently, R. dentocariosa strains were identified through colony morphology, species-specific polymerase chain reaction (PCR), and 16S ribosomal RNA gene sequencing. The identified strains were then transformed with plasmid pJRD215, and the most efficient strain was selected. Transposon insertion mutagenesis was performed to investigate the possibility of genetic modifications. RESULTS: A strain demonstrating high transforming ability, designated as R. dentocariosa LX16, was identified. This strain underwent transposon insertion mutagenesis and was screened for 5-fluoroorotic acid-resistant transposants. The insertion sites were confirmed using arbitrary primed PCR, gene-specific PCR, and Sanger sequencing. CONCLUSION: This study marks the first successful genetic modification of R. dentocariosa. Investigating R. dentocariosa at the genetic level can provide insights into its role within the oral microbiome.

Characterization of oral microbiota in 6-8-month-old small breed dogs.

BACKGROUND: Periodontitis is the most common oral disease in dogs, and its progression and severity are influenced by risk factors, such as age and body size. Recent studies have assessed the canine oral microbiota in relation to different stages of periodontitis and niches within the oral cavity. However, knowledge of the bacterial composition at different ages and body sizes, especially in puppies, is limited. This study aimed to characterize the oral microbiota in the healthy gingiva of small breed puppies using next-generation sequencing. Additionally, we assessed the impact of dental care practices and the presence of retained deciduous teeth on the oral microbiota. RESULTS: In this study, plaque samples were collected from the gingival margin of 20 small breed puppies (age, 6.9 ± 0.6 months). The plaque samples were subjected to next-generation sequencing targeting the V3-V4 region of the 16 S rRNA. The microbiota of the plaque samples was composed mostly of gram-negative bacteria, primarily Proteobacteria (54.12%), Bacteroidetes (28.79%), and Fusobacteria (5.11%). Moraxella sp. COT-017, Capnocytophaga cynodegmi COT-254, and Bergeyella zoohelcum COT-186 were abundant in the oral cavity of the puppies. In contrast, Neisseria animaloris were not detected. The high abundance of Pasteurellaceae suggests that this genus is characteristic of the oral microbiota in puppies. Dental care practices and the presence of retained deciduous teeth showed no effects on the oral microbiota. CONCLUSIONS: In this study, many bacterial species previously reported to be detected in the normal oral cavity of adult dogs were also detected in 6-8-month-old small breed dogs. On the other hand, some bacterial species were not detected at all, while others were detected in high abundance. These data indicate that the oral microbiota of 6-8-month-old small breed dogs is in the process of maturating in to the adult microbiota and may also have characteristics of the small dog oral microbiota.

TLR2-dependent and independent pyroptosis in dTHP-1 cells induced by Actinomyces oris MG-1.

In the immune system, the detection of pathogens through various mechanisms triggers immune responses. Several types of specific programmed cell deaths play a role in the inflammatory reaction. This study emphasizes the inflammatory response induced by Actinomycetes. Actinomyces spp. are resident bacteria in human oral plaque and often serve as a bridge for pathogenic bacteria, which lack affinity to the tooth surface, aiding their colonization of the plaque. We aim to investigate the potential role of Actinomyces oris in the early stages of oral diseases from a new perspective. Actinomyces oris MG-1 (A. oris) was chosen for this research. Differentiated THP-1 (dTHP-1) cells were transiently treated with A. oris to model the inflammatory reaction. Cell viability, as well as relative gene and protein expression levels of dTHP-1 cells, were assessed using CCK-8, quantitative real-time polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and Western blot assay. The treatment decreased cell viability and increased the expression of inflammatory genes such as IL-1R1 and NLRP3. It was also observed to significantly enhance the release of IL-1ß/IL-18 into the supernatant. Immunoblot analysis revealed a notable increase in the expression of N-gasdermin D persisting up to 24 h. Conversely, in models pre-treated with TLR2 inhibitors, N-gasdermin D was detectable only 12 h post-treatment and absent at 24 h. These results suggest that Actinomyces oris MG-1 induces pyroptosis in dTHP-1 cells via TLR2, but the process is not solely dependent on TLR2.

Resveratrol is an inhibitory polyphenol of epithelial-mesenchymal transition induced by Fusobacterium nucleatum.

OBJECTIVE: Resveratrol is a natural phytoalexin that has anti-inflammatory properties, reverses doxorubicin resistance, and inhibits epithelial-mesenchymal transition (EMT) in many types of cancer cells. Fusobacterium nucleatum is reportedly enriched in oral squamous cell carcinoma (OSCC) tissues compared to adjacent normal tissues, sparking interest in the relationship between F. nucleatum and OSCC. Recently, F. nucleatum was shown to be associated with EMT in OSCC. In the present study, we aimed to investigate the effects of the natural plant compound resveratrol on F. nucleatum-induced EMT in OSCC. DESIGN: F. nucleatum was co-cultured with OSCC cells, with a multiplicity of infection (MOI) of 300:1. Resveratrol was used at a concentration of 10 µM. Cell Counting Kit-8 and wound healing assays were performed to examine the viability and migratory ability of OSCC cells. Subsequently, real-time RT-PCR was performed to investigate the gene expression of EMT-related markers. Western blotting and immunofluorescence analyses were used to further analyze the expression of the epithelial marker E-cadherin and the EMT transcription factor SNAI1. RESULTS: Co-cultivation with F. nucleatum did not significantly enhance cell viability. The co-cultured cells displayed similarities to the positive control of EMT, exhibiting enhanced migration and expression changes in EMT-related markers. SNAI1 was significantly upregulated, whereas E-cadherin, was significantly downregulated. Notably, resveratrol inhibited F. nucleatum-induced cell migration, decreasing the expression of SNAI1. CONCLUSIONS: Resveratrol inhibited F. nucleatum-induced EMT by downregulating SNAI1, which may provide a target for OSCC treatment.

Complete Genome Sequence of Actinomyces oris Strain K20, Isolated from an Oral Apical Lesion.

Actinomyces oris strain K20 was isolated from oral apical lesions. Here, we report the complete circular genome sequence of this strain, obtained by means of hybrid assembly using two next-generation sequencing datasets. The strain has a 3.1-Mb genome with 2,636 coding sequences.

Single-Molecule/Cell Analyses Reveal Principles of Genome-Folding Mechanisms in the Three Domains of Life.

Comparative structural/molecular biology by single-molecule analyses combined with single-cell dissection, mass spectroscopy, and biochemical reconstitution have been powerful tools for elucidating the mechanisms underlying genome DNA folding. All genomes in the three domains of life undergo stepwise folding from DNA to 30-40 nm fibers. Major protein players are histone (Eukarya and Archaea), Alba (Archaea), and HU (Bacteria) for fundamental structural units of the genome. In Euryarchaeota, a major archaeal phylum, either histone or HTa (the bacterial HU homolog) were found to wrap DNA. This finding divides archaea into two groups: those that use DNA-wrapping as the fundamental step in genome folding and those that do not. Archaeal transcription factor-like protein TrmBL2 has been suggested to be involved in genome folding and repression of horizontally acquired genes, similar to bacterial H-NS protein. Evolutionarily divergent SMC proteins contribute to the establishment of higher-order structures. Recent results are presented, including the use of Hi-C technology to reveal that archaeal SMC proteins are involved in higher-order genome folding, and the use of single-molecule tracking to reveal the detailed functions of bacterial and eukaryotic SMC proteins. Here, we highlight the similarities and differences in the DNA-folding mechanisms in the three domains of life.

Interdental Plaque Microbial Community Changes under In Vitro Violet LED Irradiation.

Oral microbiome dysbiosis has important links to human health and disease. Although photodynamic therapy influences microbiome diversity, the specific effect of violet light irradiation remains largely unknown. In this study, we analyzed the effect of violet light-emitting diode (LED) irradiation on interdental plaque microbiota. Interdental plaque was collected from 12 human subjects, exposed to violet LED irradiation, and cultured in a specialized growth medium. Next-generation sequencing of the 16S ribosomal RNA genes revealed that α-diversity decreased, whereas ß-diversity exhibited a continuous change with violet LED irradiation doses. In addition, we identified several operational taxonomic units that exhibited significant shifts during violet LED irradiation. Specifically, violet LED irradiation led to a significant reduction in the relative abundance of Fusobacterium species, but a significant increase in several species of oral bacteria, such as Veillonella and Campylobacter. Our study provides an overview of oral plaque microbiota changes under violet LED irradiation, and highlights the potential of this method for adjusting the balance of the oral microbiome without inducing antibiotic resistance.

3D spheroid culture models for chondrocytes using polyethylene glycol-coated microfabricated chip.

As chondrocytes fail to retain their chondrogenic potential in two-dimensional monolayer cultures, several three-dimensional culture systems have been employed for investigating the physiology and pathophysiology in articular cartilage tissues. In this study, we introduced a polyethylene glycol-coated microfabricated chip that enables spheroid formation from ATDC5 cell line, commonly used as a model for in vitro chondrocyte research. ATDC5 cells cultured in our devices aggregated immediately and generated a single spheroid per well within 24 h. Most cells in spheroids cultured in differentiation medium were viable and the circular shape and smooth surface of the spheroid were maintained up to 14 d in culture. We also detected potent hypoxia conditions, a key factor in chondrogenesis, in whole lesions of ATDC5 spheroids. Expression of chondrogenesis-related genes and type X collagen protein was significantly increased in ATDC5 spheroids grown in differentiation medium, compared with monolayer-cultured ATDC5 cells. We also demonstrated that the differentiation medium-induced Akt protein phosphorylation was upregulated in ATDC5 cells cultured in our spheroid device, suggesting that enhancement of chondrogenic potential in ATDC5 spheroids results from PI3/Akt signaling activation. These results indicated that our spheroid culture system could constitute a high-throughput strategy approach towards elucidating the molecular mechanisms that regulate chondrogenesis.

Different Proteins Mediate Step-Wise Chromosome Architectures in Thermoplasma acidophilum and Pyrobaculum calidifontis.

Archaeal species encode a variety of distinct lineage-specific chromosomal proteins. We have previously shown that in Thermococcus kodakarensis, histone, Alba, and TrmBL2 play distinct roles in chromosome organization. Although our understanding of individual archaeal chromosomal proteins has been advancing, how archaeal chromosomes are folded into higher-order structures and how they are regulated are largely unknown. Here, we investigated the primary and higher-order structures of archaeal chromosomes from different archaeal lineages. Atomic force microscopy of chromosome spreads out of Thermoplasma acidophilum and Pyrobaculum calidifontis cells revealed 10-nm fibers and 30-40-nm globular structures, suggesting the occurrence of higher-order chromosomal folding. Our results also indicated that chromosome compaction occurs toward the stationary phase. Micrococcal nuclease digestion indicated that fundamental structural units of the chromosome exist in T. acidophilum and T. kodakarensis but not in P. calidifontis or Sulfolobus solfataricus. In vitro reconstitution showed that, in T. acidophilum, the bacterial HU protein homolog HTa formed a 6-nm fiber by wrapping DNA, and that Alba was responsible for the formation of the 10-nm fiber by binding along the DNA without wrapping. Remarkably, Alba could form different higher-order complexes with histone or HTa on DNA in vitro. Mass spectrometry detected HTa and Rad50 in the T. acidophilum chromosome but not in other species. A putative transcriptional regulator of the AsnC/Lrp family (Pcal_1183) was detected on the P. calidifontis chromosome, but not on that of other species studied. Putative membrane-associated proteins were detected in the chromosomes of the three archaeal species studied, including T. acidophilum, P. calidifontis, and T. kodakarensis. Collectively, our data show that Archaea use different combinations of proteins to achieve chromosomal architecture and functional regulation.

Inter-site and interpersonal diversity of salivary and tongue microbiomes, and the effect of oral care tablets.

Background: Oral microbiota has been linked to both health and diseases. Specifically, tongue-coating microbiota has been implicated in aspiration pneumonia and halitosis. Approaches altering one's oral microbiota have the potential to improve oral health and prevent diseases. Methods: Here, we designed a study that allows simultaneous monitoring of the salivary and tongue microbiomes during an intervention on the oral microbiota. We applied this study design to evaluate the effect of single-day use of oral care tablets on the oral microbiome of 10 healthy individuals. Tablets with or without actinidin, a protease that reduces biofilm formation in vitro, were tested. Results: Alpha diversity of the tongue microbiome was significantly lower than that of the salivary microbiome, using both the number of observed amplicon sequence variants (254 ± 53 in saliva and 175 ± 37 in tongue; P = 8.9e-7, Kruskal-Wallis test) and Shannon index (6.0 ± 0.4 in saliva and 5.4 ± 0.3 in tongue; P = 2.0e-7, Kruskal-Wallis test). Fusobacterium periodonticum, Saccharibacteria sp. 352, Streptococcus oralis subsp . dentisani, Prevotella melaninogenica, Granulicatella adiacens, Campylobacter concisus, and Haemophilus parainfluenzae were the core operational taxonomic units (OTUs) common to both sites. The salivary and tongue microbiomes of one individual tended to be more similar to one another than to those of other individuals. The tablets did not affect the alpha or beta diversity of the oral microbiome, nor the abundance of specific bacterial species. Conclusions: While the salivary and tongue microbiomes differed significantly in terms of bacterial composition, they showed inter- rather than intra-individual diversity. A one-day usage of oral care tablets did not alter the salivary or tongue microbiomes of healthy adults. Whether the use of oral tablets for a longer period on healthy people or people with greater tongue coating accumulation shifts their oral microbiome needs to be investigated.

Nitric Oxide Donor Modulates a Multispecies Oral Bacterial Community-An In Vitro Study.

The deterioration of human oral microbiota is known to not only cause oral diseases but also to affect systemic health. Various environmental factors are thought to influence the disruption and restoration of the oral ecosystem. In this study, we focused on the effect of nitric oxide (NO) produced by denitrification and NO synthase enzymes on dental plaque microbiota. Interdental plaques collected from 10 subjects were exposed to NO donor sodium nitroprusside (SNP) and then cultured in a specialized growth medium. Depending on the concentration of exposed SNP, a decrease in α-diversity and a continuous change in ß-diversity in the dental plaque community were shown by sequencing bacterial 16S rRNA genes. We also identified eight operational taxonomic units that were significantly altered by NO exposure. Among them, the exposure of NO donors to Fusobacterium nucleatum cells showed a decrease in survival rate consistent with the results of microbiota analysis. Meanwhile, in addition to NO tolerance, an increase in the tetrazolium salt-reducing activity of Campylobacter concisus cells was confirmed by exposure to SNP. This study provides an overview of how oral plaque microbiota shifts with exposure to NO and may contribute to the development of a method for adjusting the balance of the oral microbiome.

The Biological Effects of Interleukin-17A on Adhesion Molecules Expression and Foam Cell Formation in Atherosclerotic Lesions.

Interleukin-17A (IL-17A), a major effector cytokine secreted by T helper 17 (Th17) cells, is elevated in atherosclerosis lesions. The purpose of this study was to assess the role of IL-17A in the pathogenesis of atherosclerosis. To measure the expression of adhesion molecules, human umbilical vein endothelial cells (HUVECs) and U937 cells were stimulated with IL-17A. Western blot and real-time polymerase chain reaction analyses revealed that intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in HUVECs, and very late antigen-4 (VLA-4), lymphocyte function-associated antigen-1 (LFA-1) and macrophage-1 antigen (MAC-1) expression in U937 cells was upregulated by IL-17A. Furthermore, IL-17A stimulation resulted in mRNA and protein expression of scavenger receptor (LOX-1) in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. Oil Red O also demonstrated that IL-17A enhanced foam cell formation by PMA-activated U937 cells induced by oxidized low-density lipoprotein (ox-LDL), and this enhancement of ox-LDL-induced foam cell formation in IL-17A-treated U937 cells was downregulated by transfection of LOX-1 siRNA. These results indicated that IL-17A induced the expression of adhesion molecules, promoted the adherence of monocytes to vascular endothelial cells. IL-17A also stimulated ox-LDL-induced foam cell formation via upregulation of LOX-1 in activated macrophages. IL-17A may be responsible for the pathogenesis of atherosclerosis by inducing the adhesion of leukocytes to vascular endothelium and foam cell formation.

ß-glucan suppresses cell death of ASC deficient macrophages invaded by periodontopathic bacteria through the caspase-11 pathway.

ß-glucan is an abundant cell wall component of fungi and yeast. Dectin-1, a ß-glucan receptor, plays an important regulatory role in the natural immunity. In the present study, we investigated the effect of ß-glucan on mouse macrophages that had been invaded by the periodontopathic bacterium, Aggregatibacter actinomycetemcomitans. Exposure to curdlan, a type of ß-glucan, suppressed cell death and led to the accumulation of a sub-G1-phase population upon A. actinomycetemcomitans invasion under conditions of constitutive expression of dectin-1. Members of the nucleotide-binding domain leucine-rich repeat-containing (NLR) protein family, such as NLR protein 3 (NLRP3), NLR family apoptosis inhibitory protein (NAIP), and NLR family CARD domain-containing protein 4 (NLRC4), as well as an associated protein, caspase-11, were clearly detected in A. actinomycetemcomitans-invaded control RAW cells (c-RAW cells; negative control). Interestingly, NAIP expression was upregulated and caspase-11 expression was downregulated by dectin-1 activity in A. actinomycetemcomitans-invaded dectin-1 overexpressing RAW 264.7 cells (d-RAW cells), suggesting that dectin-1 in macrophages regulates cell death upon A. actinomycetemcomitans invasion. These results support a potential correlation between dectin-1 and regulation of cell death in macrophages.

Accumulation of hyaluronic acid in stromal cells modulates osteoclast formation by regulation of receptor activator of nuclear factor kappa-B ligand expression.

Hyaluronic acid (HA) has a pivotal role in bone and cartilage metabolism. In this study, we investigated the effect and underlying mechanisms of HA accumulation on the expression of receptor activator of nuclear factor kappa-B ligand (RANKL) induced by 1α,25(OH)2D3 and dexamethasone in stromal cells, which support osteoclastogenesis. Degradation of HA by hyaluronidase (HA'ase) treatment enhanced the expression of RANKL in ST2 cells stimulated with 1α,25(OH)2D3 and dexamethasone. Down-regulation of hyaluronan synthase 2 (HAS2) expression by siRNA also stimulated RANKL expression induced by 1α,25(OH)2D3 and dexamethasone. Results from a cell co-culture system with bone marrow cell showed that 1α,25(OH)2D3 and dexamethasone-induced RANKL expression in HA'ase treated- and HAS2 siRNA transfected-ST2 cells was down-regulated by treatment of cells with high molecular weight HA. In contrast, transforming growth factor-ß1 (TGF-ß1), which stimulates HAS2 expression and HA synthesis, down-regulated RANKL expression induced by 1α,25(OH)2D3 and dexamethasone. Interestingly, knockdown of has2 gene enhanced the expression of vitamin D receptor (VDR) and phosphorylation of signal transducers and activator of transcription 3 (STAT3) in ST2 cells stimulated by 1α,25(OH)2D3 and dexamethasone. These results indicate that accumulation of HA in bone marrow cells may affect RANKL-mediated osteoclast-supporting activity via regulation of VDR and STAT3 signaling pathways.

Docosahexaenoic acid enhances M2 macrophage polarization via the p38 signaling pathway and autophagy.

Macrophages, critical modulators of the immune response, polarize into various phenotypes, including M1 and M2. M1 macrophages are typically activated by lipopolysaccharide and produce proinflammatory cytokines. Conversely, M2 macrophages are activated by stimulation with interleukin 4 (IL)-4 and promote tissue remodeling and anti-inflammatory reactions. Recently, polyunsaturated fatty acids (PUFAs) have been shown to play important roles in the regulation of inflammation. Docosahexaenoic acid (DHA), a PUFA, has anti-inflammatory effects on chronic inflammatory disease, but its role in macrophage polarization remains unclear. In this study, we clarified the effects of DHA on macrophage polarization using U937 cells. Treatment with DHA resulted in upregulation of M2 macrophage markers and increased secretion of anti-inflammatory cytokines by U937 cells. IL-4, but not DHA, triggered phosphorylation of signal transducer and activator of transcription 6 (STAT6). DHA enhanced the expression of krüppel-like factor-4 (KLF4), a transcription factor involved in the regulation of macrophage polarization and increased the phosphorylation of p38 mitogen-activated protein kinase (MAPK). A selective inhibitor of p38 MAPK downregulated the expression of CD206 in DHA-treated U937 cells. Moreover, inhibitors of autophagy suppressed the phosphorylation of p38 MAPK and the expression of CD206 in DHA-treated U937 cells. Expression of microtubule-associated protein light chain 3-II, which is involved in autophagosome formation, was enhanced in DHA-treated U937 cells. Taken together, these results indicated that DHA enhanced the expression of M2 macrophage markers through the p38 MAPK signaling pathway and autophagy, suggesting that DHA regulates M2 macrophage polarization and plays an important role in innate immunity.

Anticancer effect of novel platinum nanocomposite beads on oral squamous cell carcinoma cells.

Nanoparticles are used in industry and medicine, because of their physiochemical properties, such as size, charge, large surface area and surface reactivity. Recently, metal nanoparticles were reported to show cell toxicity on cancer cells. In this study, we focused novel platinum nanoparticles-conjugated latex beads (P2VPs), platinum nanocomposite (PtNCP) beads, and investigated the possibility to incorporate novel anti-cancer effect of these combined nanoparticles. Oral squamous cell carcinoma cell lines, HSC-3-M3 cells were injected subcutaneously into the back of nude mice to produce a xenograft model. PtNCP beads were injected locally and examined by measuring tumor volume and comparing pathological histology. PtNCP beads treatment suppressed tumor growth and identified increasing pathological necrotic areas, in vivo. PtNCP beads inhibited the cell viability of HSC-3-M3 cells in dose-dependent manner and induced the cytotoxicity with extracellular LDH value, in vitro. Furthermore, SEM images were morphologically observed in PtNCP beads-treated HSC-3-M3 cells. The aggregation of the PtNCP beads on the cell membrane, the destructions of the cell membrane and globular structures were observed in the SEM image. Our results indicated that a potential anti-cancer effect of the PtNCP beads, suggesting the possibility as a therapeutic tool for cancer cell-targeted therapy. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2281-2287, 2019.

Ameloblastin attenuates RANKL-mediated osteoclastogenesis by suppressing activation of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1).

Ameloblastin (Ambn) is an extracellular matrix protein and member of the family of enamel-related gene products. Like amelogenin, Ambn is mainly associated with tooth development, especially biomineralization of enamel. Previous studies have shown reductions in the skeletal dimensions of Ambn-deficient mice, suggesting that the protein also has effects on the differentiation of osteoblasts and/or osteoclasts. However, the specific pathways used by Ambn to influence osteoclast differentiation have yet to be identified. In the present study, two cellular models, one based on bone marrow cells and another on RAW264.7 cells, were used to examine the effects of Ambn on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis. The results showed that Ambn suppresses osteoclast differentiation, cytoskeletal organization, and osteoclast function by the downregulation of the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts, actin ring formation, and areas of pit resorption. The expression of the osteoclast-specific genes TRAP, MMP9, cathepsin K, and osteoclast stimulatory transmembrane protein (OC-STAMP) was abolished in the presence of Ambn, while that of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), the master regulatory factor of osteoclastogenesis, was also attenuated by the downregulation of c-Fos expression. In Ambn-induced RAW264.7 cells, phosphorylation of cAMP-response element-binding protein (CREB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK), but not extracellular signal-regulated kinase 1/2 (ERK1/2), was reduced. Calcium oscillation was also decreased in the presence of Ambn, suggesting its involvement in both RANKL-induced osteoclastogenesis and costimulatory signaling. B-lymphocyte-induced maturation protein-1 (Blimp1), a transcriptional repressor of negative regulators of osteoclastogenesis, was also downregulated by Ambn, resulting in the elevated expression of v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (MafB), B-cell lymphoma 6 (Bcl6), and interferon regulatory factor-8 (Irf8). Taken together, these findings suggest that Ambn suppresses RANKL-induced osteoclastogenesis by modulating the NFATc1 axis.

Ky-2, a hybrid compound histone deacetylase inhibitor, regulated inflammatory response in LPS-driven human macrophages.

Histone deacetylase has attracted much attention as an epigenetic factor, and the modulation of histone and transcription factor acetylation status is important for regulating gene expression. Moreover, histone deacetylase inhibitors are involved in cellular growth and differentiation. In the present study, we examined the effects of Ky-2, a hybrid-compound HDAC inhibitor, on inflammatory reactions and the polarization of macrophages in vitro. Human monocyte-like THP-1 cells were polarized to macrophage-like cells using phorbol 12-myristate 13-acetate, and then polarized to M1 macrophages with LPS. Ky-2 inhibited HDAC2 expression and enhanced the acetylation of histone H3 in THP-1 cells. It also downregulated the expression of the IL-1ß-encoding gene and the LPS-induced phosphorylation of p38 mitogen-activated protein kinases in THP-1 cells. Moreover, the expression of nod-like receptor protein 3 and cleaved caspase-1 p20 was downregulated in Ky-2-treated THP-1 cells. In contrast, this agent upregulated the expression of IL-1ra in LPS-treated THP-1 cells. These results indicate that Ky-2-treatment downregulates the expression of the inflammatory cytokine, IL-1ß, in LPS-treated THP-1 cells, suggesting that Ky-2 might regulate M1 macrophage polarization through the suppression of inflammatory responses such as NLRP3 inflammasome activation.