The association between trypsin-like protease activity in the oral cavity and kidney function in Japanese workers.

AIM: To evaluate the association between trypsin-like protease (TLP) activity in the oral cavity as an indicator of periodontal health status and kidney function in Japanese workers. MATERIALS AND METHODS: This cross-sectional study included 1117 Japanese workers (mean age = 43.8 years). Tongue-swab TLP activity was quantified as a* value (the redness intensity of the matrix disc of the TLP activity assessment kit; a larger value indicates more intense enzymatic activity in the samples and poorer periodontal health status). Kidney function was assessed using the estimated glomerular filtration rate (eGFR; a lower value indicates poorer kidney function). We performed ordinal logistic regression analyses to assess the association of the a* value with three eGFR categories: ≥90, 60-89 and <60 mL/min/1.73 m2 . RESULTS: The prevalence for each eGFR category was as follows: ≥90 (31.6%), 60-89 (63.8%) and <60 mL/min/1.73 m2 (4.6%). After adjusting for potential confounders, the a* value was found to be significantly associated with reduced kidney function. The multivariable-adjusted odds ratio (95% confidence interval) for reduced kidney function was 1.12 (1.02-1.22) per unit increase in the a* value. CONCLUSIONS: Higher TLP activity was associated with reduced kidney function in Japanese workers.

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.

Controlled cell proliferation and immortalization of human dental pulp stem cells with a doxycycline-inducible expression system.

Human dental pulp stem cells are a potentially useful resource for cell-based therapies and tissue repair in dental and medical applications. However, the primary culture of isolated dental pulp stem cells has notably been limited. A major requirement of an ideal human dental pulp stem cell culture system is the preservation of efficient proliferation and innate stemness over prolonged passaging, while also ensuring ease of handling through standard, user-friendly culture methods. In this study, we have engineered a novel human dental pulp stem cell line, distinguished by the constitutive expression of telomerase reverse transcriptase (TERT), and the conditional expression of the R24C mutant cyclin-dependent kinase 4 (CDK4R24C) and Cyclin D1. We have named this cell line Tet-off K4DT hDPSCs. Furthermore, we have conducted a comprehensive comparative analysis of their biological attributes in relation to a previously immortalized human dental pulp stem cells, hDPSC-K4DT, which were immortalized by the constitutive expression of CDK4R24C, Cyclin D1 and TERT. In Tet-off K4DT cells, the expression of the K4D genes can be precisely suppressed by the inclusion of doxycycline. Remarkably, Tet-off K4DT cells demonstrated an extended cellular lifespan, increased proliferative capacity, and enhanced osteogenic differentiation potential when compared to K4DT cells. Moreover, Tet-off K4DT cells had no observable genomic aberrations and also displayed a sustained expression of stem cell markers even at relatively advanced passages. Taken together, the establishment of this new cell line holds immense promise as powerful experimental tool for both fundamental and applied research involving dental pulp stem cells.

Evaluation of a Novel Immunochromatographic Device for Detecting Porphyromonas gingivalis in Patients with Periodontal Disease.

Porphyromonas gingivalis is the most pathogenic periodontal bacterium in the world. Recently, P. gingivalis has been considered responsible for dysbiosis during the development of periodontitis. This study aimed to evaluate a novel immunochromatographic device using monoclonal antibodies against P. gingivalis in subgingival plaques. A total of 72 patients with chronic periodontitis and 53 periodontally healthy volunteers underwent clinical and microbiological examinations. Subgingival plaque samples were analyzed for the presence of P. gingivalis and compared using real-time polymerase chain reaction (PCR). In the periodontitis group, a significant positive correlation was observed between the test device scores and the real-time PCR results. The specificity, positive predictive value, negative predictive value, and accuracy of the test device for P. gingivalis, as determined by real-time PCR, were 98%, 94%, 89%, and 90%, respectively. There were significant differences in bacterial counts by real-time PCR among the groups with different ranges of device scores. Additionally, there was a significant positive correlation between the device scores for P. gingivalis and periodontal parameters. These results suggest that this novel immunochromatographic device can be effectively used for rapid detection and semi-quantification of P. gingivalis in subgingival plaques.

Ascorbic acid enhances chondrocyte differentiation of ATDC5 by accelerating insulin receptor signaling.

Chondrogenesis is strictly regulated by several factors, including cytokines, hormones, and extracellular matrix proteins. Mouse teratocarcinoma-derived lineage cells, differentiate into chondrocytes in the presence of insulin. Although ascorbic acid promotes chondrogenic differentiation, the detailed regulative mechanisms underlying its role in chondrogenesis remain unclear. Therefore, in this study, we evaluated the effects of ascorbic acid on insulin-induced chondrogenic differentiation of ATDC5 cells and the underlying intracellular signaling. The results revealed that insulin-stimulated collagen deposition, matrix formation, calcification, and expression of chondrogenic differentiation marker genes in ATDC5 cells. This enhancement by insulin was amplified with the addition of ascorbic acid. Molecular analysis revealed that the activation of insulin-induced phosphoinositide 3-kinase (PI3K)/Akt signaling was enhanced in the presence of ascorbic acid. In contrast, Wnt/ß-catenin signaling was suppressed during chondrocyte differentiation via upregulation of the Wnt agonist, secreted Frizzled-related protein 1 (sFRP-1) and 3 (sFRP-3). Notably, ascorbic acid upregulated the expression of insulin receptors and their substrates (IRS-1 and IRS-2). Furthermore, ascorbic acid reversed the suppression of IRS-1 and IRS-2 protein by insulin. These results indicate that ascorbic acid positively regulates the chondrogenic differentiation of ATDC5 cells via enhancement of insulin signaling. Our findings provide a substantial basis for further elucidation of the regulatory mechanisms of chondrocyte differentiation and the pathophysiology of OA, thus aiding in development of effective treatment strategies.

The Mechanism of Interleukin 33-Induced Stimulation of Interleukin 6 in MLO-Y4 Cells.

The differentiation and function of osteocytes are controlled by surrounding cells and mechanical stress; however, the detailed mechanisms are unknown. Recent findings suggest that IL-33 is highly expressed in periodontal tissues in orthodontic tooth movement. The present study aimed to elucidate the effect of IL-33 on the expression of regulatory factors for bone remodeling and their molecular mechanisms in the osteocyte-like cell line MLO-Y4. MLO-Y4 cells were treated with IL-33, and the activation of intracellular signaling molecules and transcriptional factors was determined using Western blot analysis and chromatin immunoprecipitation assay. IL-33 treatment enhanced the expression of IL-6 in MLO-Y4 cells, which was suppressed by the knockdown of the IL-33 receptor ST2L. Additionally, IL-33 treatment induced activation of NF-κB, JNK/AP-1, and p38 MAPK signaling pathways in MLO-Y4 cells. Moreover, pretreatment with specific inhibitors of NF-κB, p38 MAPK, and JNK/AP-1 attenuated the IL-33-induced expression of IL-6. Furthermore, chromatin immunoprecipitation indicated that IL-33 increased c-Jun recruitment to the IL-6 promoter. Overall, these results suggest that IL-33 induces IL-6 expression and regulates osteocyte function via activation of the NF-κB, JNK/AP-1, and p38 MAPK pathways through interaction with ST2L receptors on the plasma membrane.

Sleep duration and severe periodontitis in middle-aged Japanese workers.

AIM: To evaluate the association between sleep duration and severe periodontitis in Japanese workers. MATERIALS AND METHODS: This cross-sectional study included 1130 workers (mean age 43.0 years) who underwent full-mouth periodontal examinations and health check-ups and completed a self-administered questionnaire that included questions on sleep duration. Logistic regression and a restricted cubic spline model were used to analyse the data. RESULTS: Severe periodontitis was identified in 6.3% of the study population. Those with <5, 5-5.9, 6-6.9, 7-7.9, and ≥8 hr of sleep were 6.7%, 17.4%, 40.3%, 26.3%, and 8.9%, respectively. After adjusting for potential confounders, study participants who slept <5 hr were more likely to have severe periodontitis (adjusted odds ratio = 2.64; 95% confidence interval = 1.06-6.60) than those who slept 7-7.9 hr. The spline model, with a reference value of 399 min (the median sleep duration), showed a non-linear association between sleep duration and severe periodontitis, where an increased prevalence of severe periodontitis was observed only among those with a shorter sleep duration. The prevalence of severe periodontitis did not increase with longer sleep duration. CONCLUSIONS: Short sleep duration was associated with severe periodontitis in this cohort of Japanese adults.

Dectin-1-mediated suppression of RANKL-induced osteoclastogenesis by glucan from baker's yeast.

Immunoreceptors expressed on osteoclast precursor cells modify osteoclast differentiation and bone resorption activity. Dectin-1 is a lectin receptor of ß-glucan and is specifically expressed in osteoclast precursor cells. In this study, we evaluated the bioactivity of ß-glucan on receptor activator of nuclear factor-kappa B ligand (RANKL)-induced osteoclastogenesis and observed that glucan from baker's yeast inhibited this process in mouse bone marrow cells and dectin-1-overexpressing RAW264.7 (d-RAW) cells. In conjunction, RANKL-induced nuclear factor of activated T cell c1 expression was suppressed, subsequently downregulating TRAP and Oc-stamp. Additionally, nuclear factor-kappa B activation and the expression of c-fos and Blimp1 were reduced in d-RAW cells. Furthermore, glucan from baker's yeast induced the degradation of Syk protein, essential factor for osteoclastogenesis. These results suggest that glucan from baker's yeast suppresses RANKL-induced osteoclastogenesis and can be applied as a new treatment strategy for bone-related diseases.

Interruption of regular dental visits during the COVID-19 pandemic due to concerns regarding dental visits was associated with periodontitis in Japanese office workers.

OBJECTIVE: To investigate the interrelationships among concerns regarding dental visits, the status of regular dental visits, and periodontal health during the coronavirus disease 2019 (COVID-19) pandemic. BACKGROUND: Continuous oral health care and regular dental visits are important for maintaining periodontal health. Due to the possibility of contracting COVID-19, individuals have been reluctant to visit medical institutions. It is unclear how the periodontal health of the Japanese population has been affected by the interruption of regular dental visits during the COVID-19 pandemic and how concerns regarding dental visits have affected attendance at regular dental visits. METHODS: This study included 199 Japanese office workers in one municipal office at Fukuoka Prefecture, Japan (average age = 42.6 years; age range = 19-77 years; 123 men and 76 women). Periodontitis was defined based on a full-mouth periodontal examination. The status of regular dental visits during the COVID-19 pandemic and concerns regarding dental visits were obtained via questionnaire. We tested the hypothesis that concerns regarding dental visits would indirectly affect periodontal health through the interruption of regular dental visits during the COVID-19 pandemic. We used mediation analysis, in which concerns regarding dental visits (present or absent) were set as the exposure, periodontitis (present or absent) was set as the outcome, and the status of regular dental visits (continued during the COVID-19 pandemic or not) was set as the mediator. RESULTS: Of the 199 study participants, 108 had a habit of attending regular dental visits. Of these, 31 (28.7%) discontinued regular dental visits during the COVID-19 pandemic. Compared to the individuals who continued regular dental visits, those who discontinued regular dental visits had a higher prevalence of periodontitis (49.4% vs 77.4%, p < 0.05) and concerns regarding dental visits (22.1% vs 64.5%, p < 0.05). Discontinuing regular dental visits significantly mediated the association between concerns regarding dental visits and periodontitis (natural indirect effect: odds ratio = 1.68, 95% confidence interval = 1.02-2.79, proportion mediated = 64.3%). CONCLUSION: The study results showed that individuals who discontinued regular dental visits during the COVID-19 pandemic due to concerns regarding dental visits had relatively poor periodontal health.

Biological Effects of ß-Glucans on Osteoclastogenesis.

Although the anti-tumor and anti-infective properties of ß-glucans have been well-discussed, their role in bone metabolism has not been reviewed so far. This review discusses the biological effects of ß-glucans on bone metabolisms, especially on bone-resorbing osteoclasts, which are differentiated from hematopoietic precursors. Multiple immunoreceptors that can recognize ß-glucans were reported to be expressed in osteoclast precursors. Coordinated co-stimulatory signals mediated by these immunoreceptors are important for the regulation of osteoclastogenesis and bone remodeling. Curdlan from the bacterium Alcaligenes faecalis negatively regulates osteoclast differentiation in vitro by affecting both the osteoclast precursors and osteoclast-supporting cells. We also showed that laminarin, lichenan, and glucan from baker's yeast, as well as ß-1,3-glucan from Euglema gracilisas, inhibit the osteoclast formation in bone marrow cells. Consistent with these findings, systemic and local administration of ß-glucan derived from Aureobasidium pullulans and Saccharomyces cerevisiae suppressed bone resorption in vivo. However, zymosan derived from S. cerevisiae stimulated the bone resorption activity and is widely used to induce arthritis in animal models. Additional research concerning the relationship between the molecular structure of ß-glucan and its effect on osteoclastic bone resorption will be beneficial for the development of novel treatment strategies for bone-related diseases.

Rhamnolipids and surfactin inhibit the growth or formation of oral bacterial biofilm.

BACKGROUND: Bacteria survive in various environments by forming biofilms. Bacterial biofilms often cause significant problems to medical instruments and industrial processes. Techniques to inhibit biofilm formation are essential and have wide applications. In this study, we evaluated the ability of two types of biosurfactants (rhamnolipids and surfactin) to inhibit growth and biofilm formation ability of oral pathogenic bacteria such as Aggregatibacter actinomycetemcomitans, Streptococcus mutans, and Streptococcus sanguinis. RESULTS: Rhamnolipids inhibited the growth and biofilm formation ability of all examined oral bacteria. Surfactin showed effective inhibition against S. sanguinis ATCC10556, but lower effects toward A. actinomycetemcomitans Y4 and S. mutans UA159. To corroborate these results, biofilms were observed by scanning electron microscopy (SEM) and confocal microscopy. The observations were largely in concordance with the biofilm assay results. We also attempted to determine the step in the biofilm formation process that was inhibited by biosurfactants. The results clearly demonstrated that rhamnolipids inhibit biofilm formation after the initiation process, however, they do not affect attachment or maturation. CONCLUSIONS: Rhamnolipids inhibit oral bacterial growth and biofilm formation by A. actinomycetemcomitans Y4, and may serve as novel oral drug against localized invasive periodontitis.

Comparison of dental plaque reduction after use of electric toothbrushes with and without QLF-D-applied plaque visualization: a 1-week randomized controlled trial.

BACKGROUND: To evaluate the efficacy of a newly developed electric toothbrush in reducing dental plaque via a quantitative light-induced fluorescence-digital (QLF-D)-applied visualisation system in the brush head. METHODS: Participants included 20 adults aged 19 to 28 years. Participants were randomly assigned either (i) an electric toothbrush with a monitor to visualise red-fluorescent dental plaque via a camera built into the brush head (monitor usage group, n = 10) or (ii) an electric toothbrush without a monitor (monitor-non-use group, n = 10). The amount of dental plaque was assessed by personal hygiene performance (PHP) at baseline and 1 week later. RESULTS: In the monitor-usage group, PHP score was significantly lower at the 1-week follow-up than at baseline (6 vs 16; range, 0-12 vs 13-21; P = 0.029). This change was not observed in the monitor-non-use group (14 vs 13; range, 6-21 vs 2-26; P = 0.778). After 1 week, the change in PHP scores in the monitor usage group was significantly greater than that in the monitor non-use group (- 10 vs 0; range, - 21 to 9 vs - 8 to 16; P = 0.021). CONCLUSIONS: Our results clearly demonstrate that brushing teeth while looking at a monitor that depicts red-autofluorescent dental plaque via application of QLF-D improved the efficacy of dental-plaque removal relative to brushing teeth without a monitor. TRIAL REGISTRATION: Trial registration number: UMIN000033699. Name of registry: Study on effect of new devise for oral care on dental plaque clearance. Date of registration: 8th September 2018. Status of registration: Completed.

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.

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.

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.

Mechanisms involved in enhancement of osteoclast formation by activin-A.

Several growth factors in bone tissues are reported to be associated with osteoclastogenesis. Activin-A, a member of the transforming growth factor-ß (TGF-ß) family is known to be present in bone tissues and an important regulator in osteoclastogenesis with SMAD-mediated signaling being crucial for inducing osteoclast differentiation. In the present study, we examined the effect and underlying mechanisms of activin-A on osteoclast formation in vitro culture systems. Activin-A enhanced osteoclast formation in both mouse bone marrow cells and monocyte/macrophage cell line RAW 264.7 cells induced by receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) and/or macrophage stimulating factor (M-CSF). We also found that activin-A stimulated bone resorption and actin ring formation induced by RANKL and/or M-CSF. Furthermore, activin-A enhanced RANKL-induced expression of nuclear factor of activated T cell cytoplasmic 1 (NFATc1), a key regulator of osteoclastogenesis, thereby increasing osteoclastogenesis-related marker gene expression, including tartrate-resistant acid phosphatase, osteoclast stimulatory transmembrane protein, and cathepsin K. Blockage of receptor binding by follistatin, an activing-binding protein suppressed the activin-A-mediated stimulation of NFATc1. In addition, activin-A increased RANKL-induced c-fos expression without significantly affecting the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathway. Pre-treatment of the cells with a specific inhibitor of SMAD2/3 attenuated the activin-A-induced expression of NFATc1 and co-immunoprecipitation assay revealed that treatment with activin-A increased physical interaction of phosphorylated-c-fos and phosphorylated-SMAD2 protein induced by RANKL. These results suggest that activin-A enhances RANKL-induced osteoclast formation mediated by interaction of c-fos and smad2/3.

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.

Mechanisms involved in enhancement of matrix metalloproteinase-9 expression in macrophages by interleukin-33.

Endothelial transmigration of macrophages is accomplished by matrix metalloproteinase (MMP)-induced degradation of the basement membrane and extracellular matrix components. Macrophages upregulate MMP-9 expression and secretion upon immunological challenges and require its activity for migration during inflammatory responses. Interleukin (IL)-33 is a recently discovered pro-inflammatory cytokine that belongs to the IL-1 family. The aim of this study was to elucidate the mechanisms underlying IL-33-induced MMP-9 expression in the mouse monocyte/macrophage line RAW264.7. IL-33 increased MMP-9 mRNA and protein expression in RAW264.7 cells. Blockage of IL-33-IL-33 receptor (ST2L) binding suppressed IL-33-mediated induction of MMP-9. IL-33 induced phosphorylation and nuclear translocation of extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor-kappa B (NF-κB). Chromatin immunoprecipitation indicated that IL-33 increased c-fos recruitment to the MMP-9 promoter. Reporter assay findings also revealed that IL-33 stimulated the transcriptional activity of activator protein 1 (AP-1). Pre-treatment of the cells with a specific inhibitor of ERK1/2 and NF-κB attenuated the IL-33-induced activation of AP-1 subunits, transcriptional activity of AP-1, and expression of MMP-9. We also demonstrated that ERK-dependent activation of cAMP response element binding protein (CREB) is a key step for AP-1 activation by IL-33. These results indicate an essential role of ERK/CREB and NF-κB cascades in the induction of MMP-9 in monocytes/macrophages through AP-1 activation.

Ameloblastin Upregulates Inflammatory Response Through Induction of IL-1ß in Human Macrophages.

Ameloblastin (AMBN) is an enamel matrix protein that has various biological functions such as healing dental pulp and repairing bone fractures. In the present study, we clarified the effect of AMBN on the expression of an inflammatory cytokine, interleukin-1ß (IL-1ß) in lipopolysaccharide (LPS)-treated human macrophages. Real-time RT-PCR analysis showed that LPS treatment upregulated expression of the IL-1ß gene in U937 cells. Interestingly, AMBN significantly enhanced IL-1ß gene expression in LPS-treated U937 cells as well as the secretion of mature IL-1ß into culture supernatants by these cells. AMBN also activated caspase-1 p10 expression in LPS-treated U937 cells. Pretreatment with a caspase-1 inhibitor, Z-YVAD-FMK, downregulated the mature IL-1ß expression enhanced by AMBN treatment in LPS-treated U937 cells. A co-immunoprecipitation assay showed that treatment with LPS and AMBN upregulated toll-like receptor 4 (TLR4) and myeloid differentiation primary response gene 88 (MyD88) interactions, but there was no significant difference compared with LPS treatment alone in U937 cells. In contrast, western blot analysis revealed that AMBN remarkably prolonged the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), a member of the mitogen-activated protein kinase (MAPK) family. An ERK1/2-selective inhibitor, U0126, suppressed expression of the IL-1ß gene as well as its protein expression in U937 cells treated with LPS and AMBN. Taken together, these results indicate that AMBN enhances IL-1ß production in LPS-treated U937 cells through ERK1/2 phosphorylation and caspase-1 activation, suggesting that AMBN upregulates the inflammatory response in human macrophages and plays an important role in innate immunity. J. Cell. Biochem. 118: 3308-3317, 2017. © 2017 Wiley Periodicals, Inc.

Ameloblastin and enamelin prevent osteoclast formation by suppressing RANKL expression via MAPK signaling pathway.

Ameloblastin (Ambn) and enamelin (Enam) play a pivotal role in enamel mineralization. Previous studies have demonstrated that these enamel-related gene products also affect bone growth and remodeling; however, the underlying mechanisms have not been elucidated. In the present study, we examined the effects of Ambn and Enam on the receptor activator of nuclear factor kappa-B ligand (RANKL) expression induced with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and dexamethasone (DEX) on mouse bone marrow stromal cell line ST2 cells. We then verified the effect of Ambn and Enam on osteoclastogenesis. We found that pretreatment with recombinant human Ambn (rhAmbn) and recombinant human Enam (rhEnam) remarkably suppressed RANKL mRNA and protein expression induced with 1,25(OH)2D3 and DEX. Interestingly, rhAmbn and rhEnam attenuated the phosphorylation of mitogen-activated protein kinases (MAPK), including ERK1/2, JNK, and p38 in ST2 cells stimulated with 1,25(OH)2D3 and DEX. Moreover, pretreatment with specific inhibitors of ERK1/2 and p38, but not JNK, blocked RANKL mRNA and protein expression. Cell co-culture results showed that rhAmbn and rhEnam downregulated mouse bone marrow cell differentiation into osteoclasts induced with 1,25(OH)2D3 and DEX-stimulated ST2 cells. These results suggest that Ambn and Enam may indirectly suppress RANKL-induced osteoclastogenesis via downregulation of p38 and ERK1/2 MAPK signaling pathways in bone marrow stromal cells.