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Edible mushrooms are known to exert anti-inflammatory effects. In this study, the effects of ethanol extracts from edible mushrooms, such as Hericium erinaceus, and other edible mushrooms on inflammatory responses were investigated. Experiments were conducted using the inflammatory responses of human monocytes induced by lipopolysaccharide (LPS), a bacterial component, that provokes inflammation. Notably, we demonstrated that LPS mixed with ethanol and hot water extracts derived from edible mushrooms attenuated the production of inflammatory cytokines, such as interleukin (IL)-1β, -6, and -8, induced by LPS in human monocytic cell cultures. Moreover, we found that the ethanol extract of H. erinaceus contained ergosterol, which attenuated IL-8 production in LPS-stimulated cells. Subsequent component analysis of the ethanol extract of H. erinaceus revealed that ergosterol binds to lipid A to attenuate LPS-induced inflammation. Together, our findings suggest that ergosterol in ethanol extracts from edible mushrooms can prevent the induction of inflammation by binding to LPS.

Accumulating evidence suggests that specific non-coding RNAs exist in many types of malignant tissues, and are involved in cancer invasion and metastasis. However, little is known about the precise roles of non-coding RNAs in squamous cell carcinoma (SQCC) invasion and migration. Recently, the dentin matrix protein-1 (DMP-1) gene locus was identified as a transcriptionally active site in squamous cell carcinoma (SQCC) tissue and cells. However, it is unclear whether RNA associated with cell migration exist at the DMP-1 gene locus in SQCC cells. We identified a novel promoter-associated non-coding RNA in the antisense strand of DMP-1 gene locus, promoter-associated non-coding RNA (panRNA)-DMP-1, by the RACE method in SQCC cells and tissues, and characterized the functions of panRNA-DMP-1 in EGF-driven SQCC cell migration. The inhibition of endogenous panRNA-DMP-1 expression by specific siRNAs and exogenous over-expression of panRNA-DMP-1 resulted in increased and suppressed cellular migration toward EGF in SQCC cells, respectively, and nuclear expression of panRNA-DMP-1 was induced by EGF stimulation. Mechanistically, suppression of panRNA-DMP-1 expression increased EGFR nuclear localization upon EGF treatment and nuclear panRNA-DMP-1 physically interacted with EGFR, which was confirmed by RNA immunoprecipitation assay using a bacteriophage-delivered PP7 RNA labeling system. Furthermore, co-immunoprecipitation assay revealed that suppression of panRNA-DMP-1 stabilized EGFR interaction with STAT3, a known co-transcription factors of EGFR, to induce migratory properties in many cancer cells. Based on these findings, panRNA-DMP-1 is an EGFR-associating RNA that inhibits the EGF-induced migratory properties of SQCC possibly by regulating EGFR nuclear localization and EGFR binding to STAT3.

Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is constitutively produced by endothelial cells and plays a vital role in maintaining vascular homeostasis. Chronic periodontitis is an inflammatory disease characterized by bleeding of periodontal tissues that support the tooth. In this study, we aimed to determine the role of PAI-1 produced by endothelial cells in response to infections caused by the primary periodontal pathogen Porphyromonas gingivalis. We demonstrated that P. gingivalis infection resulted in significantly reduced PAI-1 levels in human endothelial cells. This reduction in PAI-1 levels could be attributed to the proteolysis of PAI-1 by P. gingivalis proteinases, especially lysine-specific gingipain-K (Kgp). We demonstrated the roles of these degradative enzymes in the endothelial cells using a Kgp-specific inhibitor and P. gingivalis gingipain-null mutants, in which the lack of the proteinases resulted in the absence of PAI-1 degradation. The degradation of PAI-1 by P. gingivalis induced a delayed wound healing response in endothelial cell layers via the low-density lipoprotein receptor-related protein. Our results collectively suggested that the proteolysis of PAI-1 in endothelial cells by gingipains of P. gingivalis might lead to the deregulation of endothelial homeostasis, thereby contributing to the permeabilization and dysfunction of the vascular endothelial barrier.

The periodontal ligament is a soft connective tissue embedded between the alveolar bone and cementum, the surface hard tissue of teeth. Periodontal ligament fibroblasts (PDLF) actively express osteo/cementogenic genes, which contribute to periodontal tissue homeostasis. However, the key factors maintaining the osteo/cementogenic abilities of PDLF remain unclear. We herein demonstrated that PPARγ was expressed by in vivo periodontal ligament tissue and its distribution pattern correlated with alkaline phosphate enzyme activity. The knockdown of PPARγ markedly reduced the osteo/cementogenic abilities of PDLF in vitro, whereas PPARγ agonists exerted the opposite effects. PPARγ was required to maintain the acetylation status of H3K9 and H3K27, active chromatin markers, and the supplementation of acetyl-CoA, a donor of histone acetylation, restored PPARγ knockdown-induced decreases in the osteo/cementogenic abilities of PDLF. An RNA-seq/ChIP-seq combined analysis identified four osteogenic transcripts, RUNX2, SULF2, RCAN2, and RGMA, in the PPARγ-dependent active chromatin region marked by H3K27ac. Furthermore, RUNX2-binding sites were selectively enriched in the PPARγ-dependent active chromatin region. Collectively, these results identified PPARγ as the key transcriptional factor maintaining the osteo/cementogenic abilities of PDLF and revealed that global H3K27ac modifications play a role in the comprehensive osteo/cementogenic transcriptional alterations mediated by PPARγ.

In the oral mechanical environment, periodontal ligament cells (PDL cells) contribute to maintaining periodontal tissue homeostasis. Recent studies showed that exosomes, which are small vesicles secreted by various types of cells, play a pivotal role in cell-to-cell communication in biological processes. We examined the secretion of exosomes from PDL cells stimulated with cyclic stretch and their role in the inflammatory response of macrophages using the human macrophage cell line THP-1 and human primary monocytes/macrophages. We prepared supernatants from human PDL cells (PDL-sup) stimulated with cyclic stretch. The treatment of macrophages with PDL-sup, but not PDL-sup from unstimulated PDL cells, inhibited the production of IL-1β in LPS/nigericin-stimulated macrophages. The pretreatment of PDL cells with GW4869, an inhibitor of exosome secretion, or siRNA for Rab27B, which controls exosome secretion, abrogated the inhibitory effects of PDL-sup. A transmission electron microscopy analysis demonstrated the existence of exosomes with diameters ranging between 30 and 100 nm in PDL-sup, suggesting that exosomes in PDL-sup contribute to this inhibition. An immunofluorescence microscopy analysis revealed that exosomes labeled with PKH67, a fluorescent dye, were incorporated by macrophages as early as 2 h after the addition of exosomes. Purified exosomes inhibited IL-1β production in LPS/nigericin-stimulated macrophages and the nuclear translocation of NF-κB as well as NF-κB p65 DNA-binding activity in LPS-stimulated macrophages, suggesting that exosomes suppress IL-1β production by inhibiting the NF-κB signaling pathway. Our results indicate that PDL cells in mechanical environments contribute to the maintenance of periodontal immune/inflammatory homeostasis by releasing exosomes.

Background and Objective Dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts and periodontal ligament cells, interplay with Hertwig's epithelial root sheath (HERS) cells during tooth root formation, in which HERS is considered to have an inductive role in initiating cementogenesis by epithelial–mesenchymal interaction. However, the specific mechanisms controlling the cementoblast/osteoblast differentiation of dental follicle cells are not fully understood. Canonical Wnt signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions. This study examined the possible expression of canonical Wnt ligand in HERS and the role of Wnt signaling during the cementoblast/osteoblast differentiation of dental follicle cells. Material and Methods The expression of Wnt3a, a representative canonical Wnt ligand, in HERS was assessed by immunohistochemistry. The differentiation and function of immortalized murine dental follicle cells were evaluated by measuring alkaline phosphatase (ALP, Alpl) activity and osteogenic gene expression. Results We identified the expression of Wnt3a in HERS during mouse tooth root development by immunohistochemistry as well as in cultured human epithelial rest cells of Malassez by real-time polymerase chain reaction, while no expression of Wnt3a was detected in cultured dental mesenchymal cells. Exposure of immortalized murine dental follicle cells to Wnt3a-induced ALP activity as well as expression of the Alpl gene. Pretreatment of cells with Dickkopf-1, a potent canonical Wnt antagonist, markedly attenuated the effect of Wnt3a on ALP expression. Furthermore, Wnt3a induced transcriptional activity of runt-related transcription factor 2 (Runx2) and expression of osterix at gene and/or protein levels. Treatment with osterix–small interfering RNA significantly inhibited Wnt3a-induced ALP expression at gene and protein levels. Conclusion These findings suggest that HERS has a potential role in stimulating cementoblast/osteoblast differentiation of dental follicle cells via the Wnt/β-catenin signaling pathway.

In March 2011, the Great East Japan Earthquake (GEJE), which was followed by a devastating tsunami, destroyed the societal and the public hygiene systems in Japanese coastal areas. Insomnia, the greatest issue among disaster victims, has detrimental effects on both physical and psychological health. Periodontitis causes chronic discomfort and inflammation, and little is known about its impact on insomnia. Three months after the earthquake, a health panel survey was conducted over four surveys, till September 2013, in which information regarding 8,015 adults was collected and used. In addition to the heath-related questionnaire, other variables including subjective symptoms of oral diseases were recorded, and the Athens Insomnia Scale was used to evaluate the severity of insomnia. The association between insomnia and periodontal disease was examined using multilevel logistic models on the panel data, after adjusting for sex, age, economic status, comorbidities, body mass index, post-traumatic stress reactions, habitual smoking and alcohol drinking, and the Kessler Psychological Distress Scale score. In addition to the higher prevalence of insomnia among GEJE victims, significant association was revealed between insomnia and gum problems (OR = 2.16, 95% CI = 1.43-3.26), and difficulty chewing (OR = 2.22, 95% CI = 1.40-3.51), after adjusting for all covariates. The present study revealed significant association between insomnia and periodontal disease among GEJE victims. This indicated that together, integrated oral health care for disaster victims would contribute not only to prevention of oral infectious diseases, but may also help alleviate other problems caused by these harmful events.

Periodontal ligament cells play important roles in the homeostasis of periodontal tissue by mechanical stress derived from mastication, such as tension, compression, fluid shear, and hydrostatic force. In the present study, we showed that cyclic tensile force increased the gene expression level of bone morphogenetic protein (BMP)-2, a crucial regulator of mineralization, in human periodontal ligament cells using real-time PCR. Signaling inhibitors, PD98059/U0126 (extracellular signal-regulated kinase (ERK) inhibitors) and SB203580/SB202190 (p38 inhibitors), revealed that tensile force-mediated BMP-2 expression was dependent on activation of the ERK1/2 and p38 mitogen-activated protein (MAP) kinase pathways. Cyclic tensile force also induced cyclooxygenase-2 (COX-2) gene expression in a manner dependent on ERK1/2 and p38 MAP kinase pathways, and induced prostaglandin E2 (PGE2) biosynthesis. NS-398, a COX-2 inhibitor, significantly reduced tensile force-mediated BMP-2 expression, indicating that PGE2 synthesized by COX-2 may be involved in the BMP-2 induction. The inhibitory effect of NS-398 was completely restored by the addition of exogenous PGE2. However, stimulation with PGE2 alone in the absence of tensile force had no effect on the BMP-2 induction, indicating that some critical molecule(s) other than COX-2/PGE2 may be required for cyclic tensile force-mediated BMP-2 induction. Collectively, the results indicate that cyclic tensile force activates ERK1/2 and p38 MAP kinase signaling pathways, and induces COX-2 expression, which is responsible for the sequential PGE2 biosynthesis and release, and furthermore, mediates the increase in BMP-2 expression at the transcriptional level.

Extracellular adenosine triphosphate (ATP) is sequentially dephosphorylated by two ectoenzymes: CD39/nucleotidase triphosphate dephosphorylase (ENTPD) and CD73/5'-ectonucleotidase (5'-NT). Adenosine, its notable metabolite, may elicit potent anti-inflammatory responses. We examined whether the CD39-adenosinergic axis may exist in gingival fibroblasts and have an effect on the expression of matrix metalloproteinase (MMP)-1, the excess production of which leads to pathological matrix degradation. We showed that transcripts of CD39, CD73, and adenosine receptors A1, A2a, and A2b, but not A3, were expressed by human gingival fibroblasts by RT-PCR. We also identified the expression of CD39 in fibroblastic cells in rat gingiva by immunohistochemistry. ATP inhibited the expression of MMP-1 triggered by interleukin-1 at gene and protein levels. However, ATP-γS, a stable ATP analog, did not. The ATP-mediated MMP-1 inhibition was restored in the presence of POM-1, a specific ENTPD inhibitor, suggesting that CD39/ENTPD was involved in the MMP-1 inhibition. ATP metabolites including adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), and adenosine inhibited MMP-1 expression, but ADP-βS, a stable ADP, did not, suggesting that adenosine converted from ATP by the action of CD39/ENTPD and CD73/5'-NT may contribute to MMP-1 inhibition. Adenosine-mediated MMP-1 inhibition was restored in the presence of H89, a protein kinase A (PKA) inhibitor. Conversely, forskolin, an enhancer of intracellular cAMP, mimicked the effect of adenosine, suggesting that the cAMP/PKA signaling pathway is involved in adenosine-mediated MMP-1 inhibition. The present findings suggest the existence of an endogenous anti-tissue destructive mechanism in gingival tissue via the CD39-adenosinergic axis.

Objective Bone morphogenetic protein (BMP)-2 promotes the osteoblastic differentiation of human periodontal ligament (PDL) cells, which play a pivotal role in periodontal regeneration. Recently, nano-sized hydroxyapatite (nano-HA) has been highlighted due to its advantageous features over micro-sized materials. Design and results We investigated the effect of nano-HA on BMP-2 expression in human PDL cells. Real time PCR analysis revealed that the expression of BMP-2 increased upon stimulation with nano-HA in dose- and time-dependent manners. An immunofluorescence assay demonstrated the synthesis of BMP-2 proteins. Concentrations of Ca 2+ as well as phosphate (Pi) in culture supernatants were unchanged, suggesting that nano-HA functioned as a nanoparticle rather than as a possible source for releasing Ca 2+ and/or Pi extracellularly, which were shown to also enhance the expression of BMP-2. Nano-HA-induced BMP-2 expression was dependent on the p38 MAP kinase pathway because increases in BMP-2 expression were inhibited by treatment with SB203580, a p38 inhibitor, and phosphorylation of p38 was detected by Western blotting. Conclusions This novel mechanism of nano-HA will be important for the rational design of future periodontal regeneration.

Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent pathway.

Extracellular phosphate (Pi) is known to play a key role in promoting osteoblastic differentiation by altering gene expression and cellular function. Importantly, it may be possible to use this knowledge as a means to deliver Pi to local sites to regenerate mineralized tissues associated with the oral cavity. Therefore, we determined the ability of Pi to regulate differentiation of pulp cells toward an odontoblast phenotype and further determined if this was in part due to an increase in the expression of bone morphogenetic protein (BMP)-2, a crucial regulator of mineralization. Results showed that Pi increased BMP-2 expression at both mRNA and protein level and BMP-2 promoter activity. Signaling inhibitors revealed that increased BMP-2 expression was dependent on cAMP/protein kinase A but not the protein kinase C signaling pathway. Treatment with 8-Br-cAMP, a cell-permeable analog of cAMP, enhanced Pi-mediated BMP-2 expression, but treatment with 8-Br-cAMP alone did not increase BMP-2, suggesting that cAMP is indispensable but not sufficient for Pi-mediated BMP-2 expression. Pi activated ERK1/2, and treatment with PD98059, an ERK1/2 inhibitor, suppressed Pi-mediated BMP-2 increase, indicating a requirement for activation of ERK1/2. ERK1/2 pathway may operate independently of cAMP-dependent signaling because MDL12,330A, an adenylate cyclase inhibitor, did not inhibit phosphorylation of ERK1/2 in response to Pi. Pulp cells expressed the sodium-dependent Pi transporter (NaPi) III type, but not NaPi-I type or NaPi-II type. Pi-mediated BMP-2 increase was inhibited in the presence of phosphonoformic acid, an inhibitor not only of NaPi transport but also of crystal nucleation. Furthermore, a similar inhibition was observed in the presence of pyrophosphate, a mineralization inhibitor. These findings demonstrate, for the first time, that Pi regulates BMP-2 expression via cAMP/protein kinase A and ERK1/2 pathways in human dental pulp cells.

Cementoblasts, tooth root lining cells, are responsible for laying down cementum on the root surface, a process that is indispensable for establishing a functional periodontal ligament. Cementoblasts share phenotypical features with osteoblasts. Elevated levels of extracellular Ca(2+) have been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of extracellular Ca(2+) signaling in cementogenesis has not been examined. Using RT-PCR, we found that elevated levels of extracellular Ca(2+) increase fibroblast growth factor (FGF)-2 gene expression with a peak at 6h. Pretreatment with a protein kinase A (PKA) inhibitor, H89, or an adenylate cyclase inhibitor, MDL-12,330A, inhibited Ca(2+)-stimulated Fgf-2 expression. In contrast, pretreatment with the protein kinase C (PKC) inhibitor GF-109203X or the phospholipase C (PLC) inhibitor U73122 did not affect the expression of Fgf-2 transcripts, suggesting that the increase in Fgf-2 expression was dependent on the PKA but not the PLC/PKC signaling pathway. Treatment with an activator of adenylate cyclase, forskolin, or a cell-permeable analog of cAMP, 8-Br-cAMP, enhanced Ca(2+)-stimulated Fgf-2 expression, but a single treatment with forskolin or 8-Br-cAMP did not, suggesting that cAMP generation is indispensable but not sufficient for Ca(2+)-stimulated FGF2 expression. Next, we examined the cation specificity of the putative receptor and showed that treatment with trivalent/divalent inorganic ions, Ca(2+), Gd(3+), Sr(2+), or Al(3+), caused a dose-dependent increase in Fgf-2 mRNA levels in a cAMP-dependent fashion, whereas Mg(2+) and the organic ions neomycin and spermine had no effect on Fgf-2 gene expression levels. These findings suggest that an extracellular Ca(2+)-sensing mechanism is present in cementoblasts and its activation leads to FGF-2 stimulation in a cAMP/PKA dependent fashion. Understanding the pathway regulating key genes involved in modulating the regeneration of oral tissues will assist in designing regenerative therapies based on reliable biological principles.

Dental pulp cells, which have been shown to share phenotypical features with osteoblasts, are capable of differentiating into odontoblast-like cells and generating a dentin-like mineral structure. Elevated extracellular Ca(2+)Ca(2+)(o) has been implicated in osteogenesis by stimulating the proliferation and differentiation of osteoblasts; however, the role of Ca(2+)(o) signaling in odontogenesis remains unclear. We found that elevated Ca(2+)(o) increases bone morphogenetic protein (BMP)-2 gene expression in human dental pulp cells. The increase was modulated not only at a transcriptional level but also at a post-transcriptional level, because treatment with Ca(2+) increased the stability of BMP-2 mRNA in the presence of actinomycin D, an inhibitor of transcription. A similar increase in BMP-2 mRNA level was observed in other human mesenchymal cells from oral tissue; periodontal ligament cells and gingival fibroblasts. However, the latter cells exhibited considerably lower expression of BMP-2 mRNA compared with dental pulp cells and periodontal ligament cells. The BMP-2 increase was markedly inhibited by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor, PD98059, and partially inhibited by the L-type Ca(2+) channels inhibitor, nifedipine. However, pretreatment with nifedipine had no effect on ERK1/2 phosphorylation triggered by Ca(2+), suggesting that the Ca(2+) influx from Ca(2+) channels may operate independently of ERK signaling. Dental pulp cells do not express the transcript of Ca(2+)-sensing receptors (CaSR) and only respond slightly to other cations such as Sr(2+) and spermine, suggesting that dental pulp cells respond to Ca(2+)(o) to increase BMP-2 mRNA expression in a manner different from CaSR and rather specific for Ca(2+)(o) among cations.

Human leukocyte elastase, a neutrophil serine protease, is considered to be a potential immunoregulatory protease. Since the PDGF receptor (PDGFR) on periodontal ligament (PDL) cells is a crucial element for various functions, such as wound healing in periodontal tissue, we investigated the effect of elastase on the expression of PDGFR on PDL cells by flow cytometry and Western blotting. We found that PDGFR-α disappeared with an increasing dose of elastase, and PDGFR-β was degraded into several fragments. Elastase degraded both receptors on fixed cells, indicating that the degradation resulted from direct proteolysis on the cell surface. Elastase also then disturbed the phosphorylation of ERK1/2, JNK/SARK, and p38, triggered by PDGF-AA and PDGF-BB, suggesting that elastase inhibited PDGFR-dependent cell activation in PDL cells. These results suggest that elastase may modulate the PDGF-mediated activity of PDL cells during periodontal wound healing.

Background and objectives: Aminopeptidase N (APN)/CD13 is a multifunctional ectoenzyme that is involved in anti-inflammatory reactions, control of immune reactions and differentiation of many cellular systems. Here, we hypothesized that CD13/APN would be expressed on human gingival fibroblasts (hGF) and would contribute to the regulation of immune responses in periodontal tissue. Methods and results: CD13/APN was expressed on hGF at the mRNA and protein levels as determined by reverse transcriptase–polymerase chain reaction (RT–PCR) and flow cytometry, respectively. Enzymatic activities accompanying the expression were assessed by colorimetrical analysis using the synthetic substrate Leu-p-nitroanilide. We examined the possible regulation of CD13/APN expression on hGF in response to T cell-derived cytokines. T helper (Th) 2 cell type cytokines such as interleukin-4 and interleukin-13, but not interleukin-2 or interleukin-15, preferentially increased the expression of proteins as well as the enzymatic activities of CD13/APN in a dose-dependent manner. Receptors for these cytokines, the interleukin-4 receptor α chain, interleukin-13 receptor α1 chain, and interleukin-2R common γ chain, were expressed on hGF assessed by RT–PCR or flow cytometry. hGF exhibited inhibitory effects for formyl-methionyl-leucyl-phenylalanine (FMLP)-induced polymorphonuclear leukocyte-activation that was evaluated by Mac-1 expression, and this inhibitory effect was partially recovered by pre-treatment with the APN-specific inhibitor bestatin. Conclusions: These findings suggested that CD13/APN expressed by hGF could contribute to the anti-inflammatory response in periodontal tissue, and may be involved in disease processes mediated by Th2 cells.

Background and objective: Periodontal ligament cells are regarded to have the capacity to differentiate into cementoblasts or osteoblasts, and are capable of forming a mineralized nodule in vitro. However, the precise mechanisms are unclear. Here we evaluated the possible involvement of growth factor receptors, such as the platelet-derived growth factor receptor (PDGFR), insulin-like growth factor-I receptor (IGF-IR), and epidermal growth factor receptor (EGFR) on periodontal ligament cells and their ligands during periodontal ligament cells differentiation in vitro. Methods: Human periodontal ligament cells were differentiated via culturing in the presence of dexamethasone, ascorbic acid, and β-glycerophosphate for mineralized nodule formation, characterized by von Kossa staining. Expressions of receptors and their ligands were analyzed by flow cytometry/reverse transcription-polymerase chain reaction. Results: During the differentiation, PDGFR-α was held at a lower level compared with the control. PDGFR-β, however, was maintained at a slightly higher level that was reversed to the control level when mineralized nodules formed. In contrast, IGF-IR and EGFR were not substantially different from the control. The mineralized nodule formation was strongly inhibited by a PDGFR kinase blocker (AG1295 and AG1296), partially inhibited by an IGF-IR kinase blocker (I-Ome-AG538 and AG1024), and not inhibited by an EGFR kinase blocker (AG99). PDGF-A, PDGF-C, PDGF-D, IGF-I, and IGF-II, but not PDGF-B, were expressed on the control as well as dexamethasone/ascorbic acid-treated periodontal ligament cells during mineralized nodule formation; however, the pattern of their expressions was quite different. Conclusion: These findings suggest that a pathway of PDGFs/PDGFR and IGFs/IGF-IR on periodontal ligament cells are involved during mineralized nodule formation, and that PDGFs and IGFs expressed by periodontal ligament cells may contribute to the formation.

Primary immune responses are initiated by dendritic cells (DC) that inform naive T helper cells about invading pathogens. DC undergo sequential events leading to irreversible maturation upon bacterial stimulation. To investigate the responses of DC during periodontal infection, we studied the effects of LPS from Porphyromonas gingivalis on DC. DC generated from human peripheral monocytes by culture with IL-4 and GM-CSF were incubated with P. gingivalis LPS (Pg LPS) or Escherichia coli LPS (Ec LPS). Flow cytometry and real-time quantitative RT-PCR analysis revealed that Pg LPS, but not Ec LPS, preferentially up-regulated CD14 and CD16 expression at protein and mRNA levels. Furthermore, Pg LPS preferentially induced the secretion of soluble CD14. CD1a, HLA-DR and CD54 were highly expressed on DC stimulated with both kinds of LPS; however, CD40, CD80, CD83 and CD86 expression on Pg LPS-stimulated DC was lower than on Ec LPS-stimulated DC. With regard to IL-6, IL-8, IL-10, IL-12 and RANTES production from DC and allogeneic T cell proliferation, Pg LPS was a weaker stimulator than Ec LPS. These results suggested that Pg LPS triggers maturation of DC with unique characteristics, which exhibited weak immunostimulatory activity and may contribute to induction of chronic inflammation at the site of periodontal infection.

BACKGROUND AND OBJECTIVES CD73/5'-nucleotidase (5'-NT) is an ectoenzyme that participates in immune/inflammatory reactions. We examined the possible expression of CD73/5'-NT on human gingival fibroblasts (hGF), which are important to the immune/inflammatory system in periodontal tissue. METHODS AND RESULTS We demonstrated that CD73/5'-NT was expressed on hGF by flow cytometry. We found that pre-treatment of hGF with 5'-AMP induced marked inhibition of granulocyte-macrophage colony-stimulating factor (GM-CSF) production from hGF upon stimulation with interleukin-1alpha (IL-1alpha) by enzyme-linked immunosorbent assay (ELISA). A specific inhibitor of 5'-NT, adenosine 5'-[alpha,beta-methylene] diphosphate blocked the inhibition of GM-CSF production, suggesting that adenosine converted from 5'-AMP acts on the inhibitory effects. The GM-CSF inhibition suggested that A3 receptor might be involved. The rank order of agonists was found to be (N6-benzyl-5'-N-ethylcarboxamidoadenosine) A3 receptor agonist > or = (2-chloroadenosine) non-selective agonist > (CGS-21680) A2A receptor agonist > adenosine > or = (N6-cyclohexyladenosine) A1 agonist. Further support for the main role of A3 receptor was the binding A3 antagonist [9-chloro-2-(2-furanyl)-5-([phenylacetyl]amino)[1,2,4]-triazolo[1,5-c]quinazdine] reversed the effect of adenosine, but no significant reverse was observed by A1 (1,3-dipropyl-8-cyclopentylxanthine), A2 [3,7-dimethyl-1-(2-propargyl)xanthine], A2A[8-(3-chlorostyryl)caffeine], and A2B (alloxazine) antagonists. The CD73/5'-NT expression was increased upon stimulation with gamma-interferon, but not other stimulants such as tumor necrosis factor-alpha, IL-4, lipopolysaccharide from Porphyromonas gingivalis and Escherichia coli, and fimbriae from P. gingivalis, and this increase was correlated with the enhanced GM-CSF inhibition by 5'-AMP but not adenosine. CONCLUSIONS These findings suggested that CD73/5'-NT on hGF exerts an anti-inflammatory effects in periodontal disease by conversion from 5'-AMP to adenosine.

Wnt signaling regulates multiple cellular events such as cell proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Canonical Wnt/β-catenin signaling can promote the differentiation of dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts, and periodontal ligament cells, toward a cementoblast/osteoblast phenotype during root formation, but little is known about the biological significance of noncanonical Wnt signaling in this process. We identified the expression of Wnt5a, a representative noncanonical Wnt ligand, in tooth root lining cells (i.e. precementoblasts/cementoblasts) and dental follicle cells during mouse tooth root development, as assessed by immunohistochemistry. Silencing expression of the Wnt5a gene in a dental follicle cell line resulted in enhancement of the Wnt3a (a representative canonical Wnt ligand)-mediated increase in alkaline phosphatase (ALP) expression. Conversely, treatment with recombinant Wnt5a inhibited the increase in ALP expression, suggesting that Wnt5a signaling functions as a negative regulator of canonical Wnt-mediated ALP expression of dental follicle cells. Wnt5a did not affect the nuclear translocation of β-catenin as well as β-catenin-mediated transcriptional activation of T-cell factor (Tcf) triggered by Wnt3a, suggesting that Wnt5a inhibits the downstream part of the β-catenin-Tcf pathway. These findings suggest the existence of a feedback mechanism between canonical and noncanonical Wnt signaling during the differentiation of dental follicle cells.

CD40 is a crucial element in the process of fibroblast activation. We demonstrated that treatment of human gingival fibroblast (HGF) with human leukocyte elastase (HLE), a neutrophil serine protease, down-regulated the expression of CD40 and binding to the CD40 ligand (CD40L) using flow cytometry. The other neutrophil serine proteases, cathepsin G and proteinase 3, exhibited markedly less activity for CD40 reduction. The CD40 reduction by HLE was also observed in skin and lung fibroblasts, but not in monocytes, macrophages, and dendritic cells. The reduction resulted from direct proteolysis by HLE on the cell surface, because HLE reduced CD40 on fixed HGF and also on cell lysates and membranes. HLE treatment of HGF decreases interleukin (IL)-8 and macrophage chemoattractant protein-1 production by HGF when stimulated by CD40L, but not by IL-1α, suggesting that HLE inhibited a CD40-dependent cell activation. These results suggest that HLE possesses an anti-inflammatory effect for the HGF-mediated inflammatory process.

The cytokine-inducing activities of fungal polysaccharides were examined in human monocytes in culture, with special reference to CD14 and Toll-like receptors (TLRs). Tumor necrosis factor alpha (TNF-alpha) production by monocytes was markedly induced in a dose-dependent manner upon stimulation with cell walls from Candida albicans and mannan from Saccharomyces cerevisiae and C. albicans, although relatively high concentrations (10 to 100 microg/ml) of stimulants were required for activation as compared with the reference lipopolysaccharide (LPS) (1 to 10 ng/ml). The yeast form C. albicans and its mannan and cell wall fractions exhibited higher TNF-alpha production than respective preparations from the hyphal form. Only slight TNF-alpha production was induced by the S. cerevisiae glucan. The TNF-alpha production triggered by reference LPS and purified fungal mannans required the presence of LPS-binding protein (LBP), and these responses were inhibited by anti-CD14 and anti-TLR4 antibodies, but not by anti-TLR2 antibody. In contrast to the activity of LPS, the activity of purified S. cerevisiae mannan was not inhibited by polymyxin B. These findings suggested that the mannan-LBP complex is recognized by CD14 on monocytes and that signaling through TLR4 leads to the production of proinflammatory cytokines in a manner similar to that induced by LPS.

Arginine-specific cysteine proteinases (gingipains-R) from periodontopathicPorphyromonas gingivaliscleaved CD14, a bacterial pattern recognition receptor, on human gingival fibroblasts (HGF). Consequently, gingipains-R reduced lipopolysaccharide-induced interleukin-8 production by HGF, indicating that gingipains-R inhibited CD14-dependent HGF activation and are involved in immune evasion by the bacterium in periodontal tissues.

Dental calculus is calcified plaque composed primarily of calcium phosphate mineral salts, and there is a clear association between the presence of calculus and the initiation/progression of periodontitis. However, it is still inconclusive whether dental calculus can be a direct causative factor. The authors examined the effect of nano/microsized calcium phosphate particles, which may be generated in the process of early precipitation and/or dissolution of calcium phosphate mineral, on the expression of interleukin (IL)-8 in human gingival epithelial cells.Primary human gingival epithelial cells and/or a human gingival carcinoma cell line (Ca9-22) were stimulated with calcium phosphate particles. Gene and protein levels were assessed by real-time polymerase chain reaction analysis and enzyme-linked immunosorbent assay, respectively. The activity of nuclear factor (NF)-κB signaling was measured by an immunofluorescence assay to evaluate NF-κB p65 nuclear translocation.The results show that nano/microsized particles stimulate IL-8 expression in human gingival epithelial cells at gene and protein levels. The activity to induce IL-8 expression depends on the particle size: particles with a diameter of 200 nm are more effective than those of 40-nm and 5-μm diameters. Calcium phosphate particles (diameter 200 nm) stimulated NF-κB activity. Pretreatment with BMS-345541, an NF-κB signaling inhibitor, inhibited the particle-mediated IL-8 gene induction, suggesting a requirement for the NF-κB signaling pathway.These findings suggest that calcium phosphate particles, which may be related to calculus development, may act as a direct causative factor in the pathogenesis of gingival epithelium.

Activated polymorphonuclear leukocytes (PMNs) release various types of proteases and express them on the cell surface. The proteases play important roles in PMN-mediated events. In the present study, flow cytometric analysis revealed that CD14 expression on human gingival fibroblasts (HGF) was markedly reduced by PMA-activated PMNs in a coculture system. We found that this reduction was caused by both secreted and cell surface proteases produced by activated PMNs. A protease responsible for the reduction was found to be human leukocyte elastase (HLE) secreted from the activated PMNs by use of various protease inhibitors, although HLE was only partially involved in CD14 reduction caused by cell-bound molecule(s) on fixed PMNs. Analysis with purified HLE revealed a time- and dose-dependent reduction of CD14 on HGF, and complete reduction was observed by 20 μg/ml HLE treatment for 30–60 min, but the other molecules such as CD26, CD59, CD157, and MHC class I on HGF were only slightly reduced. This reduction of CD14 resulted from direct proteolysis by HLE on the cell surface, because HLE reduced CD14 on fixed HGF and also on purified cell membranes. As a result of CD14 proteolysis, IL-8 production by HGF was suppressed when triggered by 10 ng/ml LPS, but not by IL-1α, indicating that HLE inhibited a CD14-dependent cell activation. These findings suggested that activated PMNs have a potential negative feedback mechanism for HGF function at the inflammatory site, particularly in periodontal tissues.

BACKGROUND AND OBJECTIVES Retinoic acid (RA) exerts a wide variety of effects on development, cellular differentiation and homeostasis in various tissues. However, little is known about the effects of RA on the differentiation of periodontal ligament cells. In this study, we investigated whether RA can affect the dexamethasone-induced differentiation of periodontal ligament cells. METHODS AND RESULTS Human periodontal ligament cells were differentiated via culturing in the presence of dexamethasone, ascorbic acid, and beta-glycerophosphate for mineralized nodule formation, as characterized by von Kossa staining. Continuous treatment with all-trans-RA inhibited the mineralization in a dose-dependent manner, with complete inhibition over 1 microm RA. Other RA analogs, 9-cis-RA and 13-cis-RA, were also effective. Furthermore, addition of RA for just the first 4 days completely inhibited the mineralization; however, as RA was added at later stages of culture, the inhibitory effect was diminished, suggesting that RA had a phase-dependent inhibition of mineralization. RA receptor (RAR)-alpha agonist (AM-580), but not retinoid X receptor agonist (methoprene acid), inhibited the mineralization, and reverse transcription-polymerase chain reaction analysis revealed that RAR-alpha was expressed on the cells, suggesting that RAR-alpha was involved in the inhibitory mechanism. This inhibition was accompanied by inhibition of alkaline phosphatase activity; however, neither expression of platelet-derived growth factor (PDGF) receptor-alpha, PDGF receptor-beta, or epidermal growth factor (EGF) receptor, nor phosphorylation of extracellular signal-regulated kinases triggered by PDGF-ascorbic acid or PDGF-BB was changed, as assessed by flow cytometry or western blot analyses. CONCLUSIONS These findings suggest that RA is a potential negative regulator for differentiation of human periodontal ligament cells.

Cysteine proteinases (gingipains) from Porphyromonas gingivalis are considered key virulence factors of severe periodontitis and host immune evasion. Since expression of intercellular adhesion molecule-1 (ICAM-1) on gingival epithelium is indispensable in polymorphonuclear leukocyte (PMN) migration at the site of periodontitis, we examined the effects of gingipains on the expression of ICAM-1 on human oral epithelial cell lines (KB and HSC-2) by flow cytometry and Western blotting. We found that three purified forms of gingipains efficiently reduced ICAM-1 expression on the cells in a time- and dose-dependent manner. Gingipains reduced the expression on fixed cells and degraded the ICAM-1 in the cell membranes, indicating that the reduction resulted from direct proteolysis. They then disturbed the ICAM-1-dependent adhesion of PMNs to the cells. These results indicate that gingipains cleave ICAM-1 on oral epithelial cells, consequently disrupting PMN-oral epithelial cell interaction, and are involved in immune evasion by the bacterium in periodontal tissues.

Cysteine proteinases (gingipains) elaborated from Porphyromonas gingivalis exhibit enzymatic activities against a broad range of host proteins and are considered key virulence factors in the onset and development of adult periodontitis and host defense evasion. In this study, we examined the ability of arginine-specific gingipains (high molecular mass Arg-specific gingipain (HRGP) and Arg-specific gingipain 2) and lysine-specific gingipain (KGP) to cleave monocyte CD14, the main receptor for bacterial cell surface components such as LPS. Binding of anti-CD14 mAb MY4 to human monocytes was almost completely abolished by 0.3 μM HRGP and KGP treatments for 15 min, and 1 μM RGP2 for 30 min. In contrast, the expressions of Toll-like receptor 4, and CD18, CD54, CD59, and HLA-A, -B, -C on monocytes were slightly increased and decreased, respectively, by 0.3 μM HRGP and KGP. This down-regulation resulted from direct proteolysis, because 1) gingipains eliminated MY4 binding even to fixed monocytes, and 2) CD14 fragments were detected in the extracellular medium by immunoblot analysis. Human rCD14 was degraded by all three gingipains, which confirmed that CD14 was a substrate for gingipains. TNF-α production by monocytes after HRGP and KGP treatments was decreased at 1 ng/ml, but not at 20 μg/ml LPS, indicating that gingipains inhibited a CD14-dependent cell activation. These results suggest that gingipains preferentially cleave monocyte CD14, resulting in attenuation of the cellular recognition of bacteria, and as a consequence sustain chronic inflammation.

A mitogenic substance on murine lymphocytes was detected in the culture supernate of Streptococcus pyogenes type 12 strain. This substance had a molecular weight of 28,000 and pI 9.2, and was designated as S. pyogenes mitogen (SPM). The proliferative response of C3H/HeN spleen cells began at 1 ng ml-1 and reached a maximal response at 100 ng ml-1 of SPM for 4 days culture. Anti-Thy 1.2 mAb and complement-treated spleen cells abrogated the proliferative response to any dose of SPM. Although the anti-major histocompatibility complex class 1 mAbs had no blocking effect on proliferation by SPM, this proliferation was substantially inhibited by the addition of either anti-I-A or anti-I-E mAb, and complete inhibition was produced by the addition of both mAbs. Fixed antigen-presenting cells still induced T cell proliferation by SPM. A significant expansion of T cells bearing V beta 13 T-cell receptor was observed up to 73% among the Thy 1.2+ cells in cultures stimulated with SPM, indicating expansion in a V beta-specific manner. Immunoblotting of IEF-separated proteins showed that anti-streptococcal pyrogenic exotoxin (SPE) C reacted with a protein of pI 6.9 and anti-SPEB did not show any reactivity. SPEA was reported to expand V beta 8.1 and 8.2 bearing murine T cells, and SPM did not. SPM also exhibited potent mitogenic activity on human T cells and V beta 21+ T cells were selectively expanded. These results lead to the conclusion that SPM was neither SPEA, B nor C, but a new protein belonging to a group of streptococcal superantigens with activity on not only human but also murine lymphocytes.

Background The production of chemokines by tissue resident cells during inflammation is considered one of the main mechanisms involved in the formation of inflammatory infiltrates. Fibroblasts are the main resident cell type in gingival and periodontal ligament tissues, and their ability to produce chemokine stromal cell-derived factor 1 alpha (SDF-1α) and its receptor CXCR4 under stimulation by gram negative bacteria, Porphyromonas gingivalis, commonly found in periodontal infections was investigated. Methods Western blots were used to assess SDF-1α and CXCR4 protein expression levels in human gingival fibroblast cells (HGF-1) induced by Lipopolysaccharide (LPS) from P. gingivalis in the presence or absence of LY294002, a highly selective inhibitor of PI-3K/Akt. RT-PCR and quantitative Real-time PCR was performed using gingival mRNAs from periodontitis patients. Immunohistochemistry was performed to analyze the expression and subcellular localization of SDF-1α and CXCR4, together with NF-kβ phosphorylation, in specimens from patients with periodontitis and in an experimental rat periodontitis model. Results We found that P. gingivalis LPS up-regulated SDF-1α and CXCR4 protein levels and elevated phosphorylation of the SDF-1α-responsive NF-kβ and Akt at 24 h in HGF-1 cells. SDF-1α and CXCR4 mRNA and protein expression levels were high in all patients with periodontitis. In the P. gingivalis-induced rat experimental periodontitis model, SDF-1α and CXCR4 immunoreactivity was higher in gingival and periodontal ligament tissues compared to the control. Conclusion Our data showed that PI-3K/Akt is an upstream participant in the P. gingivalis LPS-mediated induction of SDF-1α. Taken together, these results suggest that the chemokine SDF-1α and its receptor CXCR4 contribute to P. gingivalis-induced periodontal inflammation.