dpr and sod in Streptococcus mutans are involved in coexistence with S. sanguinis, and PerR is associated with resistance to H2O2.

Large numbers of bacteria coexist in the oral cavity. Streptococcus sanguinis, one of the major bacteria in dental plaque, produces hydrogen peroxide (H(2)O(2)), which interferes with the growth of other bacteria. Streptococcus mutans, a cariogenic bacterium, can coexist with S. sanguinis in dental plaque, but to do so, it needs a means of detoxifying the H(2)O(2) produced by S. sanguinis. In this study, we investigated the association of three oxidative stress factors, Dpr, superoxide dismutase (SOD), and AhpCF, with the resistance of S. sanguinis to H(2)O(2). The knockout of dpr and sod significantly increased susceptibility to H(2)O(2), while the knockout of ahpCF had no apparent effect on susceptibility. In particular, dpr inactivation resulted in hypersensitivity to H(2)O(2). Next, we sought to identify the factor(s) involved in the regulation of these oxidative stress genes and found that PerR negatively regulated dpr expression. The knockout of perR caused increased dpr expression levels, resulting in low-level susceptibility to H(2)O(2) compared with the wild type. Furthermore, we evaluated the roles of perR, dpr, and sod when S. mutans was cocultured with S. sanguinis. Culturing of the dpr or sod mutant with S. sanguinis showed a significant decrease in the S. mutans population ratio compared with the wild type, while the perR mutant increased the ratio. Our results suggest that dpr and sod in S. mutans are involved in coexistence with S. sanguinis, and PerR is associated with resistance to H(2)O(2) in regulating the expression of Dpr.

MK615 attenuates Porphyromonas gingivalis lipopolysaccharide-induced pro-inflammatory cytokine release via MAPK inactivation in murine macrophage-like RAW264.7 cells.

The Japanese apricot, known as Ume in Japanese, has been a traditional Japanese medicine for centuries, and is a familiar and commonly consumed food. The health benefits of Ume are now being widely recognized and have been strengthened by recent studies showing that MK615, an extract of compounds from Ume, has strong anticancer and anti-inflammatory effects. However, the potential role of MK615 in the periodontal field remains unknown. Here, we found that MK615 significantly reduced the production of pro-inflammatory mediators (tumor necrosis factor-alpha and interleukin-6) induced by Porphyromonas gingivalis lipopolysaccharide (LPS), a major etiological agent in localized chronic periodontitis, in murine macrophage-like RAW264.7 cells. MK615 markedly inhibited the phosphorylation of ERK1/2, p38MAPK, and JNK, which is associated with pro-inflammatory mediator release pathways. Moreover, MK615 completely blocked LPS-triggered NF-kappaB activation. The present results suggest that MK615 has potential as a therapeutic agent for treating inflammatory diseases such as periodontitis.

Runt-related gene 2 is involved in the inhibition of matrix metalloproteinase-13 expression by roxithromycin in human gingival epithelial cell cultures.

BACKGROUND AND OBJECTIVE: Matrix metalloproteinase (MMP)-13 has wide substrate specificity compared with other MMPs and appears to be involved in periodontitis. Previously, we reported that roxithromycin (RXM) inhibits vascular endothelial growth factor expression induced by tumour necrosis factor-alpha in human periodontal ligament cells, but little is known about the effect of RXM on MMP-13 expression in human gingival epithelial cells. We therefore examined the effect of RXM on MMP-13 mRNA expression and production in cultured human gingival epithelial cells. MATERIAL AND METHODS: Human epithelial cell lines (Ca9-22, TU4, SCCTF and HSC-3) were plated in tissue culture dishes. Then, the culture supernatants and sediments were collected and the production of MMP-13 was analysed using enzyme-linked immunosorbent assay; the expression of MMP-13 mRNA and runt-related gene 2 mRNA was assessed using reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR. We also studied the effect of Runx2 short interfering RNA (siRNA) on the induction of MMP-13. RESULTS: Roxithromycin downregulated the induction of MMP-13 in Ca9-22 cells. Roxithromycin suppressed the expression of MMP-13 mRNA not only in Ca9-22 cells, but also in other human epithelial cell lines. Roxithromycin strongly inhibited the expression of Runx2 mRNA. Furthermore, Runx2 siRNA inhibited the induction of MMP-13 in Ca9-22 cells. CONCLUSION: These results indicate that RXM suppresses MMP-13 via the downregulation of Runx2 in human gingival epithelial cell cultures.

Film thickness and fluidity of various luting cements determined using a trial indentation meter.

OBJECTIVES: The purpose of this study was to evaluate a new trial apparatus which could measure film thickness (T(h)) under a low load, using an indenter. METHODS: Five commercial luting cements were measured using this apparatus. T(h) was determined using four kinds of indenter, which were 5.0, 10.0, 15.0 and 20.0mm in diameter. For each indenter, the load per contact area of the indenter with the platen equaled 150N per 200 mm2 described in ISO No. 9917. The effects of working time (30, 60, and 90 s) and load (2.86, 6.78, 10.7, and 14.7N) on the fluidity and T(h) were examined using the indenter with a diameter of 5.0mm. The duration of loading was 10 min. The fluidity was defined as the reciprocal of the apparent viscosity and was calculated using Stefan's equation. RESULTS: The T(h) obtained using this apparatus agreed with the film thickness obtained using the test described in ISO No. 9917. For Durelon (DU), T(h) increased 1.58 times and fluidity decreased 0.26 times as the working time increased from 30 to 90 s; T(h) decreased 0.56 times and fluidity increased 1.28 times as loading increased from 2.86 to 14.7N. For Ketac cement radiopaque (KE), Panavia fluoro cement (PA), Scotchbond resin cement (SC), and Vitremer luting cement (VI), although the fluidity and T(h) were not influenced much by duration (30-90 s), T(h) decreased 0.79, 0.74, 0.95, and 0.78 times for KE, PA, SC, and VI, respectively, and fluidity decreased 0.68, 0.51, 0.30, and 0.36 times, respectively, as loading increased from 2.86 to 14.7N. SIGNIFICANCE: This apparatus was proven effective in elucidating the flow properties of luting cement.

Expression and characterization of vanilloid receptor subtype 1 in human dental pulp cell cultures.

The expression of the vanilloid receptor subtype 1 (VR1, TRPV1) was detected in human dental pulp fibroblasts (PF-10) using RT-PCR, Western blotting, and immunocytochemical analysis. As revealed by ELISA, capsaicin induced IL-6 expression in PF-10 cells, and the VR1 antagonist capsazepine dose-dependently inhibited capsaicin-induced IL-6 production, indicating that capsaicin-induced IL-6 expression is related to VR1 activation. The interaction between capsaicin and mitogen-activated protein kinases (MAPKs) was investigated. The phosphorylation of p38 MAPK and c-Jun NH2-terminal kinase (JNK) were detected after capsaicin stimulation. p38 MAPK is involved in capsaicin-induced IL-6 production, as shown by the use of specific inhibitors of this kinase. The result of EMSA showed that capsaicin inhibited tumor necrosis factor-alpha (TNF-alpha)-induced nuclear factor-kappa B (NF-kappaB) activation in PF-10 cell cultures. These results suggest that the activation of VR1 plays an important role in dental pulp inflammation.

Enhanced production of vascular endothelial growth factor by human monocytic cells stimulated with endotoxin through transcription factor SP-1.

The effect of endotoxin on the regulation of vascular endothelial growth factor (VEGF) mRNA expression in human monocytic (THP-1) cells was examined. Endotoxic lipopolysaccharide (LPS) from Escherichia coli and synthetic E. coli-type lipid A (LA-15-PP) enhanced VEGF mRNA expression. LPS-induced VEGF mRNA accumulation was regulated, at least in part, at the transcriptional level. Enhancement of VEGF gene expression by LPS was shown by gel shift analysis and use of transcription factor inhibitors to be mediated via the activation of SP-1.

Interleukin-10 inhibits expression of interleukin-6 and -8 mRNA in human dental pulp cell cultures via nuclear factor-kappaB deactivation.

The effects of interleukin (IL)-10 on the expression of IL-6 and IL-8 mRNA in human dental pulp cell cultures were investigated by using the Northern blot analysis. On stimulation with Prevotella intermedia lipopolysaccharide (PiLPS), IL-10 was produced in peripheral blood but was not detected in human dental pulp cell culture supernatants. IL-10 inhibited IL-8 mRNA expression, which is normally stimulated by PiLPS, IL-1alpha, and tumor necrosis factor-alpha and inhibited IL-6 mRNA expression, which is normally stimulated by IL-1alpha. In addition, IL-10 inhibited the activation of nuclear factor-kappaB, which is normally induced by PiLPS. We conclude that IL-10 inhibits expression of IL-6 and IL-8 mRNA in dental pulp cell cultures by inhibiting the activation of nuclear factor-kappaB.

Interleukin-10 receptor expression in human dental pulp cells in response to lipopolysaccharide from Prevotella intermedia.

Interleukin-10 receptor (IL-10R) expression in human, dental pulp, fibroblast cultures was investigated by using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. After exposure to lipopolysaccharide (LPS) from Prevotella intermedia, the IL-10R mRNA levels increased after 4 h, peaked at 7 h, and dropped back to the unstimulated level at 24 h. Maximal production of the IL-10R protein in dental pulp fibroblast cultures was detected by Western blot analysis after 12 h of LPS stimulation. In contrast, the human skin fibroblast (SF-MA) and human monocyte (U937) cell lines expressed IL-10R mRNA. Anti-CD14 antibodies inhibited P. intermedia LPS-induced IL-10R mRNA expression. These results indicate that P. intermedia LPS induces IL-10R gene expression in human, dental pulp fibroblasts in vitro.

Substance P enhances expression of lipopolysaccharide-induced inflammatory factors in dental pulp cells.

To examine how substance P (SP) is related with dental pulp inflammation, we examined the effects of SP on expression of genes for inflammatory factors in human dental pulp cell cultures. Using reverse transcriptase-polymerase chain reaction, we found that Prevotella intermedia lipopolysaccharide (LPS) induced expression of SP and SP-receptor mRNAs, and that somatostatin inhibited the LPS-induced expression of SP mRNA. We also found that SP enhanced LPS-induced stimulation of NF-kappaB binding activity. In addition, SP induced expression of cyclooxygenase-2 and interleukin-10 receptor mRNAs. In contrast, SP inhibited expression of interferon-gamma receptor mRNA. These results suggest that SP may play a regulatory role in the immunological response of dental pulp tissue to pathogenic bacteria.

Polymerization contraction stresses of resin-based composite restorations within beveled cavity preparations of Class I restorations.

PURPOSE: To evaluate internal stresses resulting from polymerization of resin-based composite materials in cavity preparations beveled at the internal line angles or the cavosurface margin, compared with those in conventional butt-joint box-shaped cavity preparations. MATERIALS AND METHODS: Contraction stress generated in resin-based composite restorations placed in an experimental cavity preparation model was determined using micro-photoelastic analysis. Three types of cavity preparations simulating Class I restorations with bevels at internal line angles (1 mm and 2 mm deep), and at the cavosurface margin (2 mm deep) were prepared in a posterior composite model in order to obtain no marginal defect with the restorative composite. The transparent restorative composite was bulk-filled into the preparations and light-cured for 80 seconds. Specimens 2 mm thick for micro-photoelastic analysis were cut perpendicular to the long axis of the preparation. Fringe patterns for directions and magnitudes of stresses were obtained using transmitted and reflected polarized light. Then, the photoelastic stress analysis was performed to examine stresses in the preparations. The data were statistically compared with the previous data in conventional butt-joint box-shaped preparations by ANOVA and Scheffé's F test (P < 0.05). RESULTS: In 1 and 2 mm deep internal beveled cavity preparations, the maximum principal stress lines were parallel to the wall at the straight internal bevels. In the preparation with a round bevel at the cavosurface margin, the stress distribution in the bulk of the preparation was similar to that in the butt-joint preparation. Maximum stress values in 1 and 2 mm deep internal beveled preparations were 12.2 +/- 0.2 MPa and 18.8 +/- 2.8 MPa, respectively. For 2 mm-deep preparation, the maximum stress values in the internal beveled preparations were significantly lower than those values in butt-joint box-shaped cavity preparations. Maximum stress values in the preparations with the bevel at the cavosurface margin were 22.7 +/- 1.0 MPa, and were not significantly different from those values observed in the butt-joint preparations.

Temperature triggered shape memory effect of transpolyisoprene-based polymer.

INTRODUCTION: Pure gutta-percha (trans-1, 4-polyisoprene [TPI]) has been used extensively as a main component of gutta-percha for root canal filling. TPI has the interesting shape memory property by cross-linking, and this polymer was commercialized under the product name of SMP-2 (Kuraray Corp, Kashima, Japan). Therefore, the purpose of this study was to examine the thermal properties and the mechanism of the shape memory function of cross-linked SMP-2. METHODS: The crystalline of the TPI was observed by x-ray diffraction. The effects of temperature on shape recovery, recovery stress, and relaxation modulus (Er[5]) were measured in cross-linked cylindrical specimens of SMP-2. Differential scanning calorimetry was used to monitor thermal events. RESULTS: On heating, a pronounced increase in recovery stress, a marked decrease in Er(5), and endothermic DSC peaks were observed over the same temperature range (38°-51°C) with shape recovery. On the other hand, on cooling, a pronounced decrease in recovery stress, a marked increase in Er(5), and an exothermic DSC peak were observed over the same temperature range (27°-33°C). CONCLUSIONS: The shape memory property of TPI is derived from its crystallinity and cross-linking ability. Fixing the deformed shape and shape recovery from the deformed shape to the original shape is relatively easy to achieve by changing the temperature of SMP-2. The shape memory function of the cross-linked SMP-2 was expected to be very useful as a root canal filling material by the modification of its some thermal properties.

Bending strength of zirconia/porcelain functionally graded materials prepared using spark plasma sintering.

OBJECTIVES: The purpose of this study was to fabricate functionally graded materials (FGMs) consisting of yttria-stabilised tetragonal zirconia polycrystal (Y-TZP) and porcelain using spark plasma sintering (SPS) and examine the influence of their microstructures and thermal stress on their bending strengths. METHODS: Two types of four-layered Y-TZP/porcelain FGMs having a constant layer thickness and a varying layer thickness, Y-TZP/porcelain composite materials having a microstructure corresponding to each layer in FGMs and monolithic materials of Y-TZP and porcelain were fabricated by SPS. The Y-TZP/porcelain volume fraction of each layer in FGMs was varied over 100/0-70/30. Three-point bending test, X-ray diffraction, density measurement, microstructure observation, and thermal stress estimation were performed to characterise the materials. RESULTS: The bending strength of the Y-TZP/porcelain composite materials decreased with the volume fraction of the porcelain. About FGMs, when the 100%Y-TZP layer was on the tensile stress side during the bending test, the bending strength was almost the same as that of the 100%Y-TZP monolithic material. On the other hand, when the 100%Y-TZP layer was on the compressive stress side, the bending strength of FGM having a constant layer thickness was almost the same as that of the 70%Y-TZP+30%porcelain composite material, while the bending strength of FGM with a varying layer thickness was significantly higher than that of the 70%Y-TZP+30%porcelain composite material. CLINICAL SIGNIFICANCE: The FGMs prepared and analyzed in this research can potentially be used for crowns and bridges as well as for inlays and onlays. CONCLUSION: The SPS method could effectively fabricate the Y-TZP/porcelain FGMs, and the bending strength results revealed that the graded structure was very efficient to raise the bending strength.

Anandamide induces matrix metalloproteinase-2 production through cannabinoid-1 receptor and transient receptor potential vanilloid-1 in human dental pulp cells in culture.

INTRODUCTION: Anandamide (N-arachidonoylethanolamine [AEA]) is one of the main endocannabinoids. Endocannabinoids are implicated in various physiological and pathologic functions, inducing not only nociception but also regeneration and inflammation. The role of the endocannabinoid system in peripheral organs was recently described. The aim of this study was to investigate the effect of AEA on matrix metalloproteinase (MMP)-2 induction in human dental pulp cells (HPC). METHODS: We examined AEA-induced MMP-2 production and the expression of AEA receptors (cannabinoid [CB] receptor-1, CB2, and transient receptor potential vanilloid-1 [TRPV1]) in HPC by Western blot. MMP-2 concentrations in supernatants were determined by enzyme-linked immunosorbent assay. We then investigated the role of the AEA receptors and mitogen-activated protein kinase in AEA-induced MMP-2 production in HPC. RESULTS: AEA significantly induced MMP-2 production in HPC. HPC expressed all 3 types of AEA receptor (CB1, CB2, and TRPV1). AEA-induced MMP-2 production was blocked by CB1 or TRPV1 antagonists and by small interfering RNA for CB1 or TRPV1. Furthermore, c-Jun N-terminal kinase inhibitor also reduced MMP-2 production. CONCLUSIONS: We demonstrated for the first time that AEA induced MMP-2 production via CB1 and TRPV1 in HPC.

Development of surface coating material for discolored tooth equipped with bleaching effect.

It is attempted to augment a coating resin with a bleaching effect to provide both short- and long-term whitening effects. Base resin containing sodium percarbonate (SPC) effectively bleached bovine teeth discolored by the Maillard reaction. SPC did not reduce Vickers hardness, but hardness in the hybrid material increased. The shear bonding strength of SPC-containing resin was low. No inflammation was apparent in hamster cheek pouch mucosa when exposed to SPC resin covered with a layer of base resin. H(2)O(2) was released into buffer from this resin, but when placed onto tooth tissue with a protective layer of base resin, penetration of H(2)O(2) into the pulp chamber was undetectable. It is concluded that SPC resin equipped with a bleaching aid can be safely used as a coating material for discolored teeth.

Hyperosmotic stress induces cell death in an odontoblast-lineage cell line.

INTRODUCTION: Osmotic stress is one of the stimulations related to dental pain caused by caries or dentin hypersensitivity. The mechanism of osmotic-induced dental pain is not completely understood. The purpose of this study was to examine the responses of odontoblasts under sucrose-induced hyperosmotic stress. METHODS: We used an odontoblast-lineage cell (OLC) line in our experiments. OLCs were stimulated with sucrose to produce hyperosmotic stress. The expressions of dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP 1) were detected by using reverse transcriptase polymerase chain reaction assay. The cell viability of OLCs was detected by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay. The responses accompanied with cell death were detected by using 4-6-diamidino-2-phenylindole staining, Western blotting of caspase-3, and annexin V assay. The expression of mitogen-activated protein kinases (MAPKs) was detected by using Western blot analysis. RESULTS: DSPP and DMP 1 were not affected by hyperosmotic stress in OLCs. Cell viability decreased over 700 mOsm for 3 hours of cell culture. The shapes of cells and nuclei became irregular and vacuolar under hyperosmotic stress. The expression of cleaved caspase-3 was increased after treatment with hyperosmotic stress. Some propidium iodide-positive cells were detected in flow cytometry analysis. Phosphorylation of 3 MAPKs was induced by hyperosmotic stress. Inhibitors of 3 MAPKs inhibited the hyperosmotic stress-induced decline in cell viability at 500 and 700 mOsm. CONCLUSIONS: Hyperosmotic stress induces cell death of OLCs with sucrose through a MAPK pathway.

Periodontal disease and type 2 diabetes mellitus: is the HMGB1-RAGE axis the missing link?

Periodontitis is a major chronic inflammatory disease associated with increased production of numerous proinflammatory cytokines, which leads to the destruction of the periodontal tissue and ultimately loss of teeth. Periodontitis has powerful and multiple influences on the occurrence and severity of systemic conditions and diseases, such as diabetes mellitus, cardiovascular disease and respiratory disease. Meanwhile, diabetes is associated with increased prevalence, severity and progression of periodontal disease. There is also abundant evidence showing that diabetes plays important etiological roles in periodontitis. High mobility group box 1 (HMGB1) was recently identified as a lethal mediator of severe sepsis and comprises a group of intracellular proteins that function as inflammatory cytokines when released into the extracellular milieu. From a clinical perspective, extracellular HMGB1 can cause multiple organ failure and contribute to the pathogenesis of sepsis, rheumatoid arthritis, cardiovascular disease and diabetes. We recently reported that HMGB1 expression in periodontal tissues was elevated in patients with severe periodontitis. In addition, the receptor for advanced glycation end-products (RAGE), a receptor for HMGB1, was strongly expressed in gingival tissues obtained from patients with type 2 diabetes and periodontitis compared with systemically healthy patients with chronic periodontitis patients. From these data, we hypothesize that HMGB1 might play a role in the development of diabetes-associated periodontitis.

MK615: a new therapeutic approach for the treatment of oral disease.

The oral cavity is inhabited by over 500 different bacterial species. Dental caries and periodontitis are major bacterial infectious diseases in the oral cavity. Prunus mume Sieb. et Zucc., which is a variety of Japanese apricot known as Ume in Japanese, has been a traditional Japanese medicine for centuries, and is a familiar and commonly consumed food. The health benefits of Ume are now being widely recognized. Recent studies showed that MK615, an extract of compounds from Ume, has strong anticancer and anti-inflammatory effects. However, the potential role of MK615 in the antimicrobial field remains unknown. Therefore, we hypothesize that MK615 has antimicrobial activities against a range of oral bacterial pathogens. Here, we show that MK615 may be a potent inhibitor of the growth of some oral bacteria and an inhibitor of biofilm formation by Streptococcus mutans, the principal etiological agent of human dental caries. Our findings suggest that MK615 has potential as a therapeutic agent for treating and preventing oral diseases such as dental caries and periodontitis.

Selective inhibition of Porphyromonas gingivalis growth by a factor Xa inhibitor, DX-9065a.

BACKGROUND: Porphyromonas gingivalis is a causative bacterium of adult periodontitis. However, there is no drug specific for P. gingivalis and for its virulence factor. OBJECTIVES: The objective of this study was to examine the effects of a new selective inhibitor of activated factor X, DX-9065a, on growth of Porphyromonas gingivalis and other periodontopathic bacteria. METHODS: We incubated P. gingivalis and other periodontopathic bacteria in the presence or absence of DX-9065a and examined the effect of DX-9065a on bacterial growth and trypsin-like activity in its cultures. We also examined the effects of DX9065a on amidolytic activity of purified trypsin-like proteinases (gingipains RgpA and RgpB), from P. gingivalis and on trypsin-like activity in gingival crevicular fluids from patients with adult periodontitis. RESULTS: DX-9065a selectively inhibited the growth of P. gingivalis and Prevotella intermedia, and its effect on P. gingivalis was bactericidal. Trypsin-like proteinase activity was detected in P. gingivalis, and the activity was strongly inhibited by DX-9065a. DX-9065a even inhibited amidolytic activity of RgpA and RgpB from P. gingivalis. Furthermore, trypsin-like proteinase activity in gingival crevicular fluids was strongly inhibited by DX-9065a. CONCLUSIONS: DX-9065a inhibits P. gingivalis growth in part through to its ability to inhibit the trypsin-like proteinase activity in P. gingivalis and may be useful for a new drug for treatment of adult periodontitis.

Substance P activates p38 mitogen-activated protein kinase to promote IL-6 induction in human dental pulp fibroblasts.

Substance P (SP) induces the expression of proinflammatory cytokines, such as interleukin (IL)-6, which are implicated in pulp inflammation. To determine the signal pathway of SP-induced IL-6, we examined the activities of the mitogen-activated protein kinases (MAPKs) in human dental pulp cell (PF-10) cultures. SP induced the phosphorylation of p38 MAPK within 5 min; this activation persisted for up to 40 min and was independent of the activation of extracellular signal-related kinases (ERK-1 and ERK-2) that were induced after SP stimulation of PF-10 cells. As shown by electrophoretic mobility shift assay p38 MAPK was not involved in SP-induced activation of nuclear factor-kappa B (NF-kappaB). However, p38 MAPK mediated SP-induced IL-6 production, as shown by the use of specific inhibitors of this kinase. Our results suggest that the activation of p38 MAPK is important for NF-kappaB-independent regulator of neurogenic inflammation in dental pulp tissues.

Neuropeptide release from dental pulp cells by RgpB via proteinase-activated receptor-2 signaling.

Dental pulp inflammation often results from dissemination of periodontitis caused mostly by Porphyromonas gingivalis infection. Calcitonin gene-related peptide and substance P are proinflammatory neuropeptides that increase in inflamed pulp tissue. To study an involvement of the periodontitis pathogen and neuropeptides in pulp inflammation, we investigated human dental pulp cell neuropeptide release by arginine-specific cysteine protease (RgpB), a cysteine proteinase of P. gingivalis, and participating signaling pathways. RgpB induced neuropeptide release from cultured human pulp cells (HPCs) in a proteolytic activity-dependent manner at a range of 12.5-200 nM. HPCs expressed both mRNA and the products of calcitonin gene-related peptide, substance P, and proteinase-activated receptor-2 (PAR-2) that were also found in dental pulp fibroblast-like cells. The PAR-2 agonists, SLIGKV and trypsin, also induced neuropeptide release from HPCs, and HPC PAR-2 gene knockout by transfection of PAR-2 antisense oligonucleotides inhibited significantly the RgpB-elicited neuropeptide release. These results indicated that RgpB-induced neuropeptide release was dependent on PAR-2 activation. The kinase inhibitor profile on the RgpB-neuropeptide release from HPC revealed a new PAR-2 signaling pathway that was mediated by p38 MAPK and activated transcription factor-2 activation, in addition to the PAR-2-p44/42 p38MAPK and -AP-1 pathway. This new RgpB activity suggests a possible link between periodontitis and pulp inflammation, which may be modulated by neuropeptides released in the lesion.