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Expression and Characterization of Vanilloid Receptor Subtype 1 in Human Dental Pulp Cell Cultures
Basic Research—Biology
Expression and Characterization of Vanilloid Receptor Subtype 1 in Human Dental Pulp Cell Cultures Rie Miyamoto, DDS, Masayuki Tokuda, DDS, PhD, Tetsuya Sakuta, DDS, PhD, Shigetaka Nagaoka, DDS, PhD, and Mitsuo Torii, DDS, PhD Abstract 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 mitogenactivated protein kinases (MAPKs) was investigated. The phosphorylation of p38 MAPK and c-Jun NH2terminal 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␣)-induced nuclear factor-kappa B (NF-B) activation in PF-10 cell cultures. These results suggest that the activation of VR1 plays an important role in dental pulp inflammation.
Key Words VR1, capsaicin, IL-6, mitogen-activated protein kinases, NF-B
From the Department of Restorative Dentistry and Endodontology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan. Address requests for reprint to Dr. Masayuki Tokuda, Department of Restorative Dentistry and Endodontology, Kagoshima University Graduate School of Medical and Dental Sciences, 8 –35-1 Sakuragaoka, Kagoshima 890-8544, Japan. E-mail address: [email protected]. Copyright © 2005 by the American Association of Endodontists
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D
ental pulp fibroblasts play a role in pulp inflammation by producing cytokines when stimulated by bacterial pathogens (1–3). Among these cytokines, interleukin (IL)-6 is produced and released in locally inflamed pulpal lesions (4). Lipopolysaccharide (LPS) induces IL-6 production in human dental pulp fibroblasts, and its production is enhanced by methyl mercaptan, a volatile sulfur compound produced by anaerobic gram-negative bacteria (5). Human dental pulp fibroblasts participate in the development and progression of pulpitis via synthesizing IL-6, which is regulated by cytokines through prostaglandin-dependent pathway (6). For these reasons, IL-6 is an indispensable, and is important for the first defense mechanism in dental pulpitis. Vanilloid receptor subtype 1 (VR1, TRPV1) is a nonselective cation channel with very high permeability to calcium ions and is mainly expressed in small-diameter sensory neurons. VR1 contains six membrane domains with an additional short hydrophobic stretch between transmembrane regions 5 and 6; it shares amino-acid sequence similarity with transient receptor potential (TRP)-related proteins. VR1 was identified as the receptor for chemicals such as capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), a major pungent ingredient in hot chili peppers. It was reported that VR1 is activated by noxious heat (⬎40°C) and by acidic conditions (⬍ pH 6), which are conditions that occur during tissue injury (7, 8). This implies that the VR1 receptor channel is a primary cellular sensor of thermal and chemical stimuli. The expression of VR1 was previously reported to be localized to neurons that convey nociceptive transmissions. There is also growing evidence for the expression of VR1 in nonneuronal cells, such as keratinocytes, epithelial cells, and cardiomyocytes (9 –11). Although VR1 has been identified in human dental pulp neurons (12), the presence of VR1 in human dental pulp fibroblasts has not been documented. Thus, we investigated the expression of VR1 and its relationship to inflammatory mediators (particularly IL-6) in human dental pulp cell (PF-10) cultures. Moreover, we examined the signal transduction of capsaicin-activated VR1 in PF-10 cell cultures.
Materials and Methods Cell Cultures Normal human dental pulp tissue was obtained from the third molar of a 21-yearold female patient with her informed consent. Dental pulp fibroblasts were prepared from the growth of the minced explants as described previously (2). The cells were cultured under 5% CO2 in ␣ -modified Eagle’s minimum essential medium (␣ -MEM; ICN Biomedicals Inc., Irvine, CA) containing 10% fetal bovine serum (FBS; BioSource International Inc., Camarillo, CA) and 200 g/ml kanamycin. These cells, designated as PF-10 cells, were homogeneous, slim, spindle-shaped fibroblasts and grew in characteristic swirls. The cells were used between the 10th and 15th passages. Confluent cells were preincubated with 1% FBS-supplemented ␣ -MEM for 18 h before the addition of other factors, as described below. HEK293 cells were obtained from the Department of Laboratory and Molecular Medicine, Kagoshima University (Kagoshima, Japan). HeLa cells were obtained from Dainippon Pharmaceutical Co. (Osaka, Japan), and MRC5, normal human embryonic lung fibroblasts were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). These commercially acquired cultures were maintained in 10% FBS-supplemented ␣ -MEM with 200 g/ml kanamycin. Neuroblastoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were maintained in 10% FBS-supplemented Eagle’s minimum essential medium (EMEM; ATCC). HepG2 cells were obtained from Dainippon Pharmaceutical Co.
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Basic Research—Biology and were maintained in 10% FBS-supplemented M199 medium (Invitrogen, Carlsbad, CA). Astrocytoma cells were obtained from SankoJunyaku Co. (Tokyo, Japan) and were maintained in 10% FBS-supplemented RPMI1640 (Sigma-Aldrich Corp., St. Louis, MO).
Biochemicals Capsaicin was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Capsazepine, a VR1 antagonist, was obtained from Tocris Cookson, Inc. (Ellisville, MO). Human recombinant tumor necrosis factoralpha (TNF-␣) was purchased from Pierce Biotechnology, Inc. (Rockford, IL). The inhibitors of p38 mitogen-activated protein kinase (MAPK) (SB203580) and of c-Jun NH2-terminal kinase (JNK) (SP600125) were purchased from Calbiochem (San Diego, CA). Reverse-Transcribed Polymerase Chain Reaction (RT-PCR) Cultured cells were grown to confluence, and total cellular RNA was prepared from the cells by the guanidinium-isothiocyanate chlorform procedure (13). One microgram of total RNA was reverse transcribed using an RNA polymerase chain reaction (PCR) kit (TAKARA BIO, Inc., Shiga, Japan) according to the manufacturer’s instructions. PCR amplification was carried out using the following oligonucleotide primers for human VR1, IL-6, and human glyceraldhyde-3-phosphate dehydrogenase (GAPDH): for VR1, 5⬘ -TGTGCCGTTTCATGTTT-3⬘ and 5⬘ -TGCAGCTTCCAGATGTT-3⬘ (14); for IL-6, 5⬘ -ATGAACTCCTTCTCCACAAG-3⬘ and 5⬘ -GTGCCTGCAGCTTCGTCAGCA-3⬘ (15); for GAPDH, 5⬘ -ATCACCATCTTCCAGGAG-3⬘ and 5⬘ -ATGGACTGTGGTCATGAG-3⬘ (16). The cDNA was denatured at 94°C for 2 min, followed by 25 cycles of denaturation at 94°C for 30 s, annealing at 62°C (VR1 and IL-6) or 57°C (GAPDH) for 30 s, and extension at 72°C for 90 s. PCR products were electrophoresed on 1.5% agarose gels and visualized by ethidium bromide. Densitometric analysis was performed using the National Institutes of Health Image software (obtained from the National Institutes of Health Web site: http://rsb.info.nih.gov/nih-image). Each gel image was imported into Image using Adobe Photoshop (Adobe System, San Jose, CA). A gel-plotting macro was used to outline the bands, and the band intensity was calculated on the uncalibrated OD setting. The results are presented as the expression ratio of VR1 or IL-6 to GAPDH. Western Blot Analysis For the analysis of VR1 expression, cells of each type were grown in 145-cm2 dishes; confluent cells were washed with 1 ⫻ phosphate buffered saline (PBS) and lysed by sonication in a buffer containing 40 mM HEPES, 1% Triton-X100, 10% glycerol, and 1 mM phenylmethanesulfonyl fluoride (PMSF). The protein concentration was measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories; Hercules, CA). Protein samples prepared from the cells were boiled for 5 min with an equal volume of 2 ⫻ sample buffer (50 mM Tris-HCl, pH 6.8, 30% glycerol, 0.01% bromophenol blue (BPB), and 2% SDS); 100 g of each protein sample was subjected to SDS-PAGE on a 10% gel for 90 min at 100 V and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membrane (Amersham Biosciences Corp., Piscataway, NJ) for 18 h at 30 V. The membranes were blocked for 1 h with 5% skim milk at 4°C and then incubated with primary antibody to VR1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell Signaling Technology, Inc., Beverly, MA). The signals were visualized with an enhanced chemiluminescence (ECL) system (Amersham Biosciences Corp.). For the detection of MAPK activation, stimulated or nonstimulated cells were washed with 1 ⫻ PBS and suspended in 100 M PMSF, 100 mM Na3VO4, 0.08% protease inhibitor cocktail (Roche Diagnostics Co., JOE — Volume 31, Number 9, September 2005
Basel, Switzerland), and 2 ⫻ sample buffer, then the cells were sonicated and boiled for 5 min. One-hundred micrograms of each protein sample was subjected to SDS-PAGE on a 10% gel for 90 min at 100 V and then electrophoretically transferred onto PVDF membrane for 18 h at 30 V. The membranes were blocked for 1 h with 5% skim milk at 4°C and then probed with primary antibodies to p38 MAPK and JNK or to the phosphorylated forms of p38 MAPK and JNK (Cell Signaling Technology Inc.), followed by incubation with HRP-conjugated antirabbit IgG and visualization with the ECL system.
Immunocytochemistry Cell of each type was cultured on glass coverslips, immersed in 4% paraformaldehyde for 18 h at 4° C, and washed three times with 1 ⫻ PBS at room temperature. Next, cells were incubated with blocking buffer (1.5% FBS in 1 ⫻ PBS) for 20 min at room temperature. VR1 was detected by incubation with rabbit polyclonal anti-human VR1 antibody for 4 h followed by incubation with goat anti-rabbit IgG conjugated with Alexa 546 dye (Molecular Probes, Inc., Eugene, OR) for 1 h at room temperature. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (0.1 g/ml) for 5 min at room temperature. After the cells were mounted on glass plates, immunofluorescence was observed using an Olympus BX51 fluorescence microscope (Olympus Corp., Tokyo, Japan). The composite images were created by DP manager software (Olympus Corp.). Enzyme-Linked Immunosorbent Assay Cells were grown in 24-well plates. After stimulation, the culture supernatants were collected and stored at ⫺80°C until use. All reagents in enzyme-linked immunosorbent assay (ELISA) system were purchased from R&D Systems, Inc. (Minneapolis, MN), and experimental procedure were conducted according to the manufacturer’s instructions. A 96-well plate was coated with monoclonal anti-human IL-6 antibody (2 g/ml) for 18 h at room temperature and blocked for 1 h at room temperature with 300 l of 1 ⫻ PBS containing 1% bovine serum albumin (BSA), 5% sucrose, and 0.05% NaN3 per well. After culture supernatants were centrifuged for 5 min at 10,000 ⫻ g to remove cellular debris, 100 l of each sample was added to a well, and the plate was incubated for 2 h at room temperature. The plate was washed three times using wash buffer (0.05% Tween-20 in 1 ⫻ PBS, pH 7.3), 100 l of biotinylated anti-human IL-6 antibody (300 g/ml) was added per well, and the plate was incubated for 2 h at room temperature. After the plate was washed three times, 100 l of streptavidin-HRP was added per well, and the plate was incubated for 20 min at room temperature. After washing three times, 100 l of substrate solution was distributed to each well, and the plate was incubated for 20 min in the dark. Then, 50 l of stop solution (1 M H2SO4) was added per well, and the absorbance at 450 nm was determined for each well using a microplate reader model 450 (Bio-Rad Laboratories, Inc.). The total protein in the medium was measured using Bio-Rad Protein Assay. The results are presented as the IL-6 concentration per total protein of each well. Electrophoretic Mobility Shift Assay (EMSA) Confluent cells were preincubated with capsaicin as indicated, followed by stimulation with 0.1 nM TNF-␣ for 15 min. Nuclear extracts were prepared as described by Dignam and others (17). Nuclear extracts (3 g) were incubated for 15 min at room temperature in a buffer that contained 10 mM Tris (pH 7.5), 100 mM potassium chloride, 5 mM MgCl2, 1 mM dithiothreitol, and 10% glycerol. A 32P-labeled, doublestranded oligonucleotide probe corresponding to the nuclear factorkappa B (NF-B) element (5⬘ -CGTGGAATTTCCTCTG-3⬘) from the IL-6 gene promoter was then added to the buffer. The DNA-protein complex VR1 Expression in Dental Pulp Cells
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Basic Research—Biology expression levels of VR1 mRNA at a PCR annealing temperature of 54°C (data not shown). The internal control showed PCR products for GAPDH at the expected size of 318 bp in each cell type (Fig. 1A). As shown in Fig. 1B, the Western blot analysis of protein isolates from cultured cells probed with the antihuman VR1 antibodies revealed a single protein band that was identical in size to those reported previously (13). The level of VR1 protein expression in PF-10 cells was weak compared with that in neuroblastoma cells. To further evaluate VR1 expression, cells were processed for VR1 immunoreactivity by using specific polyclonal antiserum for human VR1. Immunostaining photographs revealed VR1-immunoreactivity on the surface of PF-10 and neuroblastoma cells (Fig. 1C). In both cell types, immunoreactivity was absent without VR1 antibody. Taken together, these data suggest that VR1 receptors are expressed in resting cultured PF-10 cells and the level of expression in PF-10 cells is lower than that in neuroblastoma cells.
Figure 1. VR1 expression in PF-10 cells. (A) Upper panel: Representative data of RT-PCR for VR1, with GAPDH as an internal control (M, 100-bp ladder size marker; lane 1, PF-10; lane 2, MRC5; lane 3, HEK293; lane 4, HeLa; lane 5, HepG2; lane 6, neuroblastoma; lane 7, astrocytoma). The expected sizes of the products are 327 bp for VR1 and 318 bp for GAPDH. Lower panel: Bands were quantified with the NIH image software (National Institutes of Health). Data are expressed as the ratio of VR1 to GAPDH, and presented as the mean ⫾ SD of three independent experiments. (B) Western blot analysis of VR1 proteins obtained from PF-10 and neuroblastoma (NB) cells. The whole-cell lysates were immunoblotted with antihuman VR1 antibody (96 kDa). Two additional experiments gave results similar to those shown here. (C) Staining patterns of the VR1 in PF-10 (A) and neuroblastoma cells (B) with (right) and without (left) antihuman VR1 antibodies. Cells on a glass coverslip were immunostained with anti-human VR1 antibody raised in rabbits, and anti-rabbit IgG-Alexa 546 dye (red) was applied as a secondary antibody. Nuclei were counter-stained with DAPI (blue). Scale bars ⫽ 200 m. Two additional experiments gave similar results.
that formed was separated on a 5% polyacrylamide gel, the gel was dried, and radioactive bands were visualized by a Mac Bas 1000 (Hitachi, Tokyo, Japan) using an imaging plate (Fuji Photo Film Co., Tokyo, Japan).
Results VR1 Expression in PF-10 Cells We initially investigated the presence of VR1 in PF-10 cells by RT-PCR, Western blotting, and immunocytochemical analyses. Figure 1A shows the PCR products corresponding to VR1 from PF-10, MRC5, HEK293, HeLa, HepG2, neuroblastoma, and astrocytoma cells. These PCR products were detected as bands of 327 bp, the size expected for VR1. The expression of VR1 mRNA was at the same level in PF-10, MRC5, and HeLa cells; neuroblastoma, astrocytoma, HEK293, and HepG2 cells expressed high levels of VR1 mRNA. There were no differences in the 654
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Capsaicin Induces IL-6 Protein Synthesis We next evaluated the IL-6 production stimulated by capsaicin to clarify the function of VR1 in PF-10 cell cultures. Confluent cells were rendered quiescent by an 18-h incubation in ␣ -MEM containing 1% FBS, and then were stimulated with capsaicin. The concentrations of IL-6 in the cultured supernatants were measured by ELISA. The timedependent accumulation of IL-6 production upon stimulation with 100 M capsaicin is shown in Fig. 2A. IL-6 production levels began to increase after 12 h of exposure and reached a plateau at 24 h. The production of IL-6 in PF-10 cells was remarkably stimulated with 100 M capsaicin for 24 h (Fig. 2B). When PF-10 cells were treated without capsaicin, IL-6 was detected with about 200 pg/mg protein level at 8 h to 24 h. Although IL-6 production reached over 10,000 pg/mg protein when cultures were stimulated with 200 M capsaicin, the viability of the cells was about 30%; thus these findings are unclear at present (data not shown). Therefore, we used the dose of 100-M capsaicin in the subsequent IL-6 experiments. We used the VR1 antagonist capsazepine to assess whether capsaicin-induced IL-6 production is dependent on VR1. PF-10 cells were preincubated for 30 min at concentrations of capsazepine ranging from 10 M to 60 M, and then cultures were stimulated with 100 M capsaicin for 24 h. Figure 2C demonstrates that the induction of IL-6 protein synthesis by 100 M capsaicin was inhibited dose-dependently by capsazepine; the synthesis of IL-6 protein induced by 100 M capsaicin was essentially suppressed by greater than 30 M capsazepine. MAPKs Activation Stimulated with Capsaicin To investigate the effects of capsaicin on the activation of MAPKs, cells were treated with 1 M capsaicin for various time periods. Subsequently, total cell protein was extracted and subjected to Western blotting with antibodies specifically recognizing the phosphorylated or total kinases. As shown in Fig. 3A, p38 MAPK was activated after a 5-min treatment with capsaicin. Maximal activation was seen after 10 min, and activity returned to the nonstimulated level after 20 min. An activation of JNK by 1 M capsaicin was seen after 5 min, and it persisted for up to 60 min (Fig. 3B). p38 MAPK and JNK were activated when PF-10 cells were stimulated by exposure to any one of a range of capsaicin concentrations for 10 min (Fig. 3C,D). p38 MAPK was efficiently activated by 10-nM to 100 M capsaicin, whereas the levels of JNK activation were enhanced by 1 M to 100 M capsaicin (Fig. 3D). To further confirm that capsaicin induces the activation of MAPKs via VR1, we examined the effect of capsazepine on capsaicin-treated PF-10 cells. As shown in Fig. 3(E,F), capsazepine inhibited both the activation of p38 MAPK and that of JNK. JOE — Volume 31, Number 9, September 2005
Basic Research—Biology with different doses of capsaicin for 4 h and were then exposed to 0.1 nM TNF-␣ for 15 min. As shown in Fig. 5, the DNA binding activity of the NF-B element is reduced in the presence of capsaicin compared with treatment with TNF-␣ alone. Moreover, upon pretreatment with 60 M capsazepine for 30 min before stimulation with 100 M of capsaicin, the inhibition of TNF-␣ -induced NF-B activation was not observed (data not shown).
Discussion
Figure 2. Capsaicin induced IL-6 protein synthesis in PF-10 cell cultures via VR1. (A) Time course of IL-6 production in PF-10 cells stimulated with 100 M capsaicin for the indicated periods. (B) Dose response of IL-6 production in PF-10 cells stimulated with indicated concentrations of capsaicin for 24 h. (C) Capsazepine inhibits capsaicin-induced IL-6 production in PF-10 cell cultures. The PF-10 cells were either left untreated or pretreated with the indicated concentrations of capsazepine. IL-6 production in cultured supernatants was determined by ELISA as described in Materials and Methods. Results are shown as the mean ⫾ SD. Two additional experiments gave similar results.
Involvement of MAPKs in Capsaicin-Induced IL-6 Production We also investigated the involvement of p38 MAPK and JNK in capsaicin-induced IL-6 production. PF-10 cells were pretreated with specific inhibitors of p38 MAPK and JNK at three different concentrations, and then stimulated with 100 M capsaicin. As shown in Fig. 4A, a specific inhibitor of p38 MAPK (SB203580) dose-dependently inhibited the IL-6 protein synthesis induced by capsaicin, whereas a specific inhibitor of JNK (SP600125) slightly enhanced capsaicin-induced IL-6 synthesis. We examined the capsaicin-induced expression of IL-6 mRNA to confirm our data obtained at the protein level. As shown in Fig. 4B, the capsaicin-induced expression of IL-6 mRNA was inhibited by SB203580, but enhanced by SP600125. This result is in agreement with the result of a protein level. The mRNA expression of the control, GAPDH, was not affected by capsaicin or either of the inhibitors. Capsaicin Inhibits TNF-␣ -Induced NF-B Activation Finally, we examined the effects of capsaicin on NF-B activation, which is important for IL-6 transcription. PF-10 cells were pretreated JOE — Volume 31, Number 9, September 2005
We found that VR1 was expressed on human dental pulp cells in culture. The expression of VR1 has been well established in sensory neurons (7, 12). VR1 functions as a transducer of painful stimuli, conveying nociceptive information to the central nervous system by activating sensory neurons (7). VR1 also plays a role in the immunoreactive responses of human skin and keratinocytes (9, 19). Epidermal VR1 activated by capsaicin induces proinflammatory mediators such as cyclooxygenase-2, IL-8, and prostaglandin E2 in the skin (20). In the human bronchial epithelial cell line, neuropeptides and capsaicin stimulated the release of inflammatory cytokines (21). These observations suggest that VR1 exists in both neuronal and nonneuronal cells, and has a role in the inflammatory response. In the dental area, VR1 was found in the rat dental primary afferent neurons (12) and in the nerve and epithelial cells of the rat tongue and palate, which provide defense mechanisms against toxic substances in the rat oral cavity (22). Activation of VR1 induces release of neuropeptides, calcitonine gene-related protein (CGRP) and substance P (SP) in the nerve fibers and terminals (23). The release of CGRP was evoked by capsaicin, inhibited by capsazepine in bovine dental pulp (24). Bowles and others demonstrated that extracellular levels of SP are increased within symptomatic pulp tissue diagnosed with irreversible pulpitis (25). We recently reported SP enhances expression of LPS-induced inflammatory mediators in dental pulp cell cultures (26). These suggest that VR1 functions to release neuropeptides (SP and CGRP) in inflammatory pulp tissue. In the present study, we used capsaicin as a stimulant in some of our experiments to determine whether VR1 regulates IL-6 production in human dental pulp fibroblasts. The dose of 100 M capsaicin induced about 1000 pg/mg protein of IL-6 production (Fig. 2A–C). The capsaicin dose used here is high compared with the doses used in studies of inflammatory cytokine induction by capsaicin in human bronchial epithelial cell lines and in keratinocytes (20, 21). The LC50 values for capsaicin in human lung epithelial and hepatoma cells were approximately 110 and 200 M, respectively, in 24-h cultures (27). In our preliminary experiments, the viability of PF-10 cells stimulated with 100 M capsaicin was about 98%, which was not significantly different from the level of nonstimulated cells. On the basis of this result, we used the concentration of capsaicin that does not affect cell viability yet has the potential to effectively stimulate IL-6 induction. VR1 activation by capsaicin induces Ca2⫹ influx into neuronal cells (28). Consistent with neuronal VR1, VR1 induced a calcium influx in keratinocytes (19), as well as in human bronchial epithelial cells, when activated by capsaicin. Capsaicin induced IL-8 expression in a calcium-dependent manner in human keratinocytes (20). Thus, calcium influx is important for the VR1-mediated production of inflammatory mediators. However, we could not detect evidence of capsaicininduced Ca2⫹ influx in PF-10 cells by flow cytometry analysis using Fluo3-AM (Dojindo Laboratories, Tokyo, Japan) (unpublished observations). It has been reported that Ca2⫹ influx triggers paraptotic cell death, which does not fulfill the criteria for either apoptosis or necrosis (29). In our data, PF-10 cells changed morphologically and cell death occurred when cells were stimulated with greater than 200 M capsaVR1 Expression in Dental Pulp Cells
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Figure 3. Capsaicin induces the activation of p38 MAPK and JNK. (A) The PF-10 cells were stimulated with 1 M capsaicin for the indicated periods (A, B) with the indicated concentrations of capsaicin for 10 min (C, D). The PF-10 cells were pretreated with the indicated concentrations of capsazepine for 30 min and were subsequently stimulated with 1 M capsaicin for 10 min (E, F). Lysates were then analyzed by Western blotting. These data are representative of three independent experiments that gave similar results.
icin (data not shown). The further research is needed about the relationship to VR1 and cell death. The signaling pathway mechanism of VR1 activation is not well understood, but it is well known that the MAPK cascade is pivotal to the signaling pathways of many types of cells. The extracellular signal-regulated kinases (ERK-1 and ERK-2), like the classical MAPK, are mainly localized within the sub-pathways that transmit the signals for proliferation and differentiation. Likewise, p38 MAPK and JNK are activated by external stresses, such as osmotic pressure, heat stimuli, or UV irradiation (30). We investigated which MAPK could be involved in the capsaicin-induced IL-6 production through VR1. Consequently, involvement of p38 MAPK to VR1 activation was revealed by assaying the effects of specific inhibitors on IL-6 production. Ji and others reported that p38 MAPK activation mediated the inflammation-induced expression of VR1 protein in the dorsal root ganglion (31). As shown by Fig. 4, we demonstrated that capsaicin induced IL-6 production through the activation of p38 MAPK but not JNK in PF-10 cells. We also examined ERK activation using the same methodology. ERK activation was observed even in unstimulated cells and was upregulated after 5 min of stimulation with capsaicin. The capsaicin-activated ERK production was inhibited in part by capsazepine. A specific inhibitor of ERK did not inhibit capsaicin656
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induced IL-6 production (data not shown). Based upon these results, ERK may also be involved in the capsaicin-VR1 signaling pathway but not in VR1-related IL-6 production. NF-B is also an important transcriptional factor for the inflammatory signaling cascade and initiates the transcription of various proinflammatory mediators. Recently, it was reported that capsaicin inhibited NF-B activation in several cells, such as macrophage and myelomonoblastic leukemia cell, among others (32, 33). We observed the inhibition of NF-B by capsaicin in dental pulp cell cultures. Our results indicate that capsaicin inhibited TNF-␣ -induced NF-B activation in PF-10 cells. Furthermore, when PF-10 cells were preincubated with 10 M capsazepine for 30 min before treating with capsaicin, an inhibition of TNF-␣ -induced NF-B activation was not observed (data not shown). This suggests that VR1 is involved in the inhibition of NF-B by capsaicin. The mechanism of the inhibition of NF-B activation by capsaicin has not been elucidated. Further research is necessary to clarify this contradiction and the role of NF-B inactivation in capsaicin-induced IL-6 production. VR1 is activated by not only capsaicin, but also low pH or heat condition (7). Heat stimulus by hot food, tea, and so forth could trigger the inflammatory reaction of dental pulp, and could induce dental pain.
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Figure 5. NF-B activation by TNF-␣ is inhibited by capsaicin. PF-10 cells were either left untreated or pretreated with the indicated concentrations of capsaicin and subsequently stimulated with 0.1 nM TNF-␣ for 15 min. Total cell extracts were prepared and analyzed by EMSA using a 32P-labeled oligonucleotide that contained a high-affinity NF-B binding motif. The binding complex is indicated by the arrow. Two additional experiments gave similar results.
Figure 4. Capsaicin-induced IL-6 synthesis is inhibited by the p38 MAPK inhibitor, but not by the JNK inhibitor. (A) PF-10 cells were pretreated with the indicated concentrations of SB203580 (upper) or SP600125 (lower) for 1 h and were subsequently stimulated with 100 M capsaicin for 24 h. The levels of IL-6 in the cell culture supernatants were measured by ELISA as described in the Materials and Methods. The ELISA data are presented as the mean ⫾ SD (n ⫽ 3). The statistical analysis was performed with the statistical program for social science, using the Student’s t test for paired samples. Statistically significant differences are indicated by asterisks (*, p ⬍ 0.05; **, p ⬍ 0.01). Two additional experiments gave similar results. (B) IL-6 gene expression induced by capsaicin is mediated by the activation of p38 MAPK. PF-10 cells were either left untreated or pretreated with the indicated concentrations of SB203580, or SP600125 and were then exposed to 100 M capsaicin for 4 h. Upper panel indicates representative data of RT-PCR for IL-6, with GAPDH as an internal control. The expected size of the products are 544 bp for IL-6 and 318 bp for GAPDH. Lower panel indicates relative intensities that are expressed as the ratio of IL-6 to GAPDH with NIH image software. Data are presented as the mean ⫾ SD of three independent experiments.
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In another aspect, metal as restorative material has the potential to conduct heat and influence dental pulp through dentin. VR1 responds to protons, suggesting that its activity might be enhanced within the acidic environment of inflamed tissues (8). Taken together, current evidence supports the conclusion that VR1 responds to pain-producing stimuli, however, interaction of neuronal VR1 and nonneuronal VR1 remains unclear. Our results support the conclusion that the dental pulp fibroblast VR1 may act as a sensor for noxious stimuli. It can be said that VR1 and mediators that involved in the inflammatory reaction accompanying bacterial infection and tissue damage by external stimulus have a close relation, so pharmacological blockers of VR1 may provide significant relief from this type of pulpitis and consequently dental pain. Fibroblasts respond to numerous environment stimuli. These responses could be mediated by the activation of VR1, further displacing the notion that fibroblasts have the role of barriers for the teeth. Further studies are needed to elucidate the processes of fibroblast VR1 activation and the release of proinflammatory mediators in the development of dental pulp inflammation.
Acknowledgment This study was supported by a Grant-in-Aid for Specific Research (#15592024) from the Ministry of Education, Science, and Culture of Japan.
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expression in human dental pulp cell cultures stimulated with Prevotella intermedia lipopolysaccharide. J Endod 2001;27:273–7. Nagaoka S, Tokuda M, Sakuta T, et al. Interleukin-8 gene expression by human dental pulp fibroblast in cultures stimulated with Prevotella intermedia lipopolysaccharide. J Endod 1996;22:9 –12. Barkhordar RA, Hayashi C, Hussain MZ. Detection of interleukin-6 in human dental pulp and periapical lesions. Endod Dent Traumatol 1999;15:26 –7. Coil J, Tam E, Waterfield JD. Proinflammatory cytokine profiles in pulp fibroblasts stimulated with lipopolysaccharide and methyl mercaptan. J Endod 2004;30:88 –91. Lin SK, Kuo MYP, Wang JS, et al. Differential regulation of interleukin-6 and inducible cyclooxygenase gene expression by cytokines through prostaglandin-dependent and -independent mechanisms in human dental pulp fibroblasts. J Endod 2002;28:197– 201. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997; 389:816 –24. Tominaga M, Caterina MJ, Malmberg AB, et al. The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 1998;21:531– 43. Denda M, Fuziwara S, Inoue K, et al. Immunoreactivity of VR1 on epidermal keratinocyte of human skin. Biochem Biophys Res Commun 2001;285:1250 –2. Birder LA, Kanai AJ, de Groat WC, et al. Vanilloid receptor expression suggests a sensory role for urinary bladder epithelial cells. Proc Natl Acad Sci USA 2001;98: 13396 – 401. Dvorakova M, Kummer W. Transient expression of vanilloid receptor subtype 1 in rat cardiomyocytes during development. Histochem Cell Biol 2001;116:223–5. Chaudhary P, Martenson ME, Baumann TK. Vanilloid receptor expression and capsaicin excitation of rat dental primary afferent neurons. J Dent Res 2001;80:1518 – 23. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 1979;18: 5294 –9. Hayes P, Meadows HJ, Gunthorpe MJ, et al. Cloning and functional expression of a human orthologue of rat vanilloid receptor-1. Pain 2000;88:205–15. May LT, Helfgott DC, Sehgal PB. Anti- -interferon antibodies inhibit the increased expression of HLA-B7 mRNA in tumor necrosis factor-treated human fibroblasts: structural studies of the  2 interferon involved. Proc Natl Acad Sci USA 1986;83: 8957– 61. Tokunaga K, Nakamura Y, Sakata K, et al. Enhanced expression of a glyceraldehyde3-phosphate dehydrogenase gene in human lung cancers. Cancer Res 1987;47:5616 –9. Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 1983;11:1475– 89.
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18. Yiangou Y, Facer P, Ford A, et al. Capsaicin receptor VR1 and ATP-gated ion channel P2X3 in human urinary bladder. BJU Int 2001;87:774 –9. 19. Inoue K, Koizumi S, Fuziwara S, Denda S, Inoue K, Denda M. Functional vanilloid receptors in cultured normal human epidermal keratinocytes. Biochem Biophys Res Commun 2002;291:124 –9. 20. Southall MD, Li T, Gharibova LS, Pei Y, Nicol GD, Travers JB. Activation of epidermal vanilloid receptor-1 induces release of proinflammatory mediators in human keratinocytes. J Pharmacol Exp Ther 2003;304:217–22. 21. Veronesi B, Carter JD, Devlin RB, Simon SA, Oortgiesen M. Neuropeptides and capsaicin stimulate the release of inflammatory cytokines in a human bronchial epithelial cell line. Neuropeptides 1999;33:447–56. 22. Kido MA, Muroya H, Yamaza T, Terada Y, Tanaka T. Vanilloid receptor expression in the rat tongue and palate. J Dent Res 2003;82:393–7. 23. Szallasi A, Blumberg PM. Vanilloid (capsaicin) receptors and mechanisms. Pharmacol Rev 1999;51:159 –212. 24. Hargreaves KM, Jackson DL, Bowles WR. Adrenergic regulation of capsaicin-sensitive neurons in dental pulp. J Endod 2003;29:397–9. 25. Bowles WR, Withrow JC, Lepinski AM, Hargreaves KM. Tissue levels of immunoreactive substance P are increased in patients with irreversible pulpitis. J Endod 2003; 29:265–7. 26. Tokuda M, Miyamoto R, Nagaoka S, Torii M. Substance P enhances expression of lipopolysaccharide-induced inflammatory factors in dental pulp cells. J Endod 2004; 30:770 –3. 27. Reilly CA, Taylor JL, Lanza DL, Carr BA, Crouch DJ, Yost GS. Capsaicinoids cause inflammation and epithelial cell death through activation of vanilloid receptors. Toxicol Sci 2003;73:170 – 81. 28. Wood JN, Winter J, James IF, Rang HP, Yeats J, Bevan S. Capsaicin-induced ion fluxes in dorsal root ganglion cells in culture. J Neurosci 1988;8:3208 –20. 29. Jambrina E, Alonso R, Alcalde M, et al. Calcium influx through receptor-operated channel induces mitochondria-triggered paraptotic cell death. J Biol Chem 2003; 278:14134 – 45. 30. Robinson MJ, Cobb MH. Mitogen-activated protein kinase pathways. Curr Opin Cell Biol 1997;9:180 – 6. 31. Ji RR, Samad TA, Jin SX, Schmoll R, Woolf CJ. p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron 2002;36:57– 68. 32. Oh GS, Pae HO, Seo WG, et al. Capsazepine, a vanilloid receptor antagonist, inhibits the expression of inducible nitric oxide synthase gene in lipopolysaccharide-stimulated RAW264.7 macrophages through the inactivation of nuclear transcription factor-kappa B. Int Immunopharmacol 2001;1:777– 84. 33. Singh S, Natarajan K, Aggarwal BB. Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is a potent inhibitor of nuclear transcription factor-B activation by diverse agents. J Immunol 1996;157:4412–20.
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Expression and Characterization of Vanilloid Receptor Subtype 1 in Human Dental Pulp Cell Cultures Rie Miyamoto, DDS, Masayuki Tokuda, DDS, PhD, Tetsuya Sakuta, DDS, PhD, Shigetaka Nagaoka, DDS, PhD, and Mitsuo Torii, DDS, PhD Abstract 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 mitogenactivated protein kinases (MAPKs) was investigated. The phosphorylation of p38 MAPK and c-Jun NH2terminal 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␣)-induced nuclear factor-kappa B (NF-B) activation in PF-10 cell cultures. These results suggest that the activation of VR1 plays an important role in dental pulp inflammation.
Key Words VR1, capsaicin, IL-6, mitogen-activated protein kinases, NF-B
From the Department of Restorative Dentistry and Endodontology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan. Address requests for reprint to Dr. Masayuki Tokuda, Department of Restorative Dentistry and Endodontology, Kagoshima University Graduate School of Medical and Dental Sciences, 8 –35-1 Sakuragaoka, Kagoshima 890-8544, Japan. E-mail address: [email protected]. Copyright © 2005 by the American Association of Endodontists
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D
ental pulp fibroblasts play a role in pulp inflammation by producing cytokines when stimulated by bacterial pathogens (1–3). Among these cytokines, interleukin (IL)-6 is produced and released in locally inflamed pulpal lesions (4). Lipopolysaccharide (LPS) induces IL-6 production in human dental pulp fibroblasts, and its production is enhanced by methyl mercaptan, a volatile sulfur compound produced by anaerobic gram-negative bacteria (5). Human dental pulp fibroblasts participate in the development and progression of pulpitis via synthesizing IL-6, which is regulated by cytokines through prostaglandin-dependent pathway (6). For these reasons, IL-6 is an indispensable, and is important for the first defense mechanism in dental pulpitis. Vanilloid receptor subtype 1 (VR1, TRPV1) is a nonselective cation channel with very high permeability to calcium ions and is mainly expressed in small-diameter sensory neurons. VR1 contains six membrane domains with an additional short hydrophobic stretch between transmembrane regions 5 and 6; it shares amino-acid sequence similarity with transient receptor potential (TRP)-related proteins. VR1 was identified as the receptor for chemicals such as capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), a major pungent ingredient in hot chili peppers. It was reported that VR1 is activated by noxious heat (⬎40°C) and by acidic conditions (⬍ pH 6), which are conditions that occur during tissue injury (7, 8). This implies that the VR1 receptor channel is a primary cellular sensor of thermal and chemical stimuli. The expression of VR1 was previously reported to be localized to neurons that convey nociceptive transmissions. There is also growing evidence for the expression of VR1 in nonneuronal cells, such as keratinocytes, epithelial cells, and cardiomyocytes (9 –11). Although VR1 has been identified in human dental pulp neurons (12), the presence of VR1 in human dental pulp fibroblasts has not been documented. Thus, we investigated the expression of VR1 and its relationship to inflammatory mediators (particularly IL-6) in human dental pulp cell (PF-10) cultures. Moreover, we examined the signal transduction of capsaicin-activated VR1 in PF-10 cell cultures.
Materials and Methods Cell Cultures Normal human dental pulp tissue was obtained from the third molar of a 21-yearold female patient with her informed consent. Dental pulp fibroblasts were prepared from the growth of the minced explants as described previously (2). The cells were cultured under 5% CO2 in ␣ -modified Eagle’s minimum essential medium (␣ -MEM; ICN Biomedicals Inc., Irvine, CA) containing 10% fetal bovine serum (FBS; BioSource International Inc., Camarillo, CA) and 200 g/ml kanamycin. These cells, designated as PF-10 cells, were homogeneous, slim, spindle-shaped fibroblasts and grew in characteristic swirls. The cells were used between the 10th and 15th passages. Confluent cells were preincubated with 1% FBS-supplemented ␣ -MEM for 18 h before the addition of other factors, as described below. HEK293 cells were obtained from the Department of Laboratory and Molecular Medicine, Kagoshima University (Kagoshima, Japan). HeLa cells were obtained from Dainippon Pharmaceutical Co. (Osaka, Japan), and MRC5, normal human embryonic lung fibroblasts were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). These commercially acquired cultures were maintained in 10% FBS-supplemented ␣ -MEM with 200 g/ml kanamycin. Neuroblastoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were maintained in 10% FBS-supplemented Eagle’s minimum essential medium (EMEM; ATCC). HepG2 cells were obtained from Dainippon Pharmaceutical Co.
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Basic Research—Biology and were maintained in 10% FBS-supplemented M199 medium (Invitrogen, Carlsbad, CA). Astrocytoma cells were obtained from SankoJunyaku Co. (Tokyo, Japan) and were maintained in 10% FBS-supplemented RPMI1640 (Sigma-Aldrich Corp., St. Louis, MO).
Biochemicals Capsaicin was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Capsazepine, a VR1 antagonist, was obtained from Tocris Cookson, Inc. (Ellisville, MO). Human recombinant tumor necrosis factoralpha (TNF-␣) was purchased from Pierce Biotechnology, Inc. (Rockford, IL). The inhibitors of p38 mitogen-activated protein kinase (MAPK) (SB203580) and of c-Jun NH2-terminal kinase (JNK) (SP600125) were purchased from Calbiochem (San Diego, CA). Reverse-Transcribed Polymerase Chain Reaction (RT-PCR) Cultured cells were grown to confluence, and total cellular RNA was prepared from the cells by the guanidinium-isothiocyanate chlorform procedure (13). One microgram of total RNA was reverse transcribed using an RNA polymerase chain reaction (PCR) kit (TAKARA BIO, Inc., Shiga, Japan) according to the manufacturer’s instructions. PCR amplification was carried out using the following oligonucleotide primers for human VR1, IL-6, and human glyceraldhyde-3-phosphate dehydrogenase (GAPDH): for VR1, 5⬘ -TGTGCCGTTTCATGTTT-3⬘ and 5⬘ -TGCAGCTTCCAGATGTT-3⬘ (14); for IL-6, 5⬘ -ATGAACTCCTTCTCCACAAG-3⬘ and 5⬘ -GTGCCTGCAGCTTCGTCAGCA-3⬘ (15); for GAPDH, 5⬘ -ATCACCATCTTCCAGGAG-3⬘ and 5⬘ -ATGGACTGTGGTCATGAG-3⬘ (16). The cDNA was denatured at 94°C for 2 min, followed by 25 cycles of denaturation at 94°C for 30 s, annealing at 62°C (VR1 and IL-6) or 57°C (GAPDH) for 30 s, and extension at 72°C for 90 s. PCR products were electrophoresed on 1.5% agarose gels and visualized by ethidium bromide. Densitometric analysis was performed using the National Institutes of Health Image software (obtained from the National Institutes of Health Web site: http://rsb.info.nih.gov/nih-image). Each gel image was imported into Image using Adobe Photoshop (Adobe System, San Jose, CA). A gel-plotting macro was used to outline the bands, and the band intensity was calculated on the uncalibrated OD setting. The results are presented as the expression ratio of VR1 or IL-6 to GAPDH. Western Blot Analysis For the analysis of VR1 expression, cells of each type were grown in 145-cm2 dishes; confluent cells were washed with 1 ⫻ phosphate buffered saline (PBS) and lysed by sonication in a buffer containing 40 mM HEPES, 1% Triton-X100, 10% glycerol, and 1 mM phenylmethanesulfonyl fluoride (PMSF). The protein concentration was measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories; Hercules, CA). Protein samples prepared from the cells were boiled for 5 min with an equal volume of 2 ⫻ sample buffer (50 mM Tris-HCl, pH 6.8, 30% glycerol, 0.01% bromophenol blue (BPB), and 2% SDS); 100 g of each protein sample was subjected to SDS-PAGE on a 10% gel for 90 min at 100 V and electrophoretically transferred onto polyvinylidene fluoride (PVDF) membrane (Amersham Biosciences Corp., Piscataway, NJ) for 18 h at 30 V. The membranes were blocked for 1 h with 5% skim milk at 4°C and then incubated with primary antibody to VR1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell Signaling Technology, Inc., Beverly, MA). The signals were visualized with an enhanced chemiluminescence (ECL) system (Amersham Biosciences Corp.). For the detection of MAPK activation, stimulated or nonstimulated cells were washed with 1 ⫻ PBS and suspended in 100 M PMSF, 100 mM Na3VO4, 0.08% protease inhibitor cocktail (Roche Diagnostics Co., JOE — Volume 31, Number 9, September 2005
Basel, Switzerland), and 2 ⫻ sample buffer, then the cells were sonicated and boiled for 5 min. One-hundred micrograms of each protein sample was subjected to SDS-PAGE on a 10% gel for 90 min at 100 V and then electrophoretically transferred onto PVDF membrane for 18 h at 30 V. The membranes were blocked for 1 h with 5% skim milk at 4°C and then probed with primary antibodies to p38 MAPK and JNK or to the phosphorylated forms of p38 MAPK and JNK (Cell Signaling Technology Inc.), followed by incubation with HRP-conjugated antirabbit IgG and visualization with the ECL system.
Immunocytochemistry Cell of each type was cultured on glass coverslips, immersed in 4% paraformaldehyde for 18 h at 4° C, and washed three times with 1 ⫻ PBS at room temperature. Next, cells were incubated with blocking buffer (1.5% FBS in 1 ⫻ PBS) for 20 min at room temperature. VR1 was detected by incubation with rabbit polyclonal anti-human VR1 antibody for 4 h followed by incubation with goat anti-rabbit IgG conjugated with Alexa 546 dye (Molecular Probes, Inc., Eugene, OR) for 1 h at room temperature. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (0.1 g/ml) for 5 min at room temperature. After the cells were mounted on glass plates, immunofluorescence was observed using an Olympus BX51 fluorescence microscope (Olympus Corp., Tokyo, Japan). The composite images were created by DP manager software (Olympus Corp.). Enzyme-Linked Immunosorbent Assay Cells were grown in 24-well plates. After stimulation, the culture supernatants were collected and stored at ⫺80°C until use. All reagents in enzyme-linked immunosorbent assay (ELISA) system were purchased from R&D Systems, Inc. (Minneapolis, MN), and experimental procedure were conducted according to the manufacturer’s instructions. A 96-well plate was coated with monoclonal anti-human IL-6 antibody (2 g/ml) for 18 h at room temperature and blocked for 1 h at room temperature with 300 l of 1 ⫻ PBS containing 1% bovine serum albumin (BSA), 5% sucrose, and 0.05% NaN3 per well. After culture supernatants were centrifuged for 5 min at 10,000 ⫻ g to remove cellular debris, 100 l of each sample was added to a well, and the plate was incubated for 2 h at room temperature. The plate was washed three times using wash buffer (0.05% Tween-20 in 1 ⫻ PBS, pH 7.3), 100 l of biotinylated anti-human IL-6 antibody (300 g/ml) was added per well, and the plate was incubated for 2 h at room temperature. After the plate was washed three times, 100 l of streptavidin-HRP was added per well, and the plate was incubated for 20 min at room temperature. After washing three times, 100 l of substrate solution was distributed to each well, and the plate was incubated for 20 min in the dark. Then, 50 l of stop solution (1 M H2SO4) was added per well, and the absorbance at 450 nm was determined for each well using a microplate reader model 450 (Bio-Rad Laboratories, Inc.). The total protein in the medium was measured using Bio-Rad Protein Assay. The results are presented as the IL-6 concentration per total protein of each well. Electrophoretic Mobility Shift Assay (EMSA) Confluent cells were preincubated with capsaicin as indicated, followed by stimulation with 0.1 nM TNF-␣ for 15 min. Nuclear extracts were prepared as described by Dignam and others (17). Nuclear extracts (3 g) were incubated for 15 min at room temperature in a buffer that contained 10 mM Tris (pH 7.5), 100 mM potassium chloride, 5 mM MgCl2, 1 mM dithiothreitol, and 10% glycerol. A 32P-labeled, doublestranded oligonucleotide probe corresponding to the nuclear factorkappa B (NF-B) element (5⬘ -CGTGGAATTTCCTCTG-3⬘) from the IL-6 gene promoter was then added to the buffer. The DNA-protein complex VR1 Expression in Dental Pulp Cells
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Basic Research—Biology expression levels of VR1 mRNA at a PCR annealing temperature of 54°C (data not shown). The internal control showed PCR products for GAPDH at the expected size of 318 bp in each cell type (Fig. 1A). As shown in Fig. 1B, the Western blot analysis of protein isolates from cultured cells probed with the antihuman VR1 antibodies revealed a single protein band that was identical in size to those reported previously (13). The level of VR1 protein expression in PF-10 cells was weak compared with that in neuroblastoma cells. To further evaluate VR1 expression, cells were processed for VR1 immunoreactivity by using specific polyclonal antiserum for human VR1. Immunostaining photographs revealed VR1-immunoreactivity on the surface of PF-10 and neuroblastoma cells (Fig. 1C). In both cell types, immunoreactivity was absent without VR1 antibody. Taken together, these data suggest that VR1 receptors are expressed in resting cultured PF-10 cells and the level of expression in PF-10 cells is lower than that in neuroblastoma cells.
Figure 1. VR1 expression in PF-10 cells. (A) Upper panel: Representative data of RT-PCR for VR1, with GAPDH as an internal control (M, 100-bp ladder size marker; lane 1, PF-10; lane 2, MRC5; lane 3, HEK293; lane 4, HeLa; lane 5, HepG2; lane 6, neuroblastoma; lane 7, astrocytoma). The expected sizes of the products are 327 bp for VR1 and 318 bp for GAPDH. Lower panel: Bands were quantified with the NIH image software (National Institutes of Health). Data are expressed as the ratio of VR1 to GAPDH, and presented as the mean ⫾ SD of three independent experiments. (B) Western blot analysis of VR1 proteins obtained from PF-10 and neuroblastoma (NB) cells. The whole-cell lysates were immunoblotted with antihuman VR1 antibody (96 kDa). Two additional experiments gave results similar to those shown here. (C) Staining patterns of the VR1 in PF-10 (A) and neuroblastoma cells (B) with (right) and without (left) antihuman VR1 antibodies. Cells on a glass coverslip were immunostained with anti-human VR1 antibody raised in rabbits, and anti-rabbit IgG-Alexa 546 dye (red) was applied as a secondary antibody. Nuclei were counter-stained with DAPI (blue). Scale bars ⫽ 200 m. Two additional experiments gave similar results.
that formed was separated on a 5% polyacrylamide gel, the gel was dried, and radioactive bands were visualized by a Mac Bas 1000 (Hitachi, Tokyo, Japan) using an imaging plate (Fuji Photo Film Co., Tokyo, Japan).
Results VR1 Expression in PF-10 Cells We initially investigated the presence of VR1 in PF-10 cells by RT-PCR, Western blotting, and immunocytochemical analyses. Figure 1A shows the PCR products corresponding to VR1 from PF-10, MRC5, HEK293, HeLa, HepG2, neuroblastoma, and astrocytoma cells. These PCR products were detected as bands of 327 bp, the size expected for VR1. The expression of VR1 mRNA was at the same level in PF-10, MRC5, and HeLa cells; neuroblastoma, astrocytoma, HEK293, and HepG2 cells expressed high levels of VR1 mRNA. There were no differences in the 654
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Capsaicin Induces IL-6 Protein Synthesis We next evaluated the IL-6 production stimulated by capsaicin to clarify the function of VR1 in PF-10 cell cultures. Confluent cells were rendered quiescent by an 18-h incubation in ␣ -MEM containing 1% FBS, and then were stimulated with capsaicin. The concentrations of IL-6 in the cultured supernatants were measured by ELISA. The timedependent accumulation of IL-6 production upon stimulation with 100 M capsaicin is shown in Fig. 2A. IL-6 production levels began to increase after 12 h of exposure and reached a plateau at 24 h. The production of IL-6 in PF-10 cells was remarkably stimulated with 100 M capsaicin for 24 h (Fig. 2B). When PF-10 cells were treated without capsaicin, IL-6 was detected with about 200 pg/mg protein level at 8 h to 24 h. Although IL-6 production reached over 10,000 pg/mg protein when cultures were stimulated with 200 M capsaicin, the viability of the cells was about 30%; thus these findings are unclear at present (data not shown). Therefore, we used the dose of 100-M capsaicin in the subsequent IL-6 experiments. We used the VR1 antagonist capsazepine to assess whether capsaicin-induced IL-6 production is dependent on VR1. PF-10 cells were preincubated for 30 min at concentrations of capsazepine ranging from 10 M to 60 M, and then cultures were stimulated with 100 M capsaicin for 24 h. Figure 2C demonstrates that the induction of IL-6 protein synthesis by 100 M capsaicin was inhibited dose-dependently by capsazepine; the synthesis of IL-6 protein induced by 100 M capsaicin was essentially suppressed by greater than 30 M capsazepine. MAPKs Activation Stimulated with Capsaicin To investigate the effects of capsaicin on the activation of MAPKs, cells were treated with 1 M capsaicin for various time periods. Subsequently, total cell protein was extracted and subjected to Western blotting with antibodies specifically recognizing the phosphorylated or total kinases. As shown in Fig. 3A, p38 MAPK was activated after a 5-min treatment with capsaicin. Maximal activation was seen after 10 min, and activity returned to the nonstimulated level after 20 min. An activation of JNK by 1 M capsaicin was seen after 5 min, and it persisted for up to 60 min (Fig. 3B). p38 MAPK and JNK were activated when PF-10 cells were stimulated by exposure to any one of a range of capsaicin concentrations for 10 min (Fig. 3C,D). p38 MAPK was efficiently activated by 10-nM to 100 M capsaicin, whereas the levels of JNK activation were enhanced by 1 M to 100 M capsaicin (Fig. 3D). To further confirm that capsaicin induces the activation of MAPKs via VR1, we examined the effect of capsazepine on capsaicin-treated PF-10 cells. As shown in Fig. 3(E,F), capsazepine inhibited both the activation of p38 MAPK and that of JNK. JOE — Volume 31, Number 9, September 2005
Basic Research—Biology with different doses of capsaicin for 4 h and were then exposed to 0.1 nM TNF-␣ for 15 min. As shown in Fig. 5, the DNA binding activity of the NF-B element is reduced in the presence of capsaicin compared with treatment with TNF-␣ alone. Moreover, upon pretreatment with 60 M capsazepine for 30 min before stimulation with 100 M of capsaicin, the inhibition of TNF-␣ -induced NF-B activation was not observed (data not shown).
Discussion
Figure 2. Capsaicin induced IL-6 protein synthesis in PF-10 cell cultures via VR1. (A) Time course of IL-6 production in PF-10 cells stimulated with 100 M capsaicin for the indicated periods. (B) Dose response of IL-6 production in PF-10 cells stimulated with indicated concentrations of capsaicin for 24 h. (C) Capsazepine inhibits capsaicin-induced IL-6 production in PF-10 cell cultures. The PF-10 cells were either left untreated or pretreated with the indicated concentrations of capsazepine. IL-6 production in cultured supernatants was determined by ELISA as described in Materials and Methods. Results are shown as the mean ⫾ SD. Two additional experiments gave similar results.
Involvement of MAPKs in Capsaicin-Induced IL-6 Production We also investigated the involvement of p38 MAPK and JNK in capsaicin-induced IL-6 production. PF-10 cells were pretreated with specific inhibitors of p38 MAPK and JNK at three different concentrations, and then stimulated with 100 M capsaicin. As shown in Fig. 4A, a specific inhibitor of p38 MAPK (SB203580) dose-dependently inhibited the IL-6 protein synthesis induced by capsaicin, whereas a specific inhibitor of JNK (SP600125) slightly enhanced capsaicin-induced IL-6 synthesis. We examined the capsaicin-induced expression of IL-6 mRNA to confirm our data obtained at the protein level. As shown in Fig. 4B, the capsaicin-induced expression of IL-6 mRNA was inhibited by SB203580, but enhanced by SP600125. This result is in agreement with the result of a protein level. The mRNA expression of the control, GAPDH, was not affected by capsaicin or either of the inhibitors. Capsaicin Inhibits TNF-␣ -Induced NF-B Activation Finally, we examined the effects of capsaicin on NF-B activation, which is important for IL-6 transcription. PF-10 cells were pretreated JOE — Volume 31, Number 9, September 2005
We found that VR1 was expressed on human dental pulp cells in culture. The expression of VR1 has been well established in sensory neurons (7, 12). VR1 functions as a transducer of painful stimuli, conveying nociceptive information to the central nervous system by activating sensory neurons (7). VR1 also plays a role in the immunoreactive responses of human skin and keratinocytes (9, 19). Epidermal VR1 activated by capsaicin induces proinflammatory mediators such as cyclooxygenase-2, IL-8, and prostaglandin E2 in the skin (20). In the human bronchial epithelial cell line, neuropeptides and capsaicin stimulated the release of inflammatory cytokines (21). These observations suggest that VR1 exists in both neuronal and nonneuronal cells, and has a role in the inflammatory response. In the dental area, VR1 was found in the rat dental primary afferent neurons (12) and in the nerve and epithelial cells of the rat tongue and palate, which provide defense mechanisms against toxic substances in the rat oral cavity (22). Activation of VR1 induces release of neuropeptides, calcitonine gene-related protein (CGRP) and substance P (SP) in the nerve fibers and terminals (23). The release of CGRP was evoked by capsaicin, inhibited by capsazepine in bovine dental pulp (24). Bowles and others demonstrated that extracellular levels of SP are increased within symptomatic pulp tissue diagnosed with irreversible pulpitis (25). We recently reported SP enhances expression of LPS-induced inflammatory mediators in dental pulp cell cultures (26). These suggest that VR1 functions to release neuropeptides (SP and CGRP) in inflammatory pulp tissue. In the present study, we used capsaicin as a stimulant in some of our experiments to determine whether VR1 regulates IL-6 production in human dental pulp fibroblasts. The dose of 100 M capsaicin induced about 1000 pg/mg protein of IL-6 production (Fig. 2A–C). The capsaicin dose used here is high compared with the doses used in studies of inflammatory cytokine induction by capsaicin in human bronchial epithelial cell lines and in keratinocytes (20, 21). The LC50 values for capsaicin in human lung epithelial and hepatoma cells were approximately 110 and 200 M, respectively, in 24-h cultures (27). In our preliminary experiments, the viability of PF-10 cells stimulated with 100 M capsaicin was about 98%, which was not significantly different from the level of nonstimulated cells. On the basis of this result, we used the concentration of capsaicin that does not affect cell viability yet has the potential to effectively stimulate IL-6 induction. VR1 activation by capsaicin induces Ca2⫹ influx into neuronal cells (28). Consistent with neuronal VR1, VR1 induced a calcium influx in keratinocytes (19), as well as in human bronchial epithelial cells, when activated by capsaicin. Capsaicin induced IL-8 expression in a calcium-dependent manner in human keratinocytes (20). Thus, calcium influx is important for the VR1-mediated production of inflammatory mediators. However, we could not detect evidence of capsaicininduced Ca2⫹ influx in PF-10 cells by flow cytometry analysis using Fluo3-AM (Dojindo Laboratories, Tokyo, Japan) (unpublished observations). It has been reported that Ca2⫹ influx triggers paraptotic cell death, which does not fulfill the criteria for either apoptosis or necrosis (29). In our data, PF-10 cells changed morphologically and cell death occurred when cells were stimulated with greater than 200 M capsaVR1 Expression in Dental Pulp Cells
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Figure 3. Capsaicin induces the activation of p38 MAPK and JNK. (A) The PF-10 cells were stimulated with 1 M capsaicin for the indicated periods (A, B) with the indicated concentrations of capsaicin for 10 min (C, D). The PF-10 cells were pretreated with the indicated concentrations of capsazepine for 30 min and were subsequently stimulated with 1 M capsaicin for 10 min (E, F). Lysates were then analyzed by Western blotting. These data are representative of three independent experiments that gave similar results.
icin (data not shown). The further research is needed about the relationship to VR1 and cell death. The signaling pathway mechanism of VR1 activation is not well understood, but it is well known that the MAPK cascade is pivotal to the signaling pathways of many types of cells. The extracellular signal-regulated kinases (ERK-1 and ERK-2), like the classical MAPK, are mainly localized within the sub-pathways that transmit the signals for proliferation and differentiation. Likewise, p38 MAPK and JNK are activated by external stresses, such as osmotic pressure, heat stimuli, or UV irradiation (30). We investigated which MAPK could be involved in the capsaicin-induced IL-6 production through VR1. Consequently, involvement of p38 MAPK to VR1 activation was revealed by assaying the effects of specific inhibitors on IL-6 production. Ji and others reported that p38 MAPK activation mediated the inflammation-induced expression of VR1 protein in the dorsal root ganglion (31). As shown by Fig. 4, we demonstrated that capsaicin induced IL-6 production through the activation of p38 MAPK but not JNK in PF-10 cells. We also examined ERK activation using the same methodology. ERK activation was observed even in unstimulated cells and was upregulated after 5 min of stimulation with capsaicin. The capsaicin-activated ERK production was inhibited in part by capsazepine. A specific inhibitor of ERK did not inhibit capsaicin656
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induced IL-6 production (data not shown). Based upon these results, ERK may also be involved in the capsaicin-VR1 signaling pathway but not in VR1-related IL-6 production. NF-B is also an important transcriptional factor for the inflammatory signaling cascade and initiates the transcription of various proinflammatory mediators. Recently, it was reported that capsaicin inhibited NF-B activation in several cells, such as macrophage and myelomonoblastic leukemia cell, among others (32, 33). We observed the inhibition of NF-B by capsaicin in dental pulp cell cultures. Our results indicate that capsaicin inhibited TNF-␣ -induced NF-B activation in PF-10 cells. Furthermore, when PF-10 cells were preincubated with 10 M capsazepine for 30 min before treating with capsaicin, an inhibition of TNF-␣ -induced NF-B activation was not observed (data not shown). This suggests that VR1 is involved in the inhibition of NF-B by capsaicin. The mechanism of the inhibition of NF-B activation by capsaicin has not been elucidated. Further research is necessary to clarify this contradiction and the role of NF-B inactivation in capsaicin-induced IL-6 production. VR1 is activated by not only capsaicin, but also low pH or heat condition (7). Heat stimulus by hot food, tea, and so forth could trigger the inflammatory reaction of dental pulp, and could induce dental pain.
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Basic Research—Biology
Figure 5. NF-B activation by TNF-␣ is inhibited by capsaicin. PF-10 cells were either left untreated or pretreated with the indicated concentrations of capsaicin and subsequently stimulated with 0.1 nM TNF-␣ for 15 min. Total cell extracts were prepared and analyzed by EMSA using a 32P-labeled oligonucleotide that contained a high-affinity NF-B binding motif. The binding complex is indicated by the arrow. Two additional experiments gave similar results.
Figure 4. Capsaicin-induced IL-6 synthesis is inhibited by the p38 MAPK inhibitor, but not by the JNK inhibitor. (A) PF-10 cells were pretreated with the indicated concentrations of SB203580 (upper) or SP600125 (lower) for 1 h and were subsequently stimulated with 100 M capsaicin for 24 h. The levels of IL-6 in the cell culture supernatants were measured by ELISA as described in the Materials and Methods. The ELISA data are presented as the mean ⫾ SD (n ⫽ 3). The statistical analysis was performed with the statistical program for social science, using the Student’s t test for paired samples. Statistically significant differences are indicated by asterisks (*, p ⬍ 0.05; **, p ⬍ 0.01). Two additional experiments gave similar results. (B) IL-6 gene expression induced by capsaicin is mediated by the activation of p38 MAPK. PF-10 cells were either left untreated or pretreated with the indicated concentrations of SB203580, or SP600125 and were then exposed to 100 M capsaicin for 4 h. Upper panel indicates representative data of RT-PCR for IL-6, with GAPDH as an internal control. The expected size of the products are 544 bp for IL-6 and 318 bp for GAPDH. Lower panel indicates relative intensities that are expressed as the ratio of IL-6 to GAPDH with NIH image software. Data are presented as the mean ⫾ SD of three independent experiments.
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In another aspect, metal as restorative material has the potential to conduct heat and influence dental pulp through dentin. VR1 responds to protons, suggesting that its activity might be enhanced within the acidic environment of inflamed tissues (8). Taken together, current evidence supports the conclusion that VR1 responds to pain-producing stimuli, however, interaction of neuronal VR1 and nonneuronal VR1 remains unclear. Our results support the conclusion that the dental pulp fibroblast VR1 may act as a sensor for noxious stimuli. It can be said that VR1 and mediators that involved in the inflammatory reaction accompanying bacterial infection and tissue damage by external stimulus have a close relation, so pharmacological blockers of VR1 may provide significant relief from this type of pulpitis and consequently dental pain. Fibroblasts respond to numerous environment stimuli. These responses could be mediated by the activation of VR1, further displacing the notion that fibroblasts have the role of barriers for the teeth. Further studies are needed to elucidate the processes of fibroblast VR1 activation and the release of proinflammatory mediators in the development of dental pulp inflammation.
Acknowledgment This study was supported by a Grant-in-Aid for Specific Research (#15592024) from the Ministry of Education, Science, and Culture of Japan.
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