of January 19, 2016.This information is current as

SignalingReceptor-2by RgpB via Proteinase-Activated

Neuropeptide Release from Dental Pulp Cells

Ikuro MaruyamaTatsuyama, James Travis, Jan Potempa, Mitsuo Torii andImamura, Kamal Krishna Biswas, Kenji Matsushita, Shoko Salunya Tancharoen, Krishna Pada Sarker, Takahisa

http://www.jimmunol.org/content/174/9/5796doi: 10.4049/jimmunol.174.9.5796

2005; 174:5796-5804; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2005 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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Neuropeptide Release from Dental Pulp Cells by RgpB viaProteinase-Activated Receptor-2 Signaling

Salunya Tancharoen,*† Krishna Pada Sarker,‡ Takahisa Imamura,§ Kamal Krishna Biswas,†

Kenji Matsushita,¶ Shoko Tatsuyama,* James Travis,� Jan Potempa,# Mitsuo Torii,* andIkuro Maruyama1†

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. Tostudy an involvement of the periodontitis pathogen and neuropeptides in pulp inflammation, we investigated human dental pulpcell neuropeptide release by arginine-specific cysteine protease (RgpB), a cysteine proteinase of P. gingivalis, and participatingsignaling pathways. RgpB induced neuropeptide release from cultured human pulp cells (HPCs) in a proteolytic activity-depen-dent 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-2agonists, SLIGKV and trypsin, also induced neuropeptide release from HPCs, and HPC PAR-2 gene knockout by transfection ofPAR-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 releasefrom HPC revealed a new PAR-2 signaling pathway that was mediated by p38 MAPK and activated transcription factor-2activation, in addition to the PAR-2-p44/42 p38MAPK and -AP-1 pathway. This new RgpB activity suggests a possible linkbetween periodontitis and pulp inflammation, which may be modulated by neuropeptides released in the lesion. The Journal ofImmunology, 2005, 174: 5796–5804.

P orphyromonas gingivalis, a Gram-negative anaerobicbacterium, is one of the major pathogens of periodontitis,which causes periodontal tissue destruction and conse-

quent teeth loss. Cell surface-associated and secretory trypsin-likecysteine proteinases (gingipains) produced by P. gingivalis are im-portant virulence factors and are implicated in the development ofperiodontitis (1, 2). Periodontitis often leads to acute pulpitis andpulpal necrosis by dissemination of the pathogens and/or their vir-ulence components (3–10). Arginine-specific cysteine protease(RgpB)2 is a gingipain (11) and has various proinflammatory ac-tivities including vascular permeability enhancement and edemainduction (1, 2), which can occur in acute pulpitis (12, 13). RgpBmay be associated with pulpal inflammation.

Calcitonin gene-related peptide (CGRP) and substance P (SP)are neuropeptides that are widely codistributed in central and pe-

ripheral neurons (14–16). Both neuropeptides induce vasodilation(16), local blood flow increase (15), vascular permeability en-hancement (17), and pain (18, 19), thus modulating inflammation(20, 21). CGRP and SP are present in pulpal and gingival tissues(17, 22, 23) and are increased markedly in human teeth exposingpulp due to advanced caries and/or those with painful pulpitis (24,25), which suggests a modulation of pulpal inflammation by thesepeptides. CGRP and SP are released from neurons by activation ofproteinase-activated receptors (PAR) (14), a family of four sub-types of seven transmembrane domain receptors coupled with Gprotein (26). RgpB induces platelet cytosolic Ca2� increase andaggregation via PAR-1 and -4 (27) and gingival epithelial cell IL-6secretion via PAR-1 and -2 (28). RgpB may induce the release ofCGRP and SP from pulp cells through PAR activation and partic-ipate in the pulpitis that accompanies periodontitis.

To study an involvement of RgpB and the neuropeptides in den-tal pulp inflammation, we investigated neuropeptide release fromhuman pulp cells by RgpB and its mechanism including activationof PAR and the following signaling pathway.

Materials and MethodsChemicals

The synthetic PAR-2 agonist peptide H-SLIGKV-NH2, corresponding tothe tethered ligand, was purchased from Asahi Technoglass. Leupeptin waspurchased from Peptide Institute. SB202190 and SP600125 were pur-chased from Calbiochem. U0126 was purchased from Promega. Trypsin250 was purchased from Difco Laboratories. Other reagents were suppliedby Sigma-Aldrich.

Antibodies

Anti-human PAR-1 and PAR-2 mAbs and anti-PAR-2, PAR-3, and PAR-4polyclonal Abs were purchased from Santa Cruz Biotechnology. Rabbitanti-SP and anti-CGRP Abs were purchased from ICN Biomedicals andPeninsula Laboratories, respectively. mAb 1B10 was purchased fromSigma-Aldrich. MAPK assay kits (containing polyclonal Abs against p38,

Departments of *Restorative Dentistry and Endodontology and †Laboratory and Vas-cular Medicine, Kagoshima University Graduate School of Medical and Dental Sci-ence Kagoshima, Japan; ‡Department of Cell Biology and Anatomy, University ofCalgary, Faculty of Medicine, Calgary, Canada; §Division of Molecular Pathology,Kumamoto University Graduate School of Medical and Pharmaceutical Sciences,Kumamoto, Japan; ¶Department of Cardiology, Johns Hopkins University MedicalSchool, Baltimore, MD 21205; �Department of Biochemistry, University of Georgia,Athens, Georgia; and #Department of Microbiology, Faculty of Biotechnology,Jagiellonian University, Krakow, Poland

Received for publication March 8, 2004. Accepted for publication February 22, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 Address correspondence and reprint requests to Dr. Ikuro Maruyama, Department of Lab-oratory and Vascular Medicine, Kagoshima University Graduate School of Medical and Den-tal Science, Kagoshima, Japan. E-mail address: rinken@m3.kufm.kagoshima-u.ac.jp2 Abbreviations used in this paper: RgpB, arginine-specific cysteine protease; HPC,human pulp cell; PAR, proteinase-activated receptor; SP, substance P; CGRP, calci-tonin gene-related peptide; HGF, human gingival fibroblast; ATF-2, activated tran-scription factor-2; dn-ATF-2, dominant negative ATF-2; TFA, trifluoroacetic acid.

The Journal of Immunology

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00

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JNK/stress-activated protein kinase, and p44/42, phospho-p38, phospho-JNK/stress-activated protein kinase, and phospho-p44/p42) and anti-phospho-ATF-2 Ab were purchased from Cell Signaling Technology.

Purification and activation of RgpB

RgpB was isolated as previously described by Potempa et al. (29). PurifiedRgpB was activated with 10 mM L-cysteine in 0.2 M HEPES buffer, pH8.0, containing 5 mM CaCl2 at 37°C for 5 min and then kept at roomtemperature. The proteinase was diluted with 50 mM Tris-HCl, pH 7.4,containing 0.1 M NaCl and 5 mM CaCl2 immediately before use. Theamount of active enzyme in each 0purified proteinase was determined byactive site titration with D-phenylalanylprolylarginylcholoromethyl ketone(D-Phe-Pro-Arg-CH2Cl). The concentration of active RgpB was calculatedfrom the amount of the inhibitor needed for complete inactivation of theproteinase (30).

Cell culture

Human pulp cells (HPCs) were prepared as described previously (31).Briefly, normal human dental pulp tissue was obtained from extracted firstpremolars and was cultured in �-MEM (ICN Biomedicals) supplementedwith 10% FBS. The cells at subculture passage 5–9 (�80% confluent) wereused in this study. All experiments using inhibitor or/and stimulators wereperformed in �-MEM containing 0.2% FBS. Human gingival fibroblasts(HGF) were obtained from healthy gingival tissues cultured in DMEM(Life Technologies) supplemented with 10% FBS. Outgrown cells from thetissue cultures were subcultured and used at later passages 5–9. Humanalveolar epithelial cells (A549) cultured as described previously (32) wereseeded into collagen (type I)-coated tissue culture dishes and were grownto confluence in DMEM supplemented with 10% FBS at 37°C in 5% CO2.

Preparation/concentration of samples

HPCs (5 � 104 cells/well) were suspended onto 24-well cell culture plates(Nunclon) in the appropriate growth medium supplemented with 10% FBS.After an 18-h incubation in serum-free �-MEM, the 80% confluent cellswere stimulated with RgpB in the presence or absence of leupeptin. Thesupernatants (2 ml) were concentrated according to the ELISA kit protocolfor sample preparation. Briefly, cartridges of Sep-Pak C18 (Waters) wereequilibrated with acetonitrile, followed by 1% trifluoroacetic acid (TFA).Samples diluted with an equal volume of 1% TFA were slowly loaded ontothe column, and the column was washed with 10 ml of 1% TFA. Adsorbedpeptides were then eluted with 3 ml of 60% (v/v) acetonitrile. The eluantwas concentrated by microcentrifugal vacuum concentrator (MV-100;TOMY TECH) and stored at �20°C.

CGRP and SP assay

Samples reconstituted with 0.5 ml of assay buffer were assayed for CGRPand SP using ELISA kits for CGRP (BACHEM/Peninsula Laboratories)and SP (Assay Designs) according to the manufacturer’s instructions.Briefly, CGRP assay is based on the competitive binding of CGRP and thebiotinylated CGRP in standard solutions or samples to an anti-CGRP Ab.The standard CGRP solutions (0.04–1,000 ng/ml) and samples were mea-sured in duplicate. An appropriately diluted Ab against CGRP and nonbi-otinylated CGRP (either standard or 0.05 ml of unknown concentratedsamples) were mixed in a microtiter plate well. After incubation at roomtemperature for 1 h, 0.025 ml of biotinylated CGRP was poured to the welland further incubated for 2 h. Unbound biotinylated CGRP was removedfrom the well by washing five times with the assay buffer, and the well wasdried. The 0.1 ml of streptavidin-conjugated HRP was added to the welland incubated for 1 h to make an immobilized primary Ab-biotinylatedpeptide complex. After excess streptavidin-conjugated-HRP was washedaway, the bound complex was reacted with 0.1 ml of 3,3�,5,5�-tetrameth-ylbenzidine dihydrochloride for 30 min. The enzyme-substrate reactionwas stopped, and the yellow color generated was measured at 450 nm. Theminimum detection limit of the assay is 0.04–0.06 ng/ml with intra- andinterassay coefficient of variation �5% and 14%, respectively. The SPassay is based on the competitive binding to a limited quantitative SP-specific rabbit Ab between SP and alkaline phosphate-labeled SP. Thestandard solutions of SP (9.76–10,000 pg/ml) and samples were measuredin duplicate. An appropriately diluted standard SP and 0.05 ml of a samplewas mixed in a microtiter plate well. The alkaline phosphatase-conjugatedSP for 0.05 ml was then added to each well. After a 2-h incubation with0.05 ml of rabbit anti-SP at room temperature, the excess reagents werewashed away from the well, followed by an addition of p-nitrophenyl phos-phate. After a short-time incubation, the enzyme reaction was stopped, andthe yellow color generated was measured at 405 nm with a Multilabelcounter using the Wallac 1420 ARVO program. The minimum detection

limit of the assay is 8.04 pg/ml with intra- and interassay coefficient vari-ation being �6.7% and 4.2%, respectively.

In both assays, the OD values in a well were inversely proportional tothe concentrations of CGRP or SP. Peptide concentrations of samples wereestimated from the standard best fit lines made from the plots of the ODvalues of neuropeptides with known concentrations.

To confirm reliability of the ELISA, the supernatant of cells stimulatedby RgpB at 200 nM was concentrated and reconstituted with the assaybuffer as described above. The supernatant was serially 2-fold diluted, andneuropeptide concentrations of diluted samples were measured by ELISA.Best fit lines were obtained from the OD values of serial 2-fold dilutedsamples of the supernatant and compared with corresponding standard bestfit lines.

mRNA detection of the neuropeptides and PAR-1, -2, -3, and -4

Total RNA was extracted using TRIzol reagent according to the manufac-turer’s instructions (Invitrogen). First-strand cDNA was synthesized byreverse transcriptase using a commercial RT-PCR kit (Takara Biomedi-cals), and the reaction was performed following the manufacturer’s instruc-tions. Briefly, 1 �g of RNA was added to a 20-�l reaction volume con-taining 1 �l of random primers, 2 �l of 10� first-strand buffer, 0.16 �l ofRNase inhibitor (1 U/�l), 8 �l of 1 mM dNTP, and 0.14 �l of reversetranscriptase (0.25 U/�l). The reaction mix was incubated at 37°C for 60min and then at 90°C for 5 min. The resulting cDNA mixture was amplifiedwith Taq polymerase, and the following specific primers were synthesizedby Hokkaido System Science: CGRP, 5�-GAACTATTGCTAAATGCAGAACAAGCT-3� and 5�-ATTTGCTACCAGATAAGCCAATGAGA-3�(PCR product, 119 bp; GenBank accession no. X02330); SP, 5�-GACAGCGACCAGATCAAGGAGGAA-3� and 5�-CAGCATCCCGTTTGCC-3� (PCR product, 121 bp; GenBank accession no. U37529); PAR-1,5�-TACACCGGAGTGTTTGTAGT-3� and 5�-TTGAGGACGAGAGGCACTAC-3� (PCR product, 395 bp; GenBank accession no. M62424);PAR-2, 5�-GGTAAGGTTGATGGCACATC-3� and 5�-TGGTCTGCTTCACGACATAC-3� (PCR product, 509 bp; GenBank accession no.U34038); PAR-3, 5�-ATCTCATAGCTTTGTGCCTG-3� and 5�-CACGCCTGTAATCCAGCACT-3� (PCR product, 488 bp; GenBank accession no.U92971); PAR-4, 5�-AGTCTGTGCCAATGACAGTG-3� and 5�-TCATGGCAGAGCACGCGATC-3� (PCR product, 534 bp; GenBank accessionno. AF055917); GAPDH, 5�-CATCACCATCTTCCAGGAGC-3� and 5�-CATGAGTCCTTCCACGATACC-3� (PCR product, 286 bp). Amplifica-tion conditions were 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°Cfor 90 s for PAR-1 and PAR-2; 35 cycles of 94°C for 30 s, 59°C for 30 s,and 72°C for 90 s for PAR-3 and PAR-4; and 30 cycles of 94°C for 30 s,55°C for 30 s, and 72°C for 30 s with primer extension time at 72°C for30 s for CGRP; 30 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 30 swith primer extension time at 72°C for 30 s for SP; and 28 cycles of 94°Cfor 30 s, 56°C for 30 s, and 72°C for 90 s for GAPDH. To eliminate thepossibility of false positive results of neuropeptide expression by contam-inating DNA, RT-PCR was performed in the absence of reverse transcrip-tase, primers or RNA was shown as controls.

Detection of PARs on HPCs by flow cytometric analysis

HPCs and A549 cells were grown in monolayers in cell culture dishes in�-MEM or DMEM supplemented with 10% FBS, respectively. Cell mono-layers were dispersed and resuspended at a final concentration of 3 � 106

cells/ml. Cells were incubated with 20 �g/ml anti-PARs Abs or with non-immune serum for 1 h, followed by the FITC-conjugated second Ab (ICNPharmaceuticals) for 30 min. Fluorescence was analyzed with a FACSCan(Beckman Coulter).

Dental pulp tissue preparation

Dental pulp tissues (n � 20) were obtained from patients with informedconsent according to the guideline approved by the Ethical Committee atKagoshima University Dental School. Teeth were excluded from the studyif any physiological root resorption was found. After extraction, the groovewas cut off from the buccal aspect of the tooth, and the pulp tissue takencarefully from its chamber was fixed in 4% paraformaldehyde and embed-ded in paraffin.

Immunohistochemistry

Paraffin-embedded sections (5 �m) of pulp tissues were deparaffinized inxylene and rehydrated through decreasing concentrations of ethanol. Toblock endogenous peroxidase activity, we treated sections with 3% (v/v)H2O2 for 20 min and then processed them for immunostaining using pri-mary Abs and LSAB� Kit (DAKO). Sections were incubated in eachprimary Ab for 1 h at room temperature (anti-PAR-1, -2, -1B10 mouse

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mAbs, 100� dilution; anti-PAR-2 goat Ab, 50� dilution; anti-PAR-3 and-4 goat Abs, 100� dilution; anti-CGRP rabbit Ab, 250� dilution; anti-SPrabbit Ab, 200� dilution). After a washing, sections were incubated inLINK (biotinylated anti-rabbit, anti-mouse, and anti-goat Igs). Sectionswere washed and further incubated in streptavidin conjugated to HRP for30 min. As negative controls, each isotype nonimmune serum (2 �g/ml) ofthe same species was used instead of the primary Ab. The specificity of theAbs for PAR-2 was confirmed by preincubation with its specific blockingpeptides (Santa Cruz Biotechnology) (1/50 dilution) at 37°C for 1 h. Allincubation procedures were performed in a humid chamber. The peroxi-dase bound to the tissue sections was detected using 3,3�-diaminobenzidineand H2O2. The tissue sections were counterstained with hematoxylin,mounted in Aquatex (Merck) and examined with an Olympus BH 2 lightmicroscope.

Immunocytochemistry

HPCs were grown overnight in a Lab-Tech chamber slide (Nalge NuncInternational). Cells were fixed with 4% paraformaldehyde and permeabil-ized in PBS with 0.1% Triton X-100 for 15 min at room temperature.Nonspecific binding was blocked by incubating the cells with 1% normalgoat serum in PBS for 30 min. Slides were incubated in each primary Ab(anti-CGRP, -SP, or -PAR-2 Ab; 50� dilution and anti-1B10 Ab; 100�dilution) in PBS containing 0.1% Triton X-100 for 12 h at 4°C. After awashing, slides were incubated with tetramethylrhodamine (Alexa Fluor546; Molecular Probes)-conjugated goat Ab against rabbit IgG (1/100 di-lution with PBS containing 0.1% Triton X-100) for 30 min in the dark.Finally, immunoreactivity was visualized using a confocal microscope(Leica TCS4D; Leica).

Transient transfection experiments

Human sense or antisense synthetic phosphorothioate-modified oligonu-cleotides homologous to PAR-2 and c-jun were purified by HPLC. AllPAR-2 oligonucleotides used in this study were evaluated for homology topreexisting genes using the BLAST homology search program and thecurrent version of GenBank database. The PAR-2 nucleotide sequencesused were: sense, 5�-ATGCGGAGCCCCAGCGCGGC-3�; antisense, 5�-GCCGCGCTGGGGCTCCGCAT-3�, nucleotides 148–167; GenBank ac-cession no. U34038 (Sigma Genosis). The phosphorothioate-modified oli-gonucleotides are indicated by an underline. The control and antisensec-jun fully phosphorothioate-modified oligonucleotides directed against thefirst 18 bases of human c-jun mRNA were purchased from BIOMOLResearch Laboratories. The sequences used were: control c-jun oligonu-cleotide, 5�-ACTGCAAAGATGGAAACG-3�; antisense c-jun oligonucle-otide, 5�-CGTTTCCATCTTTGCAGT-3�.

Dominant negative activating transcription factor-2 (replacement ofThr69 or Thr71 with Ala, dn-ATF-2) was kindly provided by Dr. R. J. Davis(Howard Hughes Medical Institute) as described previously (33).

LipofectAMINE 2000 (Invitrogen Life Technologies) was used as acarrier of the oligonucleotides or dn-ATF-2, and we modified the manu-facturer’s protocol for transfection (34). Briefly, HPCs were washed withprewarmed (37°C) serum-free OptiMEM medium (Invitrogen Life Tech-nologies). The oligonucleotides (0.2 �g) or dn-ATF-2 (0.1 �g) were pre-mixed with 2 �l of LipofectAMINE 2000 in 50 �l of OptiMEM medium

for 30 min at 37°C and overlaid onto the washed cells. HPCs were incu-bated with either oligonucleotide-LipofectAMINE 2000 complex (for 48 h)or dn-ATF-2-LipofectAMINE 2000 complex (for 24 h) at 37°C in 5% CO2.After each transfection experiment, cells were incubated with RgpB for1 h. The supernatants were collected for ELISA. Total RNA was extractedfrom the PAR-2 oligonucleotide-transfected cells for RT-PCR. Nuclearprotein was extracted from the c-jun oligonucleotide-transfected cellsfor EMSA.

Preparation of cell lysates and Western blotting analysis

MAPKs activation was analyzed according to the method of Sarker et al.(35), with modification. In brief, 120 �l of cell suspension (5 � 105 cells/dish) were seeded onto 60-mm cell culture dishes, and cell lysates wereobtained by adding 120 �l of SDS sample buffer (containing 50 mM DTT,1 mM PMSF, and 0.5 mM Na2VO3). An equal volume of cell lysate wasapplied to each lane of a 12% SDS-polyacrylamide gel. After protein trans-fer, the nitrocellulose membrane (Schleicher & Schuell) was washed withTBST, followed by blocking with 5% nonfat milk plus 1% BSA for 1 h.

FIGURE 1. CGRP and SP in the supernatant of HPCcultured with RgpB. A, Cells were incubated with var-ious concentrations of RgpB for 1 h. B, Cells were cul-tured for 1 h in the presence of RgpB (100 nM) with orwithout 1 h of leupeptin pretreatment. The amount ofCGRP or SP was measured by ELISA. f, CGRP; �,SP. Values denote means � SD (n � 3). �, p � 0.05 vsRgpB alone. C and D, plots of the OD values obtainedfrom standard neuropeptide solutions (�) or serially di-luted supernatant of HPCs stimulated by RgpB at 200nM (�). The OOO, best fit lines of a standard neu-ropeptide; – – – –, best fit lines of supernatant values.

FIGURE 2. PAR-2 expression in HPC. A, Total RNA was extractedfrom HPC, HGF, and A549 cells, and mRNA expression of PARs wasdetected by RT-PCR. Lane M, 100-bp DNA ladder; lane 1, PAR-1 (395bp); lane 2, PAR-2 (509 bp); lane 3, PAR-3 (488 bp); lane 4, PAR-4 (534bp). B, PAR protein expression on HPC and A549 cells was analyzed byimmunofluorescent flow cytometry. The amount of PAR on the cell surfacewas shown as the geometric mean of fluorescence intensity (x-axis). Theleft peaks denote the fluorescene by nonspecific binding of control Ab. Therepresentative results of triplicate assays are shown.

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After another washing, the membrane was incubated with respective Absat 4°C overnight. After a third washing with TBST, the membrane wasincubated with HRP-conjugated anti-rabbit Ab (diluted 1/3000 in TBST)for 1 h at room temperature. Finally, the membrane was washed with TBSTand developed with an ECL kit (Amersham Pharmacia Biotech).

Preparation of nuclear extracts and EMSA

After treatment of cells with RgpB in the presence or absence of an in-hibitor, the nuclear extract was obtained from the precipitate as describedpreviously (36). Briefly, cells were washed with ice-cold Ca2�-Mg2�-freePBS and suspended in a buffer containing 1 M HEPES (pH 7.9), 1 MMgCl2, 1 M KCl, 100 mM PMSF, and 1 M DTT. The suspension wascentrifuged at 2000 � g, and the nuclei were extracted in iced low saltbuffer (1 M HEPES, 100% glycerol, 1 M MgCl2, 0.5 M EDTA, 100 mMPMSF, 1 M DTT) for 15 min. After centrifugation at 5000 � g, the su-pernatants were quickly mixed with high salt buffer (same as low salt bufferexcept 1 M KCl was added) and stored at �80°C. The DNA-binding formof AP-1 was assayed using synthetic AP-1 oligonucleotide probe 5�-GAT-CAGCATGAGTCACTTC-3�. The underlined letters are the core consen-sus sequences for the AP-1-binding sites on SP promoter (37–39). Nuclear

extracts (10 �g) were incubated with 2 � 10 4 cpm/�l [�-32P]deoxy-CTP-AP-1 oligonucleotides labeled with Klenow fragment for 40 min at 30°C.A competitive reaction was performed by adding a 100-fold molar excessof an unlabeled AP-1 probe to the reaction mixture 20 min before theaddition of the labeled AP-1 probe. A mutated AP-1 element probe wasidentical with the AP-1 oligonucleotide probe, with exception of a substi-tution of TG for CA in the AP-1-binding motif. The supershift assays wereconducted using a specific polyclonal Ab against c-Jun/AP-1 (Santa CruzBiotechnology). The reaction was identical with those described above,except for the addition of 2 �l of Ab to the binding assay system. Thecomplexes were separated by 5% nondenaturing PAGE at 175 V for 70min. The gels were dried with a gel dryer (Iwaki Glass) and visualized byexposing them to an imaging plate (BAS 1000 Mac).

Miscellaneous

All data from ELISA study were calculated from triplicate wells and areexpressed as the means � SD. The Bonferoni correction was used formultiple t test comparison, and p was determined using StatView version5.0 for the Macintosh. p � 0.05 was considered statistically significant. At

FIGURE 3. PAR-2 and neuropeptide expression indental pulp tissues and HPCs. A, Total RNA was ex-tracted from HPC, and mRNA of CGRP or SP weredetected by RT-PCR: a and c, in the absence of primers;b and d, with sense and antisense primers. The predictedsize of CGRP and SP mRNA was 119 and 121 bp, re-spectively; e, RT-PCR in the absence of DNA; f, g, andh, RT-PCR in the absence of reverse transcriptase (g andh, duplicate). The corresponding control of RNA con-dition and the reaction efficiency were confirmed by RT-PCR products of GAPDH. m, Marker. B, Immunodetec-tion of SP, CGRP, and PAR-2 in culture HPCs stainedwith fluorescence-labeled Abs. Arrowheads and arrowsindicate typical positive cells. Scale bars indicate 10�m. C, Immunohistochemical staining of dental pulptissues with Abs against SP, CGRP, PAR-2, or 1B10.Arrowheads and an arrow indicate typical positive cells.Scale bars indicate 20 �m.

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least three separate experiments were performed in each assay exceptELISA.

ResultsCGRP and SP in the supernatant of HPC

To investigate the effect of RgpB on HPCs, we cultured cells in thepresence of RgpB for 1 h and measured the amounts of CGRP andSP in the supernatant by ELISA. Both neuropeptides increased inproportion to RgpB concentrations to at least 200 nM and are atsimilar levels (Fig. 1A). Considering the molecule of CGRP is 3times bigger than that of SP (37 aa vs 11 aa), SP is released at �3times the level of CGRP on a molar basis. Leupeptin, an RgpBinhibitor (2), reduced the RgpB-induced CGRP increase signifi-cantly ( p � 0.05) and blocked the SP increase completely (Fig.1B). These results indicate that RgpB elicits a neuropeptide releasefrom HPCs in a proteolytic activity-dependent manner. The reli-ability of the ELISA was supported by the result that the best fitlines obtained from serial dilution of a representative sample par-alleled to standard best fit lines of neuropeptides (Fig. 1, C and D).The neuropeptide assays were also performed by RIA. The resultsobtained by RIA were almost identical with those by ELISA (datanot shown), and we adopted ELISA for following experiments.

Expression of PAR-2, CGRP, and SP in HPCs and humandental pulp tissues

To study whether RgpB-induced CGRP and SP release from HPCsis mediated by PAR activation, we investigated HPC PAR mRNAexpression. HPCs expressed only PAR-2 mRNA, whereas, as re-ported previously (32, 40, 41), HGF expressed PAR-1, -2, and -3mRNA and A549 cells expressed mRNA of all four PARs (Fig.2A). Consistent with the mRNA expression result, HPCs expressedonly PAR-2 protein on the cell membrane, and A549 cells ex-pressed all of the PAR proteins (Fig. 2B).

To determine whether HPCs produced the neuropeptides, weinvestigated the expression of CGRP and SP mRNA by RT-PCR.HPCs were found to express both CGRP and SP mRNA (Fig. 3A,lanes b and d), whereas no product was found when RT-PCR wasperformed in the absence of reverse transcriptase, primers or RNA,indicating production of these molecules by HPCs. The presenceof these neuropeptides was confirmed by immunocytochemicalstaining for HPCs (Fig. 3B). From the granular pattern of the flu-orescence for neuropeptides and linear fluorescence for PAR-2, itis likely that neuropeptides localize in cytoplasm granules andPAR-2 are mainly on the membrane.

To explore expression of SP, CGRP, and PAR-2 in dental pulpcells in vivo, we performed immunohistochemical staining for hu-man dental pulp tissues. Dental pulp cells are spindle-shaped stro-mal cell-like, and most of them were positive for SP (Fig. 3C,upper left), CGRP (Fig. 3C, upper right), and PAR-2 (Fig. 3C,middle left), whereas no cells were stained with nonspecific IgG(Fig. 3C, middle right). The spindle-shaped cells were also stainedwith the IgM mAb (1B10) that reacts with cell membrane surfacemolecules of human fibroblasts (Fig. 3C, lower left), whereas nocells were stained with nonspecific IgM (Fig. 3C, lower right).These results indicate that HPCs are derived from dental pulpstroma cells that have a fibroblast-like nature and express PAR-2,SP, and CGRP.

CGRP and SP release from HPCs by PAR-2 agonists

We examined whether activation of PAR-2 with two different types ofPAR-2 agonists can mimic RgpB-induced neuropeptide release.HPCs were incubated with SLIGKV, a synthetic peptide correspond-ing to the tethered ligand of PAR-2 (42). The peptide inducedneuropeptide release in a dose-dependent manner (Fig. 4A). Similar

results were obtained with trypsin (Fig. 4B), which activates PAR-2by cleaving the receptor atOSKGR362S37LIGKVO (where2des-ignates the trypsin cleavage site), exposing the N-terminal-tetheredligand SLIGKV (42, 43). RgpB appeared to be less potent than trypsinbut more effective than SLIGKV in neuropeptides release from HPCs(Figs. 1A, 4A, and B). Induction of the neuropeptide release fromHPCs by PAR-2 agonists suggests that RgpB-induced CGRP and SPrelease occurs through PAR-2 activation.

CGRP and SP in the supernatant of PAR-2-knockout HPCcultured with RgpB

To confirm the PAR-2 mediation in RgpB-induced CGRP and SPrelease, HPCs were transiently transfected with two different partsof PAR-2 sense and antisense oligonucleotides. In the PAR-2 an-tisense oligonucleotide I-transfected HPC, no PAR-2 mRNA ex-pression was observed when compared with the nontransfected orsense oligonucleotide-transfected mRNA (Fig. 5A), and the RgpB-induced CGRP and SP release was reduced to �81% and �54%( p � 0.05), respectively (Fig. 5B). In the PAR-2 sense oligonu-cleotide I-transfected HPC, the RgpB-induced CGRP and SP re-lease (Fig. 5B) was not affected. Almost identical results wereobtained in experiments using PAR-2 sense or antisense oligonu-cleotide II transfection. Neither of these oligonucleotides exerts acytotoxic effect on HPC when determined by MTT assay (data notshown). These results indicate that PAR-2 mediates most of theRgpB-induced CGRP release and more than one-half of the SPrelease in HPC.

p44/42 and p38 MAPK activation in RgpB-induced CGRP andSP release in HPC

MAPKs are involved in PAR-2-initiated signal transduction insome cells (44, 45). To study the signal transduction in RgpB-stimulated HPC, we investigated the activation of p44/42, p38MAPK, and JNK by Western blotting analysis using Abs that spe-cifically recognize each kinase or its phosphorylated form. Theratios of phosphorylated vs unphosphorylated p44/42 and p38

FIGURE 4. CGRP and SP in the supernatant of HPC cultured withPAR-2 agonists. HPC were incubated with various concentrations of eitherSLIGKV or trypsin. A, Cells were incubated with SLIGKV for 1 h. B, Cellswere incubated with various concentrations of trypsin for 1 h. The amountof CGRP or SP released was measured by ELISA. f, CGRP; �, SP.Values denote means � SD (n � 3).

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MAPK were increased by RgpB, but the ratio of JNK was notaffected by the proteinase (Fig. 6A). Phosphorylation of both ki-nases in HPC were detected within 3.5 min after exposure to RgpBand sustained until 60 min after exposure (Fig. 6A). SLIGKV andtrypsin also induced phosphorylation of p44/42 and p38 MAPKbut not JNK, in a time course similar to that seen in the case ofRgpB (Fig. 6, B and C).

To determine the contribution of p44/42 or p38 MAPK-medi-ated signaling to RgpB-induced CGRP and SP release, we exam-ined the effect of kinase inhibitors. U0126, a potent inhibitor spe-

cific for MEK1/2 (the upstream kinase of p44/42), caused �64%and �57% reduction of CGRP and SP release, respectively ( p �0.05) (Fig. 6D). SB 202190, a potent inhibitor specific for p38MAPK, inhibited �71% and �57% of RgpB-induced CGRP andSP release, respectively ( p � 0.05; Fig. 6D). SLIGKV-inducedneuropeptides release from HPC was also inhibited by U0126 orSB 202190 in similar ratios as seen in the case of RgpB (Fig. 6E).These results suggest that phosphorylation of p44/42 and p38MAPK contributes to more than one-half of the signaling pathwayinitiated by RgpB-elicited PAR-2 activation, leading to the neu-ropeptide release.

ATF-2 and AP-1 activation in RgpB-induced CGRP and SPrelease in HPC

ATF-2 and AP-1 are enhanced through phosphorylation ofMAPKs (46–49); we therefore investigated the signal transductionstep following activated-p44/42 and-p38 MAPK in RgpB-inducedneuropeptide release. RgpB-induced phosphorylated ATF-2 wasblocked by SB202190 but not by U0126 or SP600125 (Fig. 7A).The involvement of ATF-2 phosphorylation was confirmed by theresult that a pronounced reduction of RgpB-induced neuropeptiderelease was seen in cells transfected with the kinase-inactive formof ATF-2 (dn-ATF-2) (Fig. 7B).

The AP-1 DNA-binding activity strongly increased in nuclearextracts of RgpB-stimulated cells (Fig. 7C). This binding activitywas induced by the same concentration of nuclear protein ex-tracted from recombinant human vascular endothelial growth fac-tor-treated cells as shown previously (31). The AP-1 DNA-bindingcomplex was completely displaced by the excess addition of anunlebeled AP-1 oligonucleotide probe but not with the mutatedAP-1 element in the competition assay. No radioactivity stainingwas detected in the absence of nuclear protein (Fig. 7C). Theseresults indicated the specificity of the AP-1 DNA-binding activityin the nuclear extracts of RgpB-stimulated HPC. A supershift as-say demonstrated that c-jun, a component of the AP-1 bindingactivity (34), participated in the bound AP-1 complex after theaddition of anti-c-jun/AP-1 Ab to the nuclear extracts, whereas the

FIGURE 5. Effect of PAR-2 gene knockout on RgpB-induced CGRPand SP release from HPC. Either sense (S) or antisense (AS) PAR-2 oli-gonucleotide (0.2 �g) was premixed with 2 �l of LipofectAMINE 2000 in50 �l of OptiMEM medium for 30 min at 37°C. HPC were incubated withthe oligonucleotide-LipofectAMINE 2000 complex for 48 h, followed bya 1-h incubation with RgpB (100 nM). A, PAR-2 mRNA expression wasdetected by RT-PCR M, marker. B, The amount of CGRP or SP releasedwas measured by ELISA. f, CGRP; �, SP. Values denote means � SD(n � 3). �, p � 0.05 vs RgpB alone.

FIGURE 6. MAPK phosphorylation in HPC treatedwith RgpB or PAR-2 agonists and its contribution toneuropeptide release. HPC were incubated with 50 nMRgpB (A), 500 nM SLIGKV (B), or 100 nM trypsin (C)for various incubation periods (0–1 h). Activation ofp44/p42 (left panels) and p38 kinase (middle panels)and JNK (right panels) was determined by immunoblotanalysis using Abs that recognize specifically activated(phosphorylated (phos)) or unactivated forms of thesekinases. Representative results of three separate exper-iments are shown. Cells were pretreated with eitherU0126 (5 �M) or SB203580 (5 �M) for 1 h before a 1-hincubation with 50 nM RgpB (D) or 500 nM SLIGKV(E). The amount of CGRP or SP released was measuredby ELISA. f, CGRP; �, SP. Values denote means �SD (n � 3). �, p � 0.05 vs RgpB alone.

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additional of specific Ab for the p50 subunit of NF-�B transcrip-tion factor showed no effect (Fig. 7C). To ascertain the involve-ment of AP-1 neuropeptide release, nuclear protein was extractedfrom either antisense c-jun or control c-jun oligonucleotide-trans-fected cells. In the antisense c-jun oligonucleotide-transfectedcells, AP-1 DNA-binding was almost totally blocked when com-pared with the nontransfected or the control c-jun oligonucleotide-transfected cells (Fig. 7C). The AP-1 potent inhibitor, curcumin(50) suppressed RgpB-stimulated AP-1 binding activity (Fig. 7D).The result that activation of AP-1 by RgpB was blocked by MAPKinhibitors (SB202190, U0126) but not by a JNK inhibitor(SP600125; Fig. 7D) supports the hypothesis that p44/42 and p38MAPKs mediate the RgpB-induced AP-1 activation. The signifi-cant reduction of RgpB- or SLIGKV-elicited neuropeptide releaseby curcumin (Fig. 7, E and F), and by the antisense knockout ofc-jun (data not shown) suggests that PAR-2-mediated AP-1 acti-vation is associated with the RgpB-induced cell event. Together,these data demonstrate that RgpB stimulation of ATF-2/AP-1 ac-tivation is an important intermediate in its regulation of CGRP andSP release in HPC.

DiscussionThis study revealed a new activity of RgpB, induction of CGRPand SP release from HPCs (Fig. 1A), which suggests that dentalpulp inflammation is associated with the periodontal disease patho-gen P. gingivalis and is modified by neuropeptides released in thedental pulp tissue. Neuropeptides are produced and releasedmainly by the nervous system (14, 15) and also by cells other thanneurocytes, e.g., macrophages (51) and eosinophils (52). CGRPand SP are abundantly copresent in nerve fibers and perivascularplexuses in the human dental pulp tissues (22, 25). From the resultsthat CGRP- and SP-positive cells in the dental pulp stroma andHPCs were also positive for 1B10 Ab (Fig. 3C), it is likely thatHPCs are derived not from neurocytes but from the fibroblast-likecells, which are consistent with the fact that primary pulp cellsexhibited fibroblast-like nature (53). Although no study has clearlyshown neuropeptide synthesis by fibroblast-like cells, the resultthat HPCs expressed mRNA of both CGRP and SP (Fig. 3A) in-dicates production of these neuropeptides by HPCs. However, thedata that the neuropeptides increased in HPC culture medium

FIGURE 7. Transcription factor activation in RgpB-stimulated HPC and its link to the neuropeptide release. A, Cells pretreated with 2.5–10 �MSB202190 (SB), 10 �M SP600125 (SP), or 10 �M U0126 (UO) for 30 min were incubated with RgpB (50 nM) for 30 min. The phosphorylation levelof ATF-2 was determined by immunoblot analysis using Ab specific for phosphorylated ATF-2. B, dn-ATF-2 (0.1 �g) was premixed with 2 �l ofLipofectAMINE 2000 in 50 �l of OptiMEM medium for 30 min at 37°C. Cells were incubated with the dn-ATF-2/LipofectAMINE 2000 complex for 24 h,followed by a 1-h incubation with RgpB (50 nM). The amount of CGRP or SP release was measured by ELISA. C, Nuclear extracts of HPC were incubatedwith �-32P-labeled oligonucleotide probe that binds the AP-1-DNA binding site (lane 1). The nuclear extracts of RgpB-stimulated cells were preincubatedwith c-jun Ab (lane 2) or anti-NF-� B p50 Ab (p50 Ab) (lane 3), followed by the addition of labeled AP-1 probe. The nuclear extracts of HPC treated with50 ng/ml recombinant human vascular endothelial growth factor (rHuVEGF) for 4 h (lane 4) or 50 nM RgpB for 1 h (lane 5) were incubated with the labeledAP-1 probe. The nuclear extracts of RgpB-stimulated cells were incubated with the radiolabeled mutant AP-1 probe (lane 6) or the AP-1 probe aftertreatment with an excessive amount of unlabeled AP-1 probe (lane 7). The nuclear extracts of cells transfected with control c-jun (lane 8) or antisense c-jun(AS-c-jun) (lane 9) oligonucleotides for 48 h, followed by a 1-h incubation with RgpB were incubated with the labeled AP-1 probe. The labeled AP-1 probealone (lane 10). D, Cells pretreated with 5 �M SB 202190, 5 �M SP600125, 10 �M U0126, or 5 �M curcumin (C) for 1 h were incubated with 50 nMRgpB for 1 h, and nuclear extracts of the cells were incubated with the radiolabeled AP-1 probe. Cells were pretreated with curcumin (5 �M) for 1 h beforethe addition of 50 nM RgpB (E) or 500 nM SLIGKV (F) for 1 h. The amount of CGRP or SP released was measured by ELISA. f, CGRP; �, SP. Valuesdenote means � SD (n � 3). �, p � 0.05 vs RgpB alone.

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within 1 h after RgpB stimulation (Fig. 1A) and no neuropeptideswere detected in the medium before stimulation, whereas the neu-ropeptides are present in HPC cytoplasm granules (Fig. 3B), sug-gest that the RgpB effect on HPCs is to induce release rather thanproduction of these peptides.

The colocalization of PAR-2, CGRP, and SP and the mediationof the neuropeptides in PAR-2-induced edema have been reportedin neurocytes (14). The dependency of PAR-2 activation in RgpB-elicited neuropeptide release from HPC was shown by the resultsthat 1) HPC express only PAR-2 (Fig. 2), 2) PAR-2 agonists in-duce HPC neuropeptide release (Fig. 4), and 3) a pronounced re-duction of HPC neuropeptide release by RgpB occurred in PAR-2gene knockout HPC (Fig. 5B). The RgpB-induced PAR-2 activa-tion was also seen in neutrophils, oral epithelial cells, and osteo-blast-like cells (28, 54, 55). PAR-2 agonists induce CGRP and SPrelease in sensory neurons (14). It seems relevant that RgpB acti-vates HPC PAR-2 and elicits release of these neuropeptides. Lour-bakos et al. (56) have reported that RgpB stimulated PAR-2, lead-ing to neutrophil activation indicated by an increase in Ca2� withthe half-maximal response (EC50) of 49.8 � 3.9 nM. The concen-tration of RgpB in the gingival crevicular fluid of patients withadult periodontitis was �0.1 �M (57), the highest concentrationused for HPC stimulation in the present study. Accordingly, it islikely that HPC PAR-2 activation by RgpB occurs in vivo.

PAR-2 activation induces phosphorylation of p44/42 and p38MAPK in smooth muscle cells, fibroblasts (44), keratinocyte cellline (55), and cardiomyocytes (58), and JNK was also activated inthe latter two types of cells (55, 58). The result that JNK phos-phorylation was not affected in HPC neuropeptide release by RgpBas well as PAR-2 agonist (Fig. 6, A–C) suggests a similarity of thePAR-2 signaling pathway in HPC to that of smooth muscle cells orfibroblasts. As found in RgpB-elicited HPC neuropeptides release(Fig. 7, C–F), eosinophil PAR-2 activation by tryptase leads toactivation of the MAPKs/AP-1 pathway, causing cytokine release(59). The MAPKs/AP-1 pathway may be linked to PAR-2-medi-ated releasing events. ATF-2 phosphorylation induction in PAR-2-initiated signal transduction has not been reported thus far,whereas this transcription factor can mediate neuropeptides ex-pression in the sensory neurons (60). We show for the first timethat RgpB-elicited HPC neuropeptide release, in which the PAR-2-p38 MAPK-ATF-2 pathway is involved in addition to thePAR-2- p44/42 p38 MAPK and -AP-1 pathway.

CGRP and SP at a range from picomolar to micromolar havebeen reported to stimulate nonneuronal cells (61–63). The valuesof CGRP (208 pg/ml, 54 pM) and SP (182 pg/ml, 140 pM) wereobtained from 50,000 RgpB-treated HPC in 0.5 ml assay buffer,and 0.1 ml of the aliquot was used for each ELISA (Fig. 1). There-fore, released CGRP and SP contents in 10,000 HPC/0.1 ml are20.8 and 18.2 pg, respectively. Under an assumption that a HPC isa ball-like structure with a diameter of 10 �m, a HPC volume[3/4 � 3.14 � (0.0005 cm)3] is estimated to be 0.3 � 10�9 ml.Supposing that a neuropeptide evenly distributes in a HPC, CGRPand SP concentrations in a cell are 7 �g/ml (1.8 �M) and 6 �g/ml(4.6 �M), respectively. These neuropeptide concentrations shouldbe much higher in the cell granules (Fig. 3B). Accordingly, we canpredict that the concentrations of neuropeptides released fromHPCs by RgpB are high enough to exert biological activities of theneuropeptides in the dental pulp tissue. The activities of CGRP andSP to induce vasodilation, increased local blood flow, enhancedvascular permeability, and pain (14–19) would augment these ac-tivities of bradykinin released by RgpB through activation of thekallikrein/kinin pathway (2). Taken together, the new RgpB activ-ity to induce HPC neuropeptides release can be implicated in den-

tal pulp inflammation disseminated from periodontitis caused by P.gingivalis infection, which exacerbates pulpitis.

AcknowledgmentsWe thank Ko-ichi Kawahara (Department of Laboratory and VascularMedicine, Kagoshima University Graduate School of Medical and DentalScience, Kagoshima, Japan) for his useful advice, support, and discussionthroughout this study.

DisclosuresThe authors have no financial conflict of interest.

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