Hemoglobin Receptor Protein from Porphyromonas gingivalis InducesInterleukin-8 Production in Human Gingival Epithelial Cells throughStimulation of the Mitogen-Activated Protein Kinase and NF-�BSignal Transduction Pathways

Yuki Fujita,a,b Masaaki Nakayama,a Mariko Naito,c Eiki Yamachika,b Tetsuyoshi Inoue,a Koji Nakayama,c Seiji Iida,b Naoya Oharaa

‹Department of Oral Microbiologya and Department of Oral and Maxillofacial Reconstructive Surgery,b Okayama University Graduate School of Medicine, Dentistry andPharmaceutical Sciences, Okayama, Japan; Division of Microbiology and Oral Infection, Department of Molecular Microbiology and Immunology, Nagasaki UniversityGraduate School of Biomedical Sciences, Nagasaki, Japanc

Periodontitis is an inflammatory disease of polymicrobial origin affecting the tissues supporting the tooth. The oral anaerobicbacterium Porphyromonas gingivalis, which is implicated as an important pathogen for chronic periodontitis, triggers a series ofhost inflammatory responses that promote the destruction of periodontal tissues. Among the virulence factors of P. gingivalis,hemoglobin receptor protein (HbR) is a major protein found in culture supernatants. In this study, we investigated the roles ofHbR in the production of inflammatory mediators. We found that HbR induced interleukin-8 (IL-8) production in the humangingival epithelial cell line Ca9-22. p38 mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase 1/2(Erk1/2) were activated in HbR-stimulated Ca9-22 cells. Inhibitors of p38 MAPK (SB203580) and Erk1/2 (PD98059) blockedHbR-induced IL-8 production. Additionally, HbR stimulated the translocation of NF-�B-p65 to the nucleus, consistent withenhancement of IL-8 expression by activation of the NF-�B pathway. In addition, small interfering RNA (siRNA) targeting acti-vating transcription factor 2 (ATF-2) or cyclic AMP-response element-binding protein (CREB) inhibited HbR-induced IL-8 pro-duction. Moreover, pretreatment with SB203580 and PD98059 reduced HbR-induced phosphorylation of CREB and ATF-2, re-spectively. Combined pretreatment with an inhibitor of NF-�B (BAY11-7082) and SB203580 was more efficient in inhibiting theability of HbR to induce IL-8 production than pretreatment with either BAY11-7082 or SB203580 alone. Thus, in Ca9-22 cells,the direct activation of p38 MAPK and Erk1/2 by HbR caused the activation of the transcription factors ATF-2, CREB, and NF-�B, thus resulting in the induction of IL-8 production.

Periodontal disease, a chronic inflammatory disease thatcauses the destruction of periodontal tissues and alveolar

bone (1), is one of the most frequently occurring infectiousdiseases in humans (2). The anaerobic Gram-negative bacte-rium Porphyromonas gingivalis is considered an etiologicallyimportant agent for periodontal disease (3). The primary eco-logical niche of this organism is the gingival crevice, and itspresence is associated with the development of periodontalpockets. In addition, the number of P. gingivalis cells is signif-icantly reduced after periodontal treatment (4). Therefore, thisorganism is thought to play an important role in the develop-ment and progression of periodontitis.

P. gingivalis has two major types of cysteine proteinases, Arg-specific gingipain and Lys-specific gingipain, which are the prod-ucts of 3 separate genes: rgpA, rgpB, and kgp (5, 6). The proteinsencoded by rgpA and kgp have similar structures and consist of anN-terminal propeptide region, a proteolytic domain, and C-ter-minal adhesin domains: HGP15 (hemoglobin receptor protein[HbR], HA2), HGP17, HGP27, and HGP44. The adhesin do-mains are also encoded by the hemagglutinin-encoding genehagA; these regions are quite similar to the C-terminal regions(HGP44 and HbR) of the rgpA and kgp products (7, 8). HbR wasfound to have the ability to bind hemoglobin and act as a high-affinity hemophore at the P. gingivalis cell surface to capture por-phyrin from hemoglobin (9–11). The putative function of HbR ispresumed to be acquisition of heme from erythrocytes (10). HbRis located at the surface of P. gingivalis cells with RgpA, RgpB, Kgp,

and hemagglutinin domain proteins and is also released from bac-terial cells by secretion and autolysis as one of the major extracel-lular proteins detected after prolonged incubation (12, 13), sug-gesting the possibility that released HbR influences the function ofhost cells. Recently, we found that HbR bound to bone marrowmacrophages and suppressed the receptor activator of nuclear fac-tor kappa light chain enhancer of activated B cells (NF-�B) ligand(RANKL)-induced osteoclastogenesis (12). Here, we show thatHbR has the ability to promote interleukin-8 (IL-8) production inhuman gingival epithelial cells (GEC). We further demonstratethat the involvement of two major mitogen-activated protein ki-nases (MAPKs), p38 and extracellular signal-related kinase 1/2(Erk1/2), and at least 3 major transcription factors, i.e., activatingtranscription factor-2 (ATF-2), cyclic AMP response element-binding protein (CREB), and NF-�B, are necessary for adequateHbR-stimulated production of IL-8.

Received 16 September 2013 Accepted 11 October 2013

Published ahead of print 14 October 2013

Editor: B. A. McCormick

Address correspondence to Naoya Ohara, oharan@md.okayama-u.ac.jp.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.01140-12

202 iai.asm.org Infection and Immunity p. 202–211 January 2014 Volume 82 Number 1

MATERIALS AND METHODSReagents. Antibodies (Abs) against phospho-Erk1/2 (Thr202/Tyr204),Erk1/2, phospho-p38 (Thr180/Tyr182), p38, phospho-ATF-2 (Thr71),ATF-2, phospho-CREB (Ser133), and CREB were purchased from CellSignaling Technology (Danvers, MA). PD98059 and SB203580, inhibitorsof MEK1 and p38, respectively, were purchased from Wako (Osaka, Ja-pan). BAY11-7082, an inhibitor of I�B-� phosphorylation, was pur-chased from Sigma (St. Louis, MO). ATF-2 small interfering RNA(siRNA) and negative control (NC) siRNA were obtained from SantaCruz Biotechnology, Inc. (Santa Cruz, CA), and CREB siRNA was ob-tained from Ambion, Applied Biosystems (Grand Island, NY).

Purification of HbR from HbR-overexpressing Escherichia coli. Re-combinant HbR was purified essentially according to the method de-scribed by Nakayama et al. (10). Briefly, E. coli BL21(DE3) harboringpKD349 (10) and pLysS (14) was grown to an optical density (at 540 nm)of 0.5. HbR was induced with the addition of 1 mM isopropyl-�-D-thio-galactopyranoside (IPTG). After cell lysis by sonication, ammonium sul-fate was added to the cell lysate to obtain 35% saturation. The precipitatedproteins were dialyzed and applied to a column (1.6 by 25 cm) of DEAE-Sepharose (Sepharose CL-6B; GE Healthcare, Amersham, England). Pro-teins were eluted with a 0 to 1 M linear NaCl gradient. The fraction thatwas eluted at 400 mM NaCl showing the most prominent protein peakwas used in the present study. The purity of HbR in the fraction was ca.98%, as determined by densitometric analysis of SDS-PAGE gels. Lipo-polysaccharide (LPS) was removed from the sample by phase separationwith Triton X-114 by the method described by Liu et al. (15). Briefly,Triton X-114 was added to the protein preparation to a final concentra-tion of 1%. The mixture was incubated at 4°C for 30 min with constantstirring. The sample was then transferred to a 37°C water bath, incubatedfor 10 min, and centrifuged at 20,000 � g for 10 min at 25°C. The upperaqueous phase containing the protein was carefully removed and sub-jected to Triton X-114 phase separation for two more cycles. The resultingaqueous phase was assayed for endotoxin; residual LPS in the purifiedHbR preparation was measured by the Limulus test using a Limulus ColorKY Test Wako (Wako).

Preparation of protein extracts of P. gingivalis CSs. Protein extractsof culture supernatants (CSs) from P. gingivalis strain ATCC 33277 wereprepared as described by Fujimura et al. (12). Protein concentrations weredetermined by the bicinchoninic acid (BCA) colorimetric assay.

Cell culture. The human gingival epithelial cell line Ca9-22 and hu-man primary gingival epithelial (HGEP) cells were purchased fromHealth Science Research Resources Bank (Osaka, Japan) and CELLnTec(Bern, Switzerland), respectively. These cells were maintained in alphaminimal essential medium supplemented with 10% fetal calf serum (FCS;Life Technologies) at 37°C in a 5% CO2-95% air humidified atmosphere.

Cytokine assay. Cell culture supernatants from untreated cells andcells treated with 10 �g/ml of HbR were collected after incubation for 4 or12 h and assayed for the presence of 42 different cytokines and chemo-kines by using RayBio Human Cytokine Antibody Array 3 membranes(RayBiotech, Inc., Norcross, GA). Cytokines were detected by chemilu-minescence, and the results were visualized with an LAS 4000 (Image-Quant).

Detection of IL-8 production. Cells were seeded at a density of 1.0 �105 cells/well in 24-well plates. Prior to stimulation with HbR, the cellswere serum starved for 16 h. For some experiments, the cells were prein-cubated with PD98059, SB203580, or BAY11-7082 for 1 h before stimu-lation. Supernatants of cultivated Ca9-22 cells were collected after 12 h ofstimulation with HbR. The amount of IL-8 production in the superna-tants was measured by enzyme-linked immunosorbent assay (ELISA;R&D Systems, Abingdon, United Kingdom). All experiments were per-formed in triplicate, and results are reported as the averages of three sep-arate experiments.

Western blotting. Ca9-22 cells were stimulated with 10 �g/ml of HbRand incubated for the times indicated below. After incubation, cells wererinsed with phosphate-buffered saline (PBS), followed by the addition of

150 �l of SDS sample buffer containing 2 mM sodium orthovanadate and1 mM NaF. After boiling for 10 min, 20 �l of the lysates was separated bySDS-PAGE (10% gel). Separated proteins were transferred to polyvi-nylidene difluoride (PVDF) membranes, blocked with 1% fetal bovineserum (FBS; Sigma) in TBST buffer (20 mM Tris-HCl [pH 7.6], 150 mM,NaCl, and 0.1% Tween 20) for 1 h at room temperature, and then incu-bated with primary antibodies overnight at 4°C. After 2 washes withTBST, the blots were incubated with horseradish peroxidase-conjugatedsecondary antibody IgG for 1 h at room temperature and visualized usingan LAS 4000 (ImageQuant).

Immunostaining. Ca9-22 cells seeded in a Lab-Tek 8 chamber (Nunc,Roskilde, Denmark) were incubated with 10 �g/ml of HbR or PBS at 37°Cfor 2 h. After incubation, the cells were fixed with 2% paraformaldehyde inPBS. The cells were then permeabilized with 0.1% Triton X-100 in PBS,blocked with Block Ace solution (Snow Brand Milk Products, Tokyo,Japan) for 30 min, and incubated with primary antibodies (anti-phospho-ATF-2 and anti-phospho-CREB Abs). After primary antibody incuba-tion, the cells were washed and incubated with secondary antibodies con-jugated with Alexa Fluor 546 (Invitrogen, Carlsbad, CA). Stained cellswere observed using a fluorescence microscope (BZ-8000; Keyence,Osaka, Japan).

Reverse transcription (RT)-PCR. Total RNA was extracted by TRIzol(Invitrogen) from Ca9-22 cells stimulated with P. gingivalis CS (cultured for 5days) and infected with P. gingivalis, which was used as a template for cDNAsynthesis by the SuperScript first-strand synthesis system (Invitrogen). Theprimers for human IL-8 were synthesized by Invitrogen based on the nucle-otide sequence with NCBI identification code NM_000584.3. The followingsequences were used for PCR: IL-8 forward, 5=-ACACTGCGCCAACACAGAAATTA-3=, and IL-8 reverse, 5=-TTTGCTTGAAGTTTCACTGGCATC-3=. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as aninternal control, and the sequences used were as follows: GAPDH forward,5=-GCACCGTCAAGGCTGAGAAC-3=, and GAPDH reverse, 5=-TGGTGAAGACGCCAGTGGA-3=. The cDNA (0.5 �g) was mixed and PCR amplifiedaccording to the following conditions using KOD FX DNA polymerase(TOYOBO): 25 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30s, and extension at 72°C for 30 s. After amplification, each reaction mixturewas analyzed by electrophoresis on 2% agarose gels, and the gels were stainedby ethidium bromide for detection of PCR products.

Transfection with ATF-2 or CREB siRNA. Ca9-22 cells were seeded at0.5 � 105 cells per well in 24-well plates and grown overnight. ATF-2siRNA (100 �mol), CREB siRNA (100 �mol), or negative-control siRNA(NC siRNA; 100 �mol) duplexes were introduced into cells using Lipo-fectamine RNAiMAX transfection reagent (Invitrogen) according to themanufacturer’s recommendations. A mock transfection without siRNAwas also performed.

Statistical analysis. Statistical analysis of the data was performed us-ing Student’s t test.

RESULTSHbR-dependent IL-8 production in Ca9-22 cells. We first exam-ined the profile for cytokines secreted by human GEC in responseto HbR stimulation using cytokine antibody analysis (Fig. 1A).The purity of the HbR used was more than 98%, and LPS contam-ination was less than 10 pg/�g of HbR protein. Compared to un-treated cells, treatment of Ca9-22 cells with 10 �g/ml of HbR for 4or 12 h caused upregulation of IL-8. To verify this finding, IL-8protein production in response to HbR stimulation was also ex-amined. IL-8 production was significantly increased (P � 0.01) inresponse to HbR stimulation in a dose- and a time-dependentmanner compared with that in untreated cells (Fig. 1B and C). Incontrast, heat-treated HbR (incubated at 100°C for 10 min) failedto induce IL-8 production, suggesting that HbR-induced IL-8production was not due to the effects of contaminated nonproteinsubstrates (Fig. 1D). Induction of IL-8 production in response to

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HbR stimulation was also observed in HGEP cells (Fig. 1E), sug-gesting that HbR-induced IL-8 production was not a cell line-specific phenomenon.

Activation of the MAPK pathway in HbR-stimulated Ca9-22cells. HbR stimulation has been reported to increase activation ofErk1/2 and p38 MAPK in bone marrow macrophages (12). Thus,we next investigated the effects of HbR on the phosphorylation ofMAPKs in Ca9-22 cells. Phosphorylation of Erk1/2 at Thr202/Tyr204 and p38 at Thr180/Tyr182 was observed within 10 min

after addition of HbR in a dose-dependent manner (Fig. 2A andB). Heat treatment of HbR resulted in the loss of the ability tophosphorylate Erk1/2 at Thr202/Tyr204 and p38 at Thr180/Tyr182 (Fig. 2C).

Role of MAPKs in HbR-induced IL-8 production. MAPKs areknown to play a central role in the regulation of IL-8 production.In order to examine the involvement of MAPKs in HbR-inducedIL-8 production in Ca9-22 cells, chemical inhibitors specific to theMEK1/2 and p38 kinase pathways were added to cells prior to

FIG 1 Effects of HbR on IL-8 production in Ca9-22 cells. (A) Detection of IL-8 in the supernatants of HbR-treated cells. Ca9-22 cells were treated with HbR (10�g/ml) for 4 or 12 h, and the culture supernatants were reacted with the cytokine antibody membrane array. Untreated cells were used as a control. Neg, negativecontrol; Pos, positive control (included in the kit); IL-8, anti-IL-8 antibody was spotted. (B) Ca9-22 cells were incubated with HbR at the indicated concentrationsfor 12 h. IL-8 levels in the supernatant were detected by ELISA. (C) Ca9-22 cells were incubated with HbR (10 �g/ml) for the indicated times in serum-freemedium. IL-8 levels in the supernatant were detected by ELISA. (D) Ca9-22 cells were incubated with heat-treated HbR for 12 h, and IL-8 levels in the supernatantwere detected by ELISA. (E) HGEP cells were incubated with HbR at the indicated concentrations for 12 h. IL-8 levels in the supernatant were detected by ELISA.The results are representative of three separate experiments. Black and white bars indicate culture supernatants incubated with and without HbR, respectively.*, P � 0.01 (compared to the experiment without HbR).

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HbR stimulation. Pretreatment with either SB203580, a specificinhibitor of p38 MAPK activation, or PD98059, an inhibitor of theMAPK kinase MEK, only partially suppressed HbR-inducedIL-8 production. Combined pretreatment with SB203580 andPD98059 significantly abolished the ability of HbR to induce IL-8production (Fig. 3). These data suggested that activation of bothp38 and Erk1/2 was involved in HbR-induced IL-8 production.

Effect of CSs from P. gingivalis on IL-8 production. SinceHbR is a major extracellular protein in P. gingivalis (12), CSs fromP. gingivalis should have the ability to induce IL-8 production inCa9-22 cells. To clarify this possibility, Ca9-22 cells were incu-bated with 10 �g of CSs from P. gingivalis. LPS contamination wasestimated to be less than 10 pg/�g of CS. Unexpectedly, IL-8 wasnot detected in the supernatants of cultivated Ca9-22 cells incu-bated with CS (Fig. 4A). However, the mRNA for IL-8 was sub-stantially increased in response to CS stimulation or incubationwith live P. gingivalis cells (Fig. 4B). Furthermore, phosphoryla-tion of Erk1/2 at Thr202/Tyr204 and p38 at Thr180/Tyr182 wasobserved following addition of CSs or live P. gingivalis cells (Fig.4C and D). These data suggested that the IL-8 gene was tran-scribed via activation of Erk1/2 and p38 MAPK by P. gingivalis CSstimulation, leading to production of IL-8 protein. After release of

IL-8 from cells, degradation of IL-8 may occur. One possiblemechanism for this degradation event is digestion by gingipains.Since HbR is a major component of the P. gingivalis CS, it maycontribute to CS-induced IL-8 production.

Involvement of ATF-2 and CREB in HbR-induced IL-8 pro-duction. Several reports have demonstrated that activation of thetranscription factors ATF-2 and CREB is involved in MAPK-me-diated IL-8 production (16, 17). To examine the ability of HbR tostimulate IL-8 production through ATF-2/CREB-mediated path-ways in Ca9-22 cells, we examined the translocation of ATF-2 andCREB to the nucleus in HbR-treated cells and its association withIL-8 release. Interestingly, HbR induced the translocation of phos-phorylated ATF-2 and CREB to the nucleus, as visualized by im-munostaining with anti-phospho-ATF-2 and anti-phospho-CREB Abs (Fig. 5A and B). To further elucidate the roles of ATF-2and CREB in response to HbR treatment, Ca9-22 cells were trans-fected with ATF-2-siRNA or CREB-siRNA. Reduction of ATF-2and CREB expression in Ca9-22 cells treated with ATF-2-siRNAor CREB-siRNA, respectively, resulted in suppression of IL-8 ex-pression (Fig. 5C and D).

HbR induced the translocation of NF-�B to the nucleus inCa9-22 cells and inhibition of IL-8 release by BAY11-7082. Ac-tivation of the IL-8 promoter is thought to require activation ofthe transcription factor NF-�B. To examine the ability of HbR tostimulate IL-8 induction through an NF-�B-mediated pathway,we examined the translocation of NF-�B to the nucleus in HbR-treated cells and its association with IL-8 release. HbR induced thetranslocation of NF-�B-p65 to the nucleus, as visualized by im-munostaining with anti-NF-�B-p65 Abs (Fig. 6A). To confirmthat the NF-�B pathway was involved in HbR-induced produc-tion of IL-8, cells were stimulated with HbR in the presence ofBAY11-7082, an inhibitor of cytokine-induced I�B� phosphory-lation. The amount of IL-8 induced by HbR stimulation was re-duced significantly in the presence of more than 1 �M BAY11-7082 (Fig. 6B). These data indicated that HbR induced IL-8production through activation of the canonical NF-�B pathway.

FIG 2 Activation of MAP kinases by HbR. (A) Ca9-22 cells were treated withHbR (1 to 10 �g/ml) for 0, 60, or 120 min and then lysed. Proteins in the cellswere separated by SDS-PAGE, transferred to PVDF membranes, and thenimmunostained with anti-phospho-ERK1/2 (Thr202/Tyr204), anti-Erk2, an-ti-phospho-p38 (Thr180/Tyr182), or anti-p38 antibodies. (B) Ca9-22 cellswere treated with HbR (10 �g/ml) for 0 to 120 min and then lysed. The sameantibodies as used for panel A were employed for immunoblotting. (C) Ca9-22cells were treated with heat-treated HbR for 60 min and then lysed.

FIG 3 Effects of SB203580 and PD98059 on HbR-induced IL-8 secretion inCa9-22 cells. Cells were pretreated with SB203580 (3 �M), PD98059 (3 �M),or both inhibitors (3 �M each) for 1 h before a 12-h incubation with HbR (10�g/ml) in serum-free medium. IL-8 levels in the supernatant were detected byELISA. Black and white bars indicate culture supernatants incubated with andwithout HbR, respectively. Control samples (SB203580 � and PD98059 �)contained 1% dimethyl sulfoxide (final concentration). *, P � 0.01 (comparedto the experiment without any inhibitor).

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Relationships among MAPKs, ATF-2, CREB, and NF-�B inHbR-induced IL-8 production. Since we had demonstrated thatactivation of the MAPKs p38 and Erk1/2 and the transcriptionfactors ATF-2, CREB, and NF-�B was involved in HbR-inducedIL-8 production, we next investigated the link between the activa-tion of these MAPKs and transcription factors in response to HbR.Pretreatment with the p38 inhibitor SB203580 reduced HbR-in-duced CREB phosphorylation but not ATF-2 phosphorylation(Fig. 7A). In contrast, pretreatment with the Erk1/2 inhibitorPD98059 was able to reduce HbR-induced ATF-2 phosphoryla-tion but not CREB phosphorylation (Fig. 7B). These data sug-gested that HbR-stimulated p38 kinase activation acted upstreamof CREB phosphorylation and that HbR-stimulated Erk1/2 kinaseactivation acted upstream of ATF-2 phosphorylation. In order toelucidate the relationship between MAPK activation and NF-�Bactivation in HbR-induced IL-8 production, cells were stimulatedwith HbR in the presence of MAPK and NF-�B inhibitors. Com-bined pretreatment with BAY11-7082 and SB203580 was moreefficient in inhibiting HbR-induced IL-8 production than pre-treatment with BAY11-7082 or SB203580 alone (Fig. 7C). How-ever, combined pretreatment with BAY11-7082 and PD98059 didnot elicit greater HbR-induced IL-8 production than pretreat-ment with PD98059 alone. These data suggested that activation of

NF-�B occurred downstream of Erk1/2 kinase activation. Takentogether, our data suggested that HbR-induced IL-8 productionin Ca9-22 cells occurred through Erk1/2/ATF-2, NF-�B, and p38/CREB pathways, as illustrated in Fig. 8.

DISCUSSION

The present study demonstrated dose- and time-dependent ex-pression of IL-8 in human GEC stimulated with P. gingivalis HbRand analyzed the signal pathways involved in HbR-stimulatedIL-8 production.

A complex network of cytokines is involved in the initiationand progression of chronic periodontitis. Among these cytokines,IL-8, a proinflammatory chemokine, is considered one of theprincipal mediators of this inflammatory response. IL-8 can beproduced by a wide variety of cell types and plays an importantrole as a chemotactic factor in the migration and accumulation ofneutrophils, which are the predominant line of defense in thegingival crevice (18). Studies have shown that IL-8 mRNA andprotein levels are significantly increased in the chronically in-flamed periodontal tissue and gingival crevicular fluid of adultsubjects with periodontitis, relative to the levels in healthy controlsubjects (19–22). Interestingly, Jin et al. (23) reported the oppositeobservation, that IL-8 levels are lower in gingival crevicular fluid

FIG 4 P. gingivalis CS-induced IL-8 expression in Ca9-22 cells. (A) Ca9-22 cells were incubated with PBS, HbR (10 �g/ml), or P. gingivalis CS (cultured for 5days) for 6 h. Cell culture supernatants of Ca9-22 cells were collected, and IL-8 levels in culture media were measured by IL-8 ELISAs. (B) Ca9-22 cells wereincubated with P. gingivalis CS (cultured for 5 days) and infected with P. gingivalis at a multiplicity of infection of 100 for 3 h. The cells were treated with TRIzol,and total RNA was prepared for RT-PCR as described in Materials and Methods. After PCR, IL-8 and GAPDH mRNAs were detected by ethidium bromidestaining. (C and D) Ca9-22 cells were stimulated with live P. gingivalis (cell-to-bacterium ratio was 1:100) (C) or CS (10 �g/ml) (D) for 30 min and then lysed.Proteins in the cells were separated by SDS-PAGE, transferred to PVDF membranes, and then immunostained with anti-phospho-Erk1/2 (Thr202/Tyr204),anti-Erk2, anti-phospho-p38 (Thr180/Tyr182), or anti-p38 antibodies. Control samples (SB203580 � and PD98059 �) contained 1% dimethyl sulfoxide (finalconcentration).

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in periodontitis patients than in healthy controls. In the currentstudy, we examined whether the CS of P. gingivalis induced IL-8production. Amounts of IL-8 in supernatants of Ca9-22 cells ex-posed to the P. gingivalis CS were comparable to those of non-stimulated Ca9-22 cells (Fig. 4). However, IL-8 mRNA was in-creased in Ca9-22 cells exposed to the CS or infected with P.gingivalis. Moreover, phosphorylation of Erk1/2 at Thr202/Tyr204 and p38 at Thr180/Tyr182 was observed in Ca9-22 cellsupon CS stimulation, suggesting that CS had the ability to intro-duce IL-8 production from Ca9-22 cells and that loss of IL-8 oc-curred after secretion. HbR is expressed in the P. gingivalis CS andmay contribute to CS-induced IL-8 production. These resultswere consistent with observations by Zhang et al. (24); they re-

ported that IL-8 production by oral epithelial cells appeared to bereduced after infection with P. gingivalis, but infected cells ex-pressed IL-8 mRNA (24). Darveau et al. also reported that P. gin-givalis infection inhibited accumulation of IL-8 from GEC but wasassociated with a decrease in IL-8 mRNA and intracellular inva-sion by P. gingivalis (25).

Several secreted and structural components of P. gingivalis thatinduce IL-8 gene expression in host cells have been identified. Forexample, P. gingivalis LPS induces IL-8 gene expression in humangingival fibroblasts, monocytes, neutrophils, and human umbili-cal vein endothelial cells (HUVECs) (26–30). Additionally, P. gin-givalis fimbrillin-specific peptides stimulate modest IL-8 re-sponses in HUVECs (29). HtpG has also been reported to induce

FIG 5 Activation of ATF-2 and CREB by HbR and their involvement in HbR-induced IL-8 production. (A and B) Ca9-22 cells were incubated with 10 �g/mlof HbR at 37°C for 2 h. The cells were fixed using 2% paraformaldehyde and permeabilized by incubation with 0.1% Triton X-100 for 5 min. Fixed cells, stainedwith 1 �g/ml 4=,6-diamidino-2-phenylindole for 5 min, were incubated with anti-phospho-ATF (1:100) (A) or anti-phospho-CREB (1:100) (B) in Tris-bufferedsaline (TBS) containing 1% bovine serum albumin. After treatment with the respective primary antibodies, cells were incubated with either anti-rabbit polyclonalsecondary antibodies conjugated with Alexa Fluor 546 (1:1,000) or anti-mouse polyclonal secondary antibodies conjugated with Alexa Fluor 546 (1:1,000),diluted in TBS containing 1% bovine serum albumin. The graph shows a quantitative analysis of phospho-ATF (A) and phospho-CREB (B) nuclear transloca-tion. Results are means SEMs of three independent experiments. (C) Ca9-22 cells were grown overnight, and silencing of CREB or ATF-2 expression wasperformed using CREB siRNA or ATF-2 siRNA, respectively, or NC siRNA (as a control), as described in Materials and Methods. After a 24-h transfection, cellswere suspended in serum-free medium and treated with 10 �g/ml of HbR at 37°C for 12 h. Reduction of CREB and ATF-2 protein compared to the level of �-actinwas confirmed by Western blotting with anti-CREB, anti-ATF-2, or anti-�-actin antibodies. (D) Ca9-22 cells were grown overnight, and silencing of CREB orATF-2 expression was performed with CREB siRNA or ATF-2 siRNA, respectively, or NC siRNA (as a control), as described in Materials and Methods. After a24-h transfection, cells were suspended in serum-free medium and treated with 10 �g/ml of HbR at 37°C for 12 h. IL-8 production in the supernatants wasdetected by ELISA. Black and white bars indicate the amounts of IL-8 in culture supernatants incubated with and without HbR, respectively. Data arerepresentative of at least three experiments. *, P � 0.01 (compared to the control sample).

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IL-8 mRNA and protein in human monocytic and microvascularvein endothelial cells (HMVECs) (30). Moreover, RgpA has beenshown to enhance IL-8 production in LPS- or heat-killed whole-cell preparations of HUVECs (31). In contrast, gingipain from P.gingivalis has been reported to modulate the biological activities ofIL-8 by degradation and destruction of the IL-8 protein (24, 29,32). Interestingly, O’Brien-Simpson et al. (33) reported that lowconcentrations of P. gingivalis cells and RgpA-Kgp complexesstimulate the secretion of secretory intercellular adhesion mole-cule 1 (ICAM-1), IL-8, IL-6, and macrophage chemoattractantprotein (MCP) from cultured human epithelial cells and fibro-blasts. However, high concentrations of P. gingivalis cells and gin-gipains led to opposite cellular responses, i.e., reductions in thedetection levels of these mediators (33). Interestingly, similarfindings were also reported by Grenier and Tanabe; both Arg-gingipain A/B and Lys-gingipain were found to induce the secre-tion of tumor necrosis factor alpha (TNF-�) and IL-8 by macro-phages, whereas stimulation with Arg-gingipain A/B at the highestconcentration was associated with reduced detection of cytokines(34).

HbR is the major protein encoded by rgpA, kgp, and hagA andshould be one of the major inducers of the host immune response,including production of proinflammatory and anti-inflammatorycytokines. This is because HbR is located at the surface of P. gin-givalis cells and is released from bacterial cells as a major extracel-lular protein during prolonged incubation (13, 35). Moreover,HbR has the ability to tightly bind to the surface of host cells (12).

DeCarlo et al. (36) reported that anti-HbR antibodies are found inthe sera of patients with periodontal diseases, and the serum anti-body titers specific for the HbR domain vary inversely with peri-odontal disease severity, suggesting that HbR produced by P. gin-givalis cells is exposed to host immune systems.

Numerous studies have reported that infection of epithelialcells with bacterial pathogens, including Salmonella enterica sero-var Typhimurium (37, 38), Helicobacter pylori (16, 39, 40), entero-hemorrhagic E. coli (41), enteropathogenic E. coli (42), and Clos-tridium perfringens (43), results in the release of IL-8 through theactivation of MAPK pathways.

MAPKs are key components of many intracellular signalingpathways (44, 45). This superfamily includes Erk proteins, c-junN-terminal kinases (JNKs), and the p38 family of kinases (re-viewed in references 46 and 47). Numerous studies have reportedthat LPS facilitates the activation of all three of these MAPK path-ways. Moreover, P. gingivalis LPS has also been reported to acti-vate MAPK signaling pathways (48–53). In addition to LPS, fim-briae (FimA) and hemagglutinin B (HagB) from P. gingivalis havebeen reported to activate MAPK pathways (51, 54, 55).

In a previous study, we found that HbR induced the phosphor-ylation of Erk and p38, as well as NF-�B and Akt, in bone marrowmacrophages (12). Therefore, we examined whether activation ofthe MAPK pathway contributed to IL-8 production in HbR-stim-ulated human gingival cells. In this study, we used specific MAPKinhibitors, i.e., PD98059 and SB203580, to investigate the involve-ment of MAPK signaling and transcriptional elements in HbR-

FIG 6 Activation of NF-�B by HbR and its involvement in HbR-induced IL-8 production. (A) Ca9-22 cells were incubated with 10 �g/ml of HbR at 37°C for2 h. Cells were fixed using 2% paraformaldehyde and permeabilized by incubation with 0.1% Triton X-100 for 5 min. Fixed cells, stained with 1 �g/ml of4=,6-diamidino-2-phenylindole for 5 min, were incubated with anti-NF-�B-p65 antibodies (1:100) in TBS containing 1% bovine serum albumin. After incu-bation with the primary antibody, cells were incubated with anti-rabbit polyclonal antibodies conjugated with Alexa Fluor 546 (1:1,000) in TBS containing 1%bovine serum albumin. The graph shows a quantitative analysis of NF-�B nuclear translocation. Results are means SEMs of three independent experiments.*, P � 0.01 (compared to the experiment without HbR). (B) Ca9-22 cells were treated with BAY11-7082 (0, 1, 3, or 10 �M) in serum-free medium for 1 h. ThenHbR (10 �g/ml) was added to the culture, and the incubation was continued for an additional 12 h. IL-8 production in the supernatant was detected by ELISA.Data are representative of at least three experiments. *, P � 0.01 (compared to the experiment without BAY11-7082). The control sample (BAY11-7082 �)contained 1% dimethyl sulfoxide (final concentration).

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stimulated IL-8 expression in Ca9-22 cells. Our results clearlydemonstrated that both PD98059 (an Erk1/2 inhibitor) andSB203580 (a p38 inhibitor) significantly inhibited the productionof IL-8 protein (Fig. 3), suggesting that the Erk1/2 and p38 MAPK

pathways were involved in the regulation of IL-8 production inHbR-stimulated Ca9-22 cells. PD98059 and SB203580 alsostrongly abolished ATF-2 and CREB activities, respectively (Fig.7A and B), and ATF-2 siRNA and CREB siRNA reduced IL-8production in HbR-stimulated Ca9-22 cells (Fig. 5D). Theseresults suggested that the Erk1/2 and p38 MAPK pathways medi-ated the activation of ATF-2 and CREB, respectively, during theregulation of IL-8 production in HbR-stimulated Ca9-22 cells.Moreover, inhibition of IL-8 production by SB203580 in HbR-stimulated Ca9-22 cells was enhanced by cotreatment with BAY-11-7082 (an I�B� inhibitor), suggesting that the Erk1/2 pathwayalso mediated activation of NF-�B.

There are numerous reports on MAPK-mediated IL-8 produc-tion in human cells stimulated with bacterial components. How-ever, the MAPK pathways used in this process are dependent onvirulence factors and the uniqueness of each cell type. The obser-vations of our current study are comparable to those made inother investigations. Hisatsune and colleagues noted activation ofErk1/2 and p38, but not the participation of JNK, in H. pyloriVacA-induced IL-8 production by U937 cells (16). Interestingly,in contrast to our results, p38 MAPK has been shown to mediateATF-2 phosphorylation in VacA-stimulated AZ521 cells (56). Ad-ditionally, C. perfringens alpha-toxin induces the production ofIL-8 by Erk1/2/NF-�B-p65 and p38 MAPK pathways in A549 cells(43). Moreover, activation of Erk and p38, but not participation ofJNK, was also observed in Moraxella catarrhalis-induced IL-8 pro-duction by epithelial cells (57). Gaddis et al. demonstrated thathemagglutinin B (HagB) from P. gingivalis induced the activationof Erk1/2, p38 MAPK, NF-�B, and CREB to produce proinflam-matory and anti-inflammatory cytokines in dendritic cells (54).However, it has also been reported that HagB induces activation ofErk1/2, p38, and JNK, leading to IL-8 production in macrophages(55).

NF-�B-p65/RelA is known to be a critical transcriptional fac-

FIG 7 Relationships among MAP kinases, ATF-2, CREB, and NF-�B in HbR-induced IL-8 production. Ca9-22 cells were treated with SB203580 (3 �M) (A)or PD98059 (3 �M) (B) in serum-free medium for 1 h. Then HbR (10 �g/ml)was added to the culture, and the incubation was continued for up to anadditional 120 min. Proteins in the cells were separated by SDS-PAGE, trans-ferred to PVDF membranes, and then immunostained with anti-phospho-ATF-2 (Thr71), anti-ATF-2, anti-phospho-CREB (Ser133), or anti-CREB an-tibodies. (C) Ca9-22 cells were pretreated with BAY11-7082 (3 �M), SB203580(3 �M), PD98059 (3 �M), or a combination of inhibitors (3 �M each) for 1 hbefore incubation with 10 �g/ml of HbR in serum-free medium. IL-8 levels inthe supernatant were detected by ELISA. Data are representative of three ex-periments. *, P � 0.01 (compared to the experiment without any inhibitor).Control samples (SB203580 �, PD98059 �, and BAY11-7082 �) contained1% dimethyl sulfoxide (final concentration).

FIG 8 Schematic representation of proposed signaling pathways involved inHbR-induced IL-8 production. Upon exposure to HbR, Erk1/2 is activated,resulting in the activation of NF-�B and ATF-2, which, in turn, may promoteIL-8 transcription. In parallel, HbR activates the p38-MAP kinase pathway,which is also involved in HbR-mediated IL-8 upregulation through the acti-vation of CREB.

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tor for IL-8 production (58). In our study, the amount of phos-phorylated NF-�B-p65 in the nuclei of Ca9-22 cells was increasedby HbR stimulation (Fig. 6A), and BAY-11-7082 significantly re-duced HbR-induced IL-8 production (Fig. 6B), suggesting thatthe canonical NF-�B pathway was an important driver of IL-8expression.

In summary, our present findings provided the first evidencethat HbR, a major protein in culture supernatants of P. gingivalis,stimulated the activation of human GEC, inducing the productionof IL-8. The transcription factors ATF-2, CREB, and NF-�B, aswell as their upstream activators Erk and p38 MAPK, play keyroles in this response. The precise signaling pathways involved inthis process will be the subject of future studies.

ACKNOWLEDGMENT

This work was supported in part by a Grant-in-Aid for Science Researchfrom the Ministry of Education, Culture, Sports, Sciences, and Technol-ogy of Japan.

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