Microsoft Word - 김민영

Transcription

1 Td92, an outer membrane protein of Treponema denticola, induces osteoclatogenesis via PGE 2 mediated RANKL/OPG regulation Minyoung Kim The Graduate School Yonsei University Department of Applied Life Science

2 Td92, an outer membrane protein of Treponema denticola, induces osteoclatogenesis via PGE 2 mediated RANKL/OPG regulation Directed by Professor Yun-Jung Yoo The Master s Thesis Submitted to the Department of Applied Life Science and the Graduate School of Yonsei University in partial fulfillment of the requirements for the degree of Master of Dental Science Minyoung Kim July 2009

3 ACKNOWLEDGEMENT 모든일에는우연이없고오직주님의섭리만이있음을고백합니다. 아직푸릇푸릇한젊음의인생이지만, 여러나라를오가며생활하는가운데많은일들의계기와마무리, 그리고다음페이지로의진행은저의의지가아니었음을깨닫습니다. 스쳐지나가는생각들조차주님께서살피고계심을안다는것은큰축복이아닐수없습니다. 그러므로이논문의첫감사는당연히제인생을주관하고계신주님께드려야할것입니다. 이논문은제가치위생사로서평소많은관심을가졌던치주염의발병기전에관한연구의기록입니다. 주님께서창조하신세계의일부를공부하며인간으로서제가알고있는것의한계를절실히느꼈지만, 그것을탓하기보다이모든눈에보이지않는것들의움직임을세밀히주관하고계신그분의창조성에감탄하는시간이더많았습니다. 그무엇하나이유없이움직이는것들은없었습니다. 그움직임들을이해하기위해선저의지혜가아닌창조주하나님께서주시는지혜가필요했습니다. 어려움들가운데주님을기억하게하신역사를감사드립니다. 모든생명의근본, 지혜의근본은하나님이십니다. 아직부족하지만이것이제가석사기간동안가장깊이배운점인것같습니다. 저에게좋아하고궁금해하던분야를공부하는기쁨을누릴수있게도움을주신유윤정지도교수님께마음깊이감사를드립니다. 꾸준한운동으로건강한자기관리의모범을보이신차정헌교수님, 저의건강또한많이챙겨주셔서늘든든했습니다. 부드러운다독임과날렵한지적으로더좋은논문이태어날수있게도움을주신서울대치대최봉규교수님께도깊은감사를드립니다. 미국에서꼭한번뵙고싶습니다. 우리실험실의분위기메이커박은정포닥선생님, 양파즙으로매일거듭나고계신홍규오빠, 토종한국인임이밝혀진동기진문이, 언제나어디서나주님앞에무릎꿇어기도하고있다는성환이, 위력이가히위력적인성실한성일이, 다들고맙고또고마워요. 차한잔의여유를즐길줄아는멋진효진, 유럽학회의보따리친구였던단아한아란, 뭐든지다있어보이는생화학방의귀염둥이주아와지희, 아래층의한미모하시는제진아, 이주현선생님, 벌서보고싶어집니다. 단비같은말씀으로주말마다저의지친영혼을살아나게하신최용태목사님, 똑소리나는우리이은숙전쏴님, 어두운곳을좋아하는것같지만은근아기자기한워비스트미디어팀의팀원들, 기도로함께해줘서고맙고사랑합니다. 사랑하는베프수영아, 결혼축하해!! 우린결혼도비슷하게하는구나 ~ 영화죽은시인의사회의한국판주인공이라할수있는김성일선생님. 학생들에대한식지않는가르침의열정은대한민국에서가장뜨거우실것같습니다. 학생으로서느꼈던그감동은 10 년이지난지금도생생합니다. 선생님께이논문을드릴수있어기쁩니다. 지병인이비인후과질환이나을수있게저의석사기간동안정말신경많이써주신세브란스병원이비인후과의명의김창훈교수님께도감사를잊을수없습니다.

4 한국에서치위생사면허를취득할수있게모든여건들을도와주신연세대학교치위생과정원균교수님, 김남희교수님, 장선옥선생님께감사드립니다. 혹독한겨울의국시맹공부였지만, 선생님들의따뜻한배려에모든것이넉넉히좋았습니다. 막내딸의긴공부인생을줄곧기도로응원해주신사랑하는엄마, 아빠,, 두분의아낌없는지원으로제능력으로는경험할수없었던더큰세상에서많은것을배울수있었습니다. 사랑해요엄마, 아빠 ~ 언니와오빠, 그리고완전귀여운조카준혁이에게도그동안고맙다는말제대로못했는데저를많이배려해주고이해해줘서고마워요. 우리집의엔돌핀준혁이는고모에게큰힘이되었어요 ~ 엄마, 아빠못지않게저를많이사랑해주신할아버지, 할머니께도이논문을자랑스럽게드리고싶습니다. 그리고이제곧저의시아버님, 시어머님, 도련님이되실대전의아버님, 어머님, 호준이, 그리고시할머님, 시할아버님. 논문때문에자주못뵈러가서제얼굴까먹으셨을텐데곧보여드리러갈게요 ~ 마지막으로, 저의지침을위로해주고집념을응원해주고무엇보다그긴시간동안사랑으로제곁을지켜준신랑호림에게사랑을전합니다 년 7 월 13 일 신촌동에서민영 Special Thanks to.. I give special thanks to Dr. Henderson, Donna Cleere, Jnita Collins, Jane McFarland, Karen Lester, Lynnette Hayhurst, and Dr. Fong of Amarillo College Dental Hygiene Program. I would like to appreciate to all the professors who provided me every necessary need for me to focus on dental hygiene studying. You have given me a dream to become such a great professor like you and it is a big part of my life that cannot be compared with anything else. Even if I cannot contact you as well as I wish, please remember that you are always in my heart. Also, I would like to say that I miss my best friend Nikki Darrah who always stood next to me and shared God s love. I miss all of you Jinsil, Grace, Sarah, Rhett, Charity, Laura, Yiling, and Rachel who were my dorm friends during the best time of my life at ORU.

5 TABLE OF CONTENTS ABSTRACT (In English) 1 I. INTRODUCTION 4 II. MATERIALS AND METHODS 9 1. Chemicals 9 2. Preparation of Td SDS-PAGE analysis Preparation of primary osteoblasts Osteoclast formation assay ELISA for cytokines and PGE Statistical analysis 15 III. RESULTS Effect of Td92 on osteoclast formation Effect of Td92 on RANKL and OPG expression in osteoblasts Effect of Td92 on PGE 2 expression in osteoblasts Effect of NS398 and indomethacin on RANKL/OPG expression - i -

6 altered by Td Effect of NS398 and indomethacin on osteoclast formation induced by Td92 25 IV. DISCUSSION 26 V. REFERENCES 36 ABSTRACT (In Korean) 46 - ii -

7 LIST OF FIGURES Figure 1. Expression of Td92 in Escherichia coli Figure 2. Effect of Td92 on osteoclast formation Figure 3. Effect of Td92 on RANKL and OPG expression in osteoblasts Figure 4. Effect of NS398 and indomethacin on Td92-regulated RANKL and OPG expression in osteoblasts Figure 5. Effect of NS398 and indomethacin on Td92-induced osteoclast formation Figure 6. Td92 induces osteoclastogenesis by regulating RANKL/OPG/PGE 2 - iii -

8 ABSTRACT Td92, an outer membrane protein of Treponema denticola, induces osteoclatogenesis via PGE 2 mediated RANKL/OPG regulation Minyoung Kim Department of Applied Life Science The Graduate School, Yonsei University (Directed by Professor Yun-Jung Yoo) Periodontitis is a chronic inflammatory disease in periodontium which causes significant alveolar bone loss. Osteoclasts are bone-resorbing multinucleated cells. Osteoblasts regulate osteoclast differentiation by receptor activator of NF-кB ligand (RANKL) and osteoprotegerin (OPG) expression

9 Treponema denticola is one of the oral bacteria involved in periodontitis. Td92, Tp92 homolog of T. denticola, is a surface-exposed outer membrane protein that stimulates production of various proinflammatory mediators. However, the role of Td92 on alveolar bone resorption still remains unclear. To elucidate the role of Td92 on bone resorption, the effect of Td92 on osteoclast differentiation was evaluated in co-cultures of mouse calvariae-derived osteoblasts and bone marrow cells. The expression of RANKL, OPG, and PGE 2 in osteoblasts was estimated by ELISA. Td92 induced osteoclast formation in co-cultures. In osteoblasts, RANKL and PGE 2 expression was upregulated while OPG expression was down-regulated by Td92. OPG inhibited Td92-induced osteoclast formation. NS398 or indomethacin, prostaglandin synthesis inhibitors, also inhibited Td92-induced osteoclast formation. The effect of Td92 on expressions of RANKL, OPG, and PGE 2 in osteoblasts was blocked by NS398 or indomethacin. These results suggest that Td92 promotes osteoclast formation through the regulation of RANKL and OPG productions via PGE 2 dependent mechanism

10 Key words: Treponema denticola, Td92, osteoclast formation, periodontitis - 3 -

11 Td92, an outer membrane protein of Treponema denticola, induces osteoclatogenesis via PGE 2 mediated RANKL/OPG regulation Minyoung Kim Department of Applied Life Science The Graduate School, Yonsei University (Directed by Professor Yun-Jung Yoo) I. INTRODUCTION Osteoclasts are tartrate-resistant acid phosphatase (TRAP) positive multinucleated cells with bone-resorbing activity. The osteoclast formation is induced by receptor activator of NF-ĸB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) expressed by osteoblast/stromal cells (1, 2). In bone, RANKL is an essential cytokine for osteoclastogenesis and it presents - 4 -

12 in soluble- and membrane-bound form (3). RANKL binds to RANK, a receptor of RANKL, expressed on osteoclast precursors and by this interaction, osteoclast precursors differentiate into osteoclasts in the presence of macrophage-colony stimulating factor (M-CSF). Osteoprotegerin (OPG) produced by osteoblasts blocks osteoclastogenesis by interfering RANKL- RANK interaction (4). The levels of RANKL and OPG expression are regulated by several bone-resorbing factors such as 1α,25-dihydroxyvitamin D ₃(1α,25(OH) ₂D ₃), parathyroid hormone (PTH), prostaglandin E ₂(PGE ₂ ) and lipopolysaccharide (LPS). Periodontitis is a chronic inflammatory disease in the periodontal tissue with bacterial etiology. Alveolar bone is a periodontal tissue to support teeth. Also, alveolar bone loss observed in periodontitis is a non-reversible condition induced by stimulation of osteoclast formation. Therefore, understanding the mechanisms of bone resorption induced by periodontal pathogens can be an important knowledge for the prevention and treatment of periodontitis. It was reported that many of periodontal pathogens, such as - 5 -

13 Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella nigrescens show the activity of bone resorption. LPS of P. gingivalis or P. nigrescens also have been implicated as an inducer of osteoclast formation in periodontitis (5, 6). A. actinomycetemcomitans has several osteoclastogenic factors such as LPS, capsular polysaccharides (CPs), and chaperone (cpn60) (7). Therefore, various components of oral bacteria including LPS, appear to be a potent activator of osteoclastogenesis in periodontitis (8-11). An elevated level of Treponema species in subgingival plaque of periodontitis patients supports the hypothesis that Treponema species play an important role in periodontitis. There are 10 species of oral spirochetes which can be cultivated up to present time and Treponema denticola is one them. T. denticola is known to be involved in the early-onset periodontitis, chronic periodontitis, and necrotizing ulcerative gingivitis (12). T. denticola has various virulence factors such as proteolytic enzymes and cytolytic factors which involved in pathogenesis of periodontitis (13-17). Tp92 is a 92kDa T

14 pallidum antigen that resides on the treponemal surface and it has been reported to have immunoprotective capabilities of inducing opsonization and phagocytosis, thus may be a useful vaccine candidate for syphilis (18). Tp92 homologs (88 to 92 kda) were recently reported as highly conserved surface proteins of four representative oral spirochetes (Treponema denticola, T. lecithinolyticum, T. maltophilum, and T. socranskii subsp. socranskii) and they were shown to have amino-acid sequence identities of 37.9 to 49.3% and similarities of 54.5 to 66.9% to Tp92 (19). Tp92 homologs have been demonstrated to contribute to cell attachment, inflammation, and tissue destruction by inducing various proinflammatory factors. Td92 is a Tp92 homolog of T. denticola that is a highly conserved surface protein of T. denticola (19). Td92 is considered to be involved in cytopathogenic process of periodontal disease through binding to epithelial cells and inducing the expression of pro-inflammatory factors such as Interleukin (IL)-6, IL-8, COX- 2, and PGE 2 in human monocytic cell line and periodontal ligament (PDL) cells (19). However, it is not yet to be identified for the role of Td92 in bone - 7 -

15 resorption. Therefore, the effect of Td92 on osteoclast formation was evaluated in co-cultures of mouse calvaria-derived osteoblasts and bone marrow cells. Also, the involvement of RANKL, OPG, and PGE 2 in Td92- induced osteoclast formation was evaluated in osteoblast culture

16 II. MATERIALS AND METHODS 1. Chemicals LPS (Escherichia coli O26:B6), Indomethacin, CelLytic M, and Protease Inhibitor Cocktail were purchased from Sigma (St. Louis, MO, USA). NS398 was purchased from Calbiochem (San Diego, CA, USA). Human rm-csf and Human ropg were obtained from Peprotech (Rocky Hill, NJ, USA). Collagenase was purchased from Wako pure Chemicals (Osaka, Japan). Minimum Essential Medium alpha (α-mem), Dispase, Fetal bovine serum (FBS), Dulbecco s phosphate-buffered Saline (PBS), 100X Antibiotic- Antimycotic (Ab), and 25% Trypsin-EDTA were purchased from Gibco BRL (Grand Lsland, NY, USA). The chemicals used for TRAP staining is as follows: Sodium Acetate Trihydrate, Fast Red Violet LB Salt, and Naphthol AS-MX phosphate were purchased from Sigma (St. Louis, MO, USA). Sodium (+)- Tartrate Dihydrate was purchased from Wako pure Chemicals (Osaka, Japan). Acetic Acid was purchased Junsei Chemicals (Tokyo, Japan)

17 Mice were obtained from Sankyo Laboratory Animal Center (Tokyo, Japan). Animal studies were performed after the experimental protocols approved by animal ethics committee of Yonsei University College of Dentistry. 2. Preparation of Td92 Recombinant Tp92 homolog of T. denticola (Td92) was thankfully given from B.K. Choi in Seoul National University. The Tp92 gene homolog of T. denticola was amplified from the genomic DNA by PCR. PCR was performed in a total volume of 50 μl containing 15 pmol of each primer, 1.25 U of Ex Taq polymerase (Perkin Elmer Cetus, Foster City, CA). The PCR products were cloned in E. coli by using the TA cloning vector pcr2.1-topo, and the inserts were isolated and cloned in E. coli M15 by using the expression vector pqe-30 as described previously (20). After the induction of E. coli with 1mM isopropyl-β-d-thiogalactopyranoside (IPTG), histidine-tagged recombinant proteins were purified by sonication, solubilizaiton with a detergent, and subsequent renaturation, followed by affinity chromatography using nickel

18 nitrilotriacetic acid agarose (Qiagen, Valencia, CA) as described previously (20). Endotoxin present in E. coli, which may potentially contaminate the recombinant proteins, were removed using polymyxinb-agarose according to the instructions of the manufacturer (Sigma chemicals Co., St. Louis, MO, USA). The endotoxin decontamination of Td92 was verified using CHO/CD14/TLR4cells. 3. SDS-PAGE and immunoblot assay The expression of the Td92 in E. coli was verified by immunoblotting using a monoclonal mouse antihistidine Ab (Qiagen, Alencia, CA, USA). E. coli M15 cells transformed with the recombinant plasmids were cultured in Luria- Bertani broth containing antibiotics and induced with IPTG. The E. coli lysates (20 μg of protein) were subjected to SDS-PAGE and subsequently transferred onto nitrocellulose membranes. The membranes were blocked with 2% bovine serum albumin (BSA) for 1h and allowed to react with antihistidine Ab for 1h. After being washed with PBS-0.2% Tween 20, the membranes were allowed

19 to react with alkaline phsphatase-labeled anti-mouse IgG for 1 h. After being washed with PBS-0.2% Tween 20, the membranes were developed with 5- bromo-4-chloro-3-indolylphosphate (165 μg/ml) and nitroblue tetrasolium (330 μg/ml). The expected molecular size of the prepared Td92 was approximately 92kDa (Fig. 1). 4. Preparation of primary osteoblasts Mouse osteoblasts were isolated and cultured as described previous studies (21, 22). Twenty-five to thirty newborn ddy mice (1-day-old) were used for one preparation of osteoblasts. Mice were sacrificed in 70% alcohol and calvariae including frontal and parietal bones were detached anatomically. Calvariae were then collected in α-mem and washed in 10 ml of α MEM containing 0.2% collagenase and 0.1% dispase briefly to remove debris and blood cells. Calvariae were incubated in collagenase-dispase solution for 10 min at 37, in a 250 x g shaking water bath. The first supernatant was discarded and 10 ml of fresh solution was added and incubated at 37, in a 250 x g shaking water

20 bath for 20 min. The supernatant was collected and further incubation with fresh solution was repeated for four times. The last four supernatants were collected as a primary osteoblast population. Primary osteoblasts were cultured in α-mem supplemented with 10% FBS and 1% Ab mixture for 3 days. Cells were detached by trypsin-edta, centrifuged, suspended in 90% FBS and 10% dimethyl sulfoxide, and stored at Osteoclast formation assay Primary osteoblasts stored at -80 were cultured in α-mem supplemented with 10% FBS and 1% Ab. Bone marrow cells were obtained from tibiae of 6- wk-old male ddy mice and cultured in the presence of M-CSF (50 ng/ml) for 16 hours prior to co-culture. Primary osteoblasts (8 x 10 3 cells) were cocultured with bone marrow cells (8 x 10 4 cells) for 6 days in 200 μl α-mem containing 10% FBS and 1% Ab in 96-well plates (NUNC, Denmark). Cocultures were treated with μg/ml Td92 or μg/ml LPS in the absence or presence of OPG (100 ng/ml), NS398 (1 μm) or indomethacin (1-13 -

21 μm). The medium was refreshed on 3rd day. Osteoclast formation was evaluated by TRAP, a marker enzyme of osteoclasts, staining. TRAP staining was performed as described previously (23). Cells were fixed with 10% formaldehyde and with ethanol/acetone (1:1) solution. 100 μl of TRAP staining solution was added to each well and stained for 5 min. TRAP positive multinucleated cells containing more than 3 nuclei were counted as osteoclasts. The results obtained from a typical experiment of three independents are expressed as the mean SD of three cultures. 6. ELISA for cytokines and PGE 2 Primary osteoblasts (2x10 4 cells) were cultured in α-mem containing 10% FBS and 1% Ab medium in 48-well culture plate until confluence. Cells were then further incubated in the presence or absence of Tp92 homolog (10 μg/ml) or LPS (0.01 μg/ml) for 3 days. Some cultures were treated in combination with NS398 (1 μm) or indomethacin (1 μm). The cell lysates were harvested to determine the concentration of RANKL and culture supernatants were used

22 to determine the concentration of OPG or PGE 2. To collect cell lysates, cultures wells were washed with DPBS and treated with CelLytic M for 15 min. The collected cells were centrifuged at 16,000 x g for 15 min to pellet cellular debris. Each sample was quantified using Bio-Rad Protein Assay kit prior to ELISA. The results obtained from a typical experiment of three independents are expressed as the mean SD of two cultures. 7. Statistical analysis Statistical analysis was performed by a Student s t-test of Sigma Plot 8.0 to express the difference between the two groups. P value < 0.05 was considered to be statistically significant

23 A kda M ㅡ B kda M ㅡ Figure 1. Expression of td92 in E. coli. Expression of histidine-tagged recombinant proteins was analyzed by SDS-PAGE (10% polyacrylamide gel) (A) and immunoblot assay with anti-histidine antibody (B). M: Protein size marker; : uninduced E. coli cell lysates; +: IPTG-induced E. coli cell lysates

24 II. RESULTS 1. Effect of Td92 on osteoclast formation To determine the effect of T. denticola Tp92 homolog (Td92) on osteoclast formation, co-cultures composed of osteoblasts and bone marrow cells were treated with Td92 (1 to 10 μg/ml), and TRAP-positive multinucleated cells were counted. Td92 stimulated the formation of TRAP-positive osteoclasts and the maximal number of osteoclasts was observed at 5-10 g/ml (Fig. 2A and B). LPS, a positive control, showed the maximal effect of osteoclast formation at a concentration of g/ml (Fig. 2A and C). Endotoxin decontamination of Td92 was verified by heat treatment of Td92 at 99 C for 30 min. Heated Td92 (10 μg/ml) did not induce osteoclast formation while heated LPS still induced osteoclast formation in a similar manner with noneheated LPS (Fig. 2D), suggesting Td92 is a heat labile stimulator of osteoclast formation

25 A None Td92 10 μg/ml LPS 0.1 μg/ml B C TRAP+ MNCs/well TRAP+ MNCs/well Td92 ( μ g/ml) LPS ( μ g/ml) D None Td92 LPS TRAP+ MNCs/well Figure 2. Effect of Td92 on osteoclast formation. Mouse calvariae-derived osteoblasts and bone marrow cells were co-cultured in the absence or presence of Td92 ( μg/ml) or LPS ( μg/ml) for 6 days and the cells were stained for TRAP (A). TRAP-positive multinucleated cells containing more than three nuclei were counted as osteoclasts (B, C)., P < 0.05 vs. none-treated cells. Co-cultures were treated with Td92 (10 μg/ml, open bar), heated Td92 (10 μg/ml, oblique lined bar), LPS (0.1 μg/ml, open bar) or heated LPS (0.1 μg/ml, oblique lined bar) for 6 days (D). TRAP-positive multinucleated cells containing more than three nuclei were counted as osteoclasts., P < 0.05 vs. Td92 or LPS treated-cells.

26 2. Effect of Td92 on RANKL and OPG expression in osteoblasts In order to assess the effect of Td92 on RANKL and OPG expression, Td92 (10 μg/ml) or heated Td92 was treated in osteoblasts. Also, the levels of RANKL in cell lysates and OPG in culture supernatants were assayed by ELISA. Td92 increased RANKL expression similar to LPS and its expression was 4.7-fold higher compared to none-treated group (Fig. 3A). Heated Td92 did not increase RANKL expression. In contrast to Td92, heat treatment of LPS did not block the LPS-induced RANKL expression. Td92 decreased OPG expression similar to LPS and its expression was 2.4-fold lower compared to none-treated group (Fig. 3B). Heated Td92 recovered the Td92-induced OPG decrease. In contrast to Td92, heat treatment of LPS did not recover the LPSinduced down-regulated OPG expression (Fig. 3C). To confirm the involvement of RANKL in Td92- induced osteoclast formation, co-cultures were treated with Td92 in the presence or absence of OPG. The osteoclast formation stimulated by Td92 was completely inhibited by the addition of OPG (Fig. 3D). These results suggest that the expression level of RANKL/

27 OPG in osteoblasts is crucially involved in Td92-stimulated osteoclast formation

28 A B Concentration of RANKL (pg/ μ g protein) None Td92 Heated Td92 LPS Heated LPS Concentration of OPG (pg/ml) None Td92 Heated Td92 LPS Heated LPS C None Td92 -OPG +OPG TRAP+ MNCs/well LPS 0 Td92 LPS OPG Figure 3. Effect of Td92 on RANKL and OPG expression in osteoblasts. Calvariaederived osteoblasts were cultured with Td92 (10 μg/ml), heated Td92 (10 μg/ml), LPS (0.1 μg/ml) or heated LPS (0.1 μg/ml) for 3 days. The concentration of RANKL (A) and OPG (B) in cell lysates or culture supernatants, respectively, was determined by ELISA., P < 0.05 vs. none-treated cells,, P < 0.05 vs. Td92 or LPS-treated cells. Osteoblasts and bone marrow cells were co-cultured with Td92 (10 μg/ml) or LPS (0.1 μg/ml) in the absence or presence of OPG (100 ng/ml) for 6 days (C). TRAP-positive multinucleated cells containing more than three nuclei were counted as osteoclasts., P < 0.05 vs. none-treated cells,, P < 0.05 vs. Td92 or LPS-treated cells.

29 3. Effect of Td92 on PGE 2 expression in osteoblasts In order to assess the involvement of PGE 2 in Td92- induced osteoclast formation, osteoblasts were stimulated with Td92 and the level of PGE 2 expression was measured by ELISA. Osteoblasts treated with Td92 or LPS showed increased level of PGE 2 expression (Fig. 4A). The increased PGE 2 expression, furthermore, was completely inhibited with the addition of NS398 or indomethacin, inhibitors of prostaglandin synthesis (Fig. 4A). The effect of NS398 or indomethacin on cell viability of osteoblasts was evaluated by MTT assay. NS398 or indomethacin did not affect cell viability (data not shown). 4. Effect of NS398 and indomethacin on RANKL/OPG expression altered by Td92 To investigate whether PGE 2 is involved in regulating RANKL and OPG expression in osteoblasts, osteoblasts were treated with Td92 in the absence or presence of NS398 or indomethacin. RANKL and OPG expression level was measured by ELISA. The RANKL expression induced by Td92 or LPS was

30 down-regulated by NS398 or indomethacin (Fig. 4B). Down-regulated OPG expression by Td92 or LPS was recovered by NS398 or indomethacin (Fig. 4C). These results indicate that RANKL and OPG expression altered by Tp92 in osteoblasts are closely related to the production of PGE

31 A B C Concenration of PGE 2 (pg/ml) Td92 LPS NS398 Indomethacin Concentration of RANKL (pg/ g/protein) 0 Td92 LPS NS398 Indomethacin Concentration of OPG (pg/ml) 0 Td92 LPS NS398 Indomethacin μ Figure 4. Effect of NS398 and indomethacin on Td92-regulated RANKL and OPG expression in osteoblasts. Osteoblasts were cultured with Td92 (10 μg/ml) or LPS (0.1 μg/ml) in the absence or presence of NS398 (1 μm) or indomethacin (1 μm). The cultures were incubated for 3 days, and the concentration of PGE 2 (A), RANKL (B), and OPG (C). was determined by ELISA., P < 0.05 vs. none-treated cells., P < 0.05 vs. Td92 or LPStreated cells

32 5. Effect of NS398 and indomethacin on osteoclast formation induced by Td92 To confirm the involvement of PGE 2 in Td92-induced osteoclast formation, Td92 was added to co-culture in the absence or presence of NS398 or indomethacin. Osteoclast formation induced by Td92 was inhibited by the addition of NS398 (1 μm) or indomethacin (1 μm) (Fig. 5). These results indicate that PGE 2 is critically involved in Td92-induced osteoclastogenesis

33 - +NS398 +Indomethacin None Td92 LPS TRAP+ MNCs/well Td92 LPS NS398 Indomethacin Figure 5. Effect of NS398 and indomethacin on Td92-induced osteoclast formation. Osteoblasts and bone marrow cells were co-cultured with Td92 (10 μg/ml) or LPS (0.1 μg/ml) in the absence or presence of NS398 (1 μm) or indomethacin (1 μm) for 6 days. TRAP-positive multinucleated cells containing more than three nuclei were counted as osteoclasts., P < 0.05 vs. none-treated cells., P < 0.05 vs. Td92 or LPS-treated cells.

34 III. DISCUSSION Td92 has been proposed to contribute to the inflammation and osteoclastogenesis by inducing the production of tumor necrosis factor (TNF), IL-1β, IL-6, IL-8, and PGE 2 in THP-1 and PDL cells (19). Although it is possible that Td92 have osteoclastogenic ability by inducing osteoclastogenic cytokines, the exact role of Td92 on bone cells with the regulation of RANKL and OPG expressions remains unclear. For the first time, this study demonstrated that Td92 has stimulatory effect on osteoclastogenesis via RANKL/OPG/PGE 2 regulation. Outer membrane proteins (OMPs) of T. denticola include major surface proteins (Msp), hemolysin/agglutinin, dentilisin, OppA, and HbpA/HbpB. Msp of T. denticola has pore-forming activity and adhesive activity (24, 25). Pore-forming activity of Msp was demonstrated via depolarization and increased conductance of the HeLa cell membranes (26). Msp retards Ca 2+ release from endoplasmic reticulum stores and inhibits consequent Ca 2+ influx by uncoupling store-operated channels (27). In the

35 animal model, Msp was elevated followed by greater Th-2-titled immune response and greator bone resorption was consequently observed in T. denticola infected model (28, 29). Dentilisin, chymotrypsin-like protease (CTLP), adheres to and lyses epithelial cells (30). Dentilisin also reduce proinflammatory cytokines such as IL-1β, IL-6, and TNF-α by degradation and penetrates epithelial cell layer through disrupting transepithelial resistance (TER) by likely degradading the tight junctional proteins such as ZO-1 (31, 32). T. denticola OppA, a solute binding protein involved in peptide uptake and environmental signaling in a wide range of bacteria, was demonstrated to bind to plasminogen and fibronectin (33). These previous studies suggest that these OMPs of T. denticola are valuable candidate for the virulence factor with binding activity, cytotoxic/antigenic activity, and cell stimulatory activity during host-t. denticola interaction in periodontitis. In the present study, Td92, one of the OMPs of T. denticola, was demonstrated as a stimulator of osteoclastogenesis. Outer membrane proteins (OMPs) detected in bacterial outer membrane is one of the first class molecules implicated in host

36 bacterium interaction in the process of bacterial infection. Therefore, Td92, with other OMPs, could contribute to tissue destruction in periodontitis. LPS, a representative potent inflammatory stimulator of gramnegative bacteria, binds to Toll-like receptor 4 (TLR4) and induces myeloid differentation protein 88 (MyD88), which then initiates the activation of downstream signaling pathways, leading to osteoclast formation through RANKL/OPG regulation in osteoblasts (8). Therefore, LPS is generally used as a positive control in the study of osteoclast formation of periodontitis. Prior to experiments, E. coli LPS from different manufactures and strains were tested and the maximal osteoclastogenic effect of LPS was seemed to shift in the range of 0.01 to 1 g/ml depends on manufactures or the origins of E. coli (data not shown). In this study, 0.1 g/ml of LPS from E. coli 026:B6 was used with maximal osteoclastogenic effect. When Td92 was compared with LPS, the osteoclastogenic activity of Td92 was similar to that of LPS. Regarding periodontitis, RANKL and OPG are important cytokines which stimulates or inhibits bone destruction, respectively. Osteoblasts

37 infected with P. gingivalis exhibited elevated RANKL expression (34) and osteoblasts treated with LPS from P. nigrescens showed decreasing of OPG expression. The prevalence of A. actinomycetemcomitans, P. gingivalis, and T. forsythensis and level of RANKL in gingival crevicular fluid (GCF) of periodontitis patients was shown to be positively correlated. Other studies also demonstrated the increased RANKL expression level in GCF/gingival tissues of diseased sites is correlated to the incidence of periodontitis (35-38). RANKL has been proposed to be both Soluble- and membrane-bound form in association with osteoclastogenesis. Hofbauer et al. reported direct cell-to-cell contact with osteoblasts would allow continuous exposure of the membranebound RANKL to osteoclast precursors whereas the soluble form would explain the stimulatory effect of conditioned medium harvested from osteoblasts (4). Suda et al. reported direct cell-to-cell contact between osteoblasts and osteoclast progenitors is involved in osteoclastogenesis (39). Hikita et al. described the ectodomain shedding of membrane-bound RANKL (mrna) in vivo by RANKL sheddases such as matrix metalloproteinase

38 (MMP14) (40). In order to determine which forms of RANKL expression is mainly involved in osteoclast formation by Td92, the level of RANKL in both supernatant and cell lysates harvested from osteoblast culture was examined. Cell lysates showed increased level of RANKL with Td92 treatment. However, culture supernatants did not show the detective level of RANKL in any of treated groups (data not shown). These results indicate that Td92 primarily induces membrane-bound RANKL in osteoblasts. This study also showed that Td92 markedly decreased the expression level of OPG. To confirm the involvement of RANKL/OPG expressions in Td92-induced osteoclastogenesis, OPG, a decoy receptor of RANKL, was added in Td92 treated co-cultures. Osteoclast formation induced by Td92 was significantly inhibited by OPG. This result reveals that Td92 is a potent virulence factor in bone-resorptive periodontitis by inducing osteoclast formation through RANKL up-regulation and OPG down-regulation. Due to the Td92 is a recombinant protein, this study verified the endotoxin decontamination by utilizing NF-ĸB reporter cell line CHO/CD14/TLR4 cells

39 and heat-treatment. Td92 did not induce the NF-ĸB reporter in CHO cells to express membrane CD25 through TLR4-dependent NF-ĸB activation. However, LPS, a ligand of TLR4, induced NF-ĸB-regulated CD25 expression in flow cytometry (data not shown). When Td92 was heat-treated, osteoclast formation was completely inhibited and heat treatment of Td92 reversed the RANKL/OPG regulative activity of Td92. However, heat-treated LPS still possessed the same osteoclast formation activity and RANKL/OPG regulatory activity of none-heated LPS. Therefore, the effect of Td92 on osteoclast formation or RANKL/OPG regulation could be concluded as the effect of recombinant protein, not LPS. Recent studies have shown the increased GCF-PGE 2 concentrations at the site of periodontitis, which notify PGE 2 is one of the major pathogenic molecules in periodontitis (41, 42). In bone, PGE 2 has been demonstrated to play a critical role as a mediator of RANKL-dependent osteoclastogenesis (43). Elevation of PGE 2 in LPS-induced osteoblasts suppressed OPG expression (39). Therefore, correlation between PGE 2 and RANKL/OPG is one of the

40 main focuses in the study of alveolar bone resorption of periodontitis. In the present study, Td92 significantly induced PGE 2 production in osteoblasts and this induction was completely inhibited by NS398/indomethacin, the inhibitors of PGE 2 synthesis. In addition, up-regulated RANKL expression by Td92 was completely inhibited by NS398/indomethacin. In contrast, down-regulated OPG expression by Td92 was recovered by NS398/indomethacin. NS398/indomethacin also further inhibited osteoclast formation induced by Td92. These results indicate that PGE 2 is critically involved in Td92-induced osteoclastogenesis by regulating RANKL/OPG expression in osteoblasts. In osteoblasts, activated TLR4, a signal-transducing receptor for LPS, induces COX-2 which then stimulates PGE 2 (39). In this pathway, PGE 2 inhibits OPG expression and this finally induces osteoclastogenesis (44). Other studies also reported that LPS activates several downstream signaling pathways, which cause osteoclast differentiation through expression of RANKL in osteoblasts (45, 46). Although similarities between Td92 and LPS in inducing osteoclastogenesis were shown in this study, the precise

41 mechanisms of Td92 on alveolar bone resorption in periodontitis need further investigation within other osteoblast signaling pathways. Moreover, to better understand coincidence or differences among the other Tp92 homologs and the roles of Treponema species in alveolar bone resorption of periodontitis, Tp92 homologs of other Treponema species should be further investigated. This study showed that Td92 induces osteoclastogenesis by upregulating RANKL expression and down-regulating OPG expression and the regulation of RANKL/OPG is mediated by PGE 2 (Fig 6.). Therefore, this effect of Td92 may contribute to alveolar bone resorption of periodontitis

42 Osteoblast Td92 NS398 Indomethacin PGE 2 RANKL OPG Pre-osteoclast Osteoclast Figure 6. Td92 induces osteoclastogenesis by regulating RANKL/OPG/PGE

43 (1) Takahashi N, Akatsu T, Udagawa N, et al. Osteoblastic cells are involved in osteoclast formation. Endocrinology 1988; 123: (2) Udagawa N, Takahashi N, Akatsu T, et al. Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells. Proc Natl Acad Sci U S A 1990; 87: (3) Hofbauer LC, Kuhne CA, Viereck V. The OPG/RANKL/RANK system in metabolic bone diseases. J Musculoskelet Neuronal Interact 2004; 4: (4) Hofbauer LC, Khosla S, Dunstan CR, Lacey DL, Boyle WJ, Riggs BL. The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption. J Bone Miner Res 2000; 15: (5) Kiji M, Nagasawa T, Hormdee D, et al. Internal prostaglandin synthesis augments osteoprotegerin production in human gingival fibroblasts

44 stimulated by lipopolysaccharide. Clin Exp Immunol 2007; 149: (6) Chung YH, Chang EJ, Kim SJ, et al. Lipopolysaccharide from Prevotella nigrescens stimulates osteoclastogenesis in cocultures of bone marrow mononuclear cells and primary osteoblasts. J Periodontal Res 2006; 41: (7) Ohguchi Y, Ishihara Y, Ohguchi M, et al. Capsular polysaccharide from Actinobacillus actinomycetemcomitans inhibits IL-6 and IL-8 production in human gingival fibroblast. J Periodontal Res 2003; 38: (8) Yang S, Takahashi N, Yamashita T, et al. Muramyl dipeptide enhances osteoclast formation induced by lipopolysaccharide, IL-1 alpha, and TNF-alpha through nucleotide-binding oligomerization domain 2- mediated signaling in osteoblasts. J Immunol 2005; 175: (9) Wada N, Maeda H, Yoshimine Y, Akamine A. Lipopolysaccharide stimulates expression of osteoprotegerin and receptor activator of NF

45 kappa B ligand in periodontal ligament fibroblasts through the induction of interleukin-1 beta and tumor necrosis factor-alpha. Bone 2004; 35: (10) Daly CG, Seymour GJ, Kieser JB. Bacterial endotoxin: a role in chronic inflammatory periodontal disease? J Oral Pathol 1980; 9: (11) Ulevitch RJ, Tobias PS. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Annu Rev Immunol 1995; 13: (12) Sela MN. Role of Treponema denticola in periodontal diseases. Crit Rev Oral Biol Med 2001; 12: (13) Uitto VJ, Grenier D, Chan EC, McBride BC. Isolation of a chymotrypsinlike enzyme from Treponema denticola. Infect Immun 1988; 56: (14) Holt SC, Bramanti TE. Factors in virulence expression and their role in periodontal disease pathogenesis. Crit Rev Oral Biol Med 1991; 2:

46 (15) Syed SA, Makinen KK, Makinen PL, Chen CY, Muhammad Z. Proteolytic and oxidoreductase activity of Treponema denticola ATCC grown in an aerobic and anaerobic gaseous environment. Res Microbiol 1993; 144: (16) Rosen G, Naor R, Kutner S, Sela MN. Characterization of fibrinolytic activities of Treponema denticola. Infect Immun 1994; 62: (17) Rosen G, Naor R, Rahamim E, Yishai R, Sela MN. Proteases of Treponema denticola outer sheath and extracellular vesicles. Infect Immun 1995; 63: (18) Cameron CE, Lukehart SA, Castro C, Molini B, Godornes C, Van Voorhis WC. Opsonic potential, protective capacity, and sequence conservation of the Treponema pallidum subspecies pallidum Tp92. J Infect Dis 2000; 181: (19) Jun HK, Kang YM, Lee HR, Lee SH, Choi BK. Highly conserved surface proteins of oral spirochetes as adhesins and potent inducers of proinflammatory and osteoclastogenic factors. Infect Immun 2008; 76:

47 (20) Lee SH, Kim KK, Choi BK. Upregulation of intercellular adhesion molecule 1 and proinflammatory cytokines by the major surface proteins of Treponema maltophilum and Treponema lecithinolyticum, the phylogenetic group IV oral spirochetes associated with periodontitis and endodontic infections. Infect Immun 2005; 73: (21) Choi HG, Kim JM, Kim BJ, Yoo YJ, Cha JH. Mouse strain-dependent osteoclastogenesis in response to lipopolysaccharide. J Microbiol 2007; 45: (22) Choi BK, Moon SY, Cha JH, Kim KW, Yoo YJ. Prostaglandin E(2) is a main mediator in receptor activator of nuclear factor-kappab liganddependent osteoclastogenesis induced by Porphyromonas gingivalis, Treponema denticola, and Treponema socranskii. J Periodontol 2005; 76: (23) Suda T, Jimi E, Nakamura I, Takahashi N. Role of 1 alpha,

48 dihydroxyvitamin D3 in osteoclast differentiation and function. Methods Enzymol 1997; 282: (24) Cullen PA, Haake DA, Adler B. Outer membrane proteins of pathogenic spirochetes. FEMS Microbiol Rev 2004; 28: (25) Egli C, Leung WK, Muller KH, Hancock RE, McBride BC. Pore-forming properties of the major 53-kilodalton surface antigen from the outer sheath of Treponema denticola. Infect Immun 1993; 61: (26) Mathers DA, Leung WK, Fenno JC, Hong Y, McBride BC. The major surface protein complex of Treponema denticola depolarizes and induces ion channels in HeLa cell membranes. Infect Immun 1996; 64: (27) Wang Q, Ko KS, Kapus A, McCulloch CA, Ellen RP. A spirochete surface protein uncouples store-operated calcium channels in fibroblasts: a novel cytotoxic mechanism. J Biol Chem 2001; 276: (28) Lee SF, Andrian E, Rowland E, Marquez IC. Immune response and

49 alveolar bone resorption in a mouse model of Treponema denticola infection. Infect Immun 2009; 77: (29) Ishihara K, Okuda K. Molecular pathogenesis of the cell surface proteins and lipids from Treponema denticola. FEMS Microbiol Lett 1999; 181: (30) Fenno JC, Hannam PM, Leung WK, Tamura M, Uitto VJ, McBride BC. Cytopathic effects of the major surface protein and the chymotrypsinlike protease of Treponema denticola. Infect Immun 1998; 66: (31) Miyamoto M, Ishihara K, Okuda K. The Treponema denticola surface protease dentilisin degrades interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor alpha. Infect Immun 2006; 74: (32) Chi B, Qi M, Kuramitsu HK. Role of dentilisin in Treponema denticola epithelial cell layer penetration. Res Microbiol 2003; 154: (33) Fenno JC, Tamura M, Hannam PM, Wong GW, Chan RA, McBride BC. Identification of a Treponema denticola OppA homologue that binds

50 host proteins present in the subgingival environment. Infect Immun 2000; 68: (34) Okahashi N, Inaba H, Nakagawa I, et al. Porphyromonas gingivalis induces receptor activator of NF-kappaB ligand expression in osteoblasts through the activator protein 1 pathway. Infect Immun 2004; 72: (35) Liu D, Xu JK, Figliomeni L, et al. Expression of RANKL and OPG mrna in periodontal disease: possible involvement in bone destruction. Int J Mol Med 2003; 11: (36) Lu HK, Chen YL, Chang HC, Li CL, Kuo MY. Identification of the osteoprotegerin/receptor activator of nuclear factor-kappa B ligand system in gingival crevicular fluid and tissue of patients with chronic periodontitis. J Periodontal Res 2006; 41: (37) Mogi M, Otogoto J, Ota N, Togari A. Differential expression of RANKL and osteoprotegerin in gingival crevicular fluid of patients with periodontitis. J Dent Res 2004; 83:

51 (38) Vernal R, Chaparro A, Graumann R, Puente J, Valenzuela MA, Gamonal J. Levels of cytokine receptor activator of nuclear factor kappab ligand in gingival crevicular fluid in untreated chronic periodontitis patients. J Periodontol 2004; 75: (39) Suda K, Udagawa N, Sato N, et al. Suppression of osteoprotegerin expression by prostaglandin E2 is crucially involved in lipopolysaccharide-induced osteoclast formation. J Immunol 2004; 172: (40) Hikita A, Yana I, Wakeyama H, et al. Negative regulation of osteoclastogenesis by ectodomain shedding of receptor activator of NF-kappaB ligand. J Biol Chem 2006; 281: (41) Preshaw PM, Heasman PA. Prostaglandin E2 concentrations in gingival crevicular fluid: observations in untreated chronic periodontitis. J Clin Periodontol 2002; 29: (42) Soder B, Jin LJ, Wickholm S. Granulocyte elastase, matrix metalloproteinase-8 and prostaglandin E2 in gingival crevicular fluid in

52 matched clinical sites in smokers and non-smokers with persistent periodontitis. J Clin Periodontol 2002; 29: (43) Suzawa T, Miyaura C, Inada M, et al. The role of prostaglandin E receptor subtypes (EP1, EP2, EP3, and EP4) in bone resorption: an analysis using specific agonists for the respective EPs. Endocrinology 2000; 141: (44) Ohshiba T, Miyaura C, Ito A. Role of prostaglandin E produced by osteoblasts in osteolysis due to bone metastasis. Biochem Biophys Res Commun 2003; 300: (45) Zou W, Bar-Shavit Z. Dual modulation of osteoclast differentiation by lipopolysaccharide. J Bone Miner Res 2002; 17: (46) Sato N, Takahashi N, Suda K, et al. MyD88 but not TRIF is essential for osteoclastogenesis induced by lipopolysaccharide, diacyl lipopeptide, and IL-1alpha. J Exp Med 2004; 200:

53 ABSTRACT (IN KOREAN) Treponema denticola 의외막단백질인 Td92 의파골세포형성유도능 ( 지도교수유윤정 ) 연세대학교대학원응용생명과학과 김민영 치주염은치주조직의만성염증성질환으로치조골의흡수를야기한 다. 파골세포는골흡수기능을갖는다핵의세포이다. 조골세포는 파골세포분화조절인자인 receptor activator or NF- кb (RANKL) 와 osteoprotegerin (OPG) 를발현하고이를통해파골세포의분화 를조절한다. Treponema denticola 는치주염을일으키는원인균 들중하나이다. T. dencitola 의주외막단백질인 Tp92 homolog

54 (Td92) 는다른구강나선균의 Tp92 homolog 와상동성을보이며 염증인자와세포결합에관여하는것으로보고되어있으나 Td92 의치 조골의흡수에대한영향은아직알려져있지않다. 본연구에서는 Td92 의골흡수유도능을평가하기위해생쥐의두개골에서분리한 조골세포와경골에서분리한골수세포를혼합배양하여 Td92 의파골 세포의형성능을평가하였다. 또한효소면역측정법을이용하여조골 세포에서 Td92 의 RANKL, OPG 와 PGE 2 의생성유도능을분석하였 다. Td92 는혼합배양에서파골세포의형성을농도의존적으로유도하였 다. Td92 는조골세포의 RANKL 및 PGE 2 발현을증가시켰으며 OPG 발현은감소시켰다. OPG 와 PGE 2 형성억제인자인 NS398 및 indomethacin 은 Td92 에의한파골세포형성을억제하였다. 또한, NS398 및 indomethacin 은조골세포에서 Td92 에의한 RANKL 및

55 OPG 발현의변화를억제하였다. 이들결과는 T. denticola 의주외막 단백질인 Td92 가 PGE 2 를매개로조골세포의 RANKL 및 OPG 의 발현을조절하여파골세포형성을유도함을시사한다. 이를통해치 주염의주요원인균인 T. denticola 의주외막단백질이치조골의흡 수를일으키는치주염의병변에밀접한관련이있음을확인하였다. 핵심되는말 : Treponema denticola, 파골세포형성, Td92, 치주염