DRSRDH_2008_v22n6_1566.hwp

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1 동의생리병리학회지제 22 권 6 호 Korean J. Oriental Physiology & Pathology 22(6): , 2008 김정중 김동주 이병기 김광진 이명수 1 이재훈 1 김헌수 2 이창훈 1 변승재 3 장성조 4 송정훈 5 오재민 이준석 6 김광미 전철홍 7 * 원광대학교의과대학해부학교실, 1 : 내과학교실, 2 : 해부병리학교실, 3 : 일반외과학교실, 4 : 신경외과학교실, 5: 성형외과학교실, 6 : 산부인과학교실, 7 : 정형외과학교실 Effects of Curcumin on Osteoclasts Jeong Joong Kim, Dong Joo Kim, Byung Ki Lee, Kwang Jin Kim, Myeung Su Lee 1, Jae Hoon Lee 1, Hun Soo Kim 2, Chang Hoon Lee 1, Seung Jae Byun 3, Sung Jo Jang 4, Jeong Hoon Song 5, Jae Min Oh, Jun Seok Lee 6, Kwang Mee Kim, Churl Hong Chun 7 * Departments of Anatomy, 1:Departments of Internal Medicine, 2:Departments of Pathology, 3:Departments of Neurosurgery, 4:Departments of Reconstructive Surgery, 5:Departments of Plastic Surgery, 6:Departments of Obstetrics and Gynecology, 7:Departments of Orthopaedic Surgery, College of Medicine, Wonkwang University Bone is a dynamic tissue that is constantly resorbed by osteoclasts and then replaced by osteoblasts. Osteoclasts, multinucleated cells of monocyte/macrophage lineage, are responsible for bone disorders, including osteoporosis and rheumatoid arthritis. In this study, we examined the effect of the curcumin on osteoclast survival and bone resorption. We found that curcumin significantly inhibited RANKL-mediated osteoclast survival. DAPI staining revealed that curcumin induced the apoptotic features of osteoclasts. Although curcumin did not suppress the phosphorylation of Akt and ERK in osteoclasts treated with RANKL, curcumin induced the cleavage of pro-caspase-9 and -3 its active forms. Also, curcumin inhibited the formation of actin rings of osteoclasts. RANKL-mediated bone resorption was inhibited by the addition of curcumin. Together with the results of this study, these findings suggest that the curcumin inhibited the survival of osteoclasts by activating caspase-9 and -3 and suppressed the bone resorptive activity. Thus, curcumin may be developed as antiresorptive drugs for the treatment of bone-related disorders. Key words : bone, osteoclast, curcumin 서론 골 (bone) 항상성은골을흡수하는다핵형파골세포와골을형성하는조골세포의조화에의해양질의골이유지된다. 그러나만성염증, 에스트로젠 (estrogen) 의결핍, 파골세포유도인자의증가등으로파골세포의과도한증식및활성이유도되는데, 이로인하여치주염및골다공증이수반된다. 따라서파골세포의조절은골질환치료에중요하다할수있다 1,2). 다핵형파골세포는조혈모세포에서유래되어전구세포인단핵구및대식세포로부터많은과정을통해분화된다 (Takahashi et al., 1999). Receptor activator of NF-κB ligand(rankl; TRANCE, OPGL, * 교신저자 : 전철홍, 익산시신용동 원광대학교의과대학 cch@wonkwang.ac.kr, Tel : 접수 : 2008/10/27 수정 : 2008/11/20 채택 : 2008/12/02 ODF로도불림 ) 는 tumor necrosis factor (TNF) 계열의싸이토카인으로조골세포의세포막에서발현되며 RANK를발현하는전구세포와의결합으로파골세포의분화, 생존, 기능을촉진한다 3). RANKL이전구세포의 RANK에결합하면 NF-κB, Mi transcription factor (Mitf), PU.1, c-fos, nuclear factor of activated T cells (NFAT)c1과같은파골세포분화유도인자가발현된다 2). 특히, NFATc1은파골세포의분화에중요한조절자로작용하며 TRAP, OSCAR, DC-STAMP, MMP-9등분화및세포간의융합그리고골흡수에중요한여러인자발현을유도한다 4). 세포사멸은생물체의생리학적항상성을유지하기위한중요한과정이다. 세포사멸체계의불균형은 Alzheimer와같은신경학적질병과암발생등의생리학적질병이유발된다 5,6). 세포사멸의주요한경로는 extrinsic( 세포사멸인자에의존적인신호전달체계 ) 과 intrinsic(mitochondria에의존적인신호전달체계 )

2 신호전달경로로나뉜다 7,8). Mitochondria에의한세포사멸은 cytochrome c의방출로개시되는데, cytochrome c 방출은 Bak, Bax, Bad, Bid, Bim과같은세포사멸유도인자와 Bcl-2와 Bcl-xL 같은세포사멸억제인자의균형에의해조절된다 9,10). 세포사멸유도인자에의해방출된 cytochrome c는 datp, apoptotic proteinase-activating factor-1과연합되어 apoptosome이형성되는데, 이 apoptosome은 caspase-9와 caspase-3을활성화시켜세포사멸을유도한다 11-13). 식물은감염및질병으로부터방어하기위해수많은대사물질을만들어내는데식물에서추출된대사산물들은여러형태로인간의여러질병을위한약재로이용되고있다. 특히 curcumin (diferuloymethane) 은강황 (Curcuma longa) 에서유래된주요성분으로식료품, 화장품, 의약품등널리사용되고있다. curcumin은의약품으로널리알려져있는데, 호흡기관련질병 ( 천식, 알레르기 ) 과여러암 ( 대장암, 폐암, 유방암, 위암, 간암, 식도암 ) 동물모델에서의미있는효과가검증되었다 14). 최근 curcumin이 NF-κB 신호경로를억제하여 RANKL에의한파골세포분화를억제한다고보고되었다 15). 그러나파골세포의생존및골흡수에 curcumin의효과는알려지지않았다. 본연구에서우리는파골세포의생존과기능에 curcumin의효과를검증하였다. Curcumin은 caspase-9와 caspase-3의활성을촉진하여파골세포사멸을유도하였고파골세포의골흡수를억제하였다. 재료및방법 1. 시료본연구에사용한 curcumin은강황에서순수분리하였다. Human RANKL과 M-CSF는 Peprotech (London, UK) 사에서구입하였다. 1α, 25-dihydroxyvitamin D3 (VitD3), prostaglandin E2 (PGE2), 4,6-diamidino-2-phenylindole (DAPI), tartrate-resistant acid phosphatase (TRAP) 는 Sigma Aldrich (St. Louis, MO, USA) 사에서구입하였다. phospho (p)-akt, Akt, p-erk, ERK, cleaved-caspase-9, cleaved-caspase-3에대한항체는 Cell signaling Technology (Beverly, MA, USA) 사의제품을사용하였다. Rhodamine-conjugated phalloidin은 Molecular Probes (Eugene, OR, USA) 사에서구입하였다. Hydroxyapatitecoated plate는오스코텍사 (Korea) 의제품을사용하였다. 2. 파골세포배양골수세포는 5주령 ICR 생쥐의대퇴골과경골에서이전에보고된논문과동일한방법으로분리하였다 16). 조골세포는생후 1 일령생쥐의두개골을 0.1% collagenase로용해하여이전에보도된논문과동일한방법으로분리하였다 16). 성숙파골세포를얻기위해, 골수세포와조골세포는 10% FBS와항생제 (penicillin/streptomycin) 를첨가한 α-minimum essential medium (MEM) 배양액에부유하고 VitD3 (10-8 M) 와 PGE2 (10-6 M) 를처리한후 collagen으로코팅한 90-mm 배양접시에넣어배양기에서 6일간동조배양하였다. 3. 세포생존분석동조배양으로얻은성숙파골세포는 collagenase를처리하여 10% FBS와항생제가포함된 α-mem에부유하고 48-well plate에첨가하여 4시간배양하였다. 4시간배양후, trypsin을처리하여조골세포를제거하고순수파골세포를얻었다. 파골세포는 curcumin과 RANKL을처리하고 24 시간배양하였다. 배양후, 세포는 10% formalin으로고정하고 0.1% Triton X-100으로세포막을투과한후 TRAP 용액으로세포를염색하였다. 붉은색으로염색된 TRAP 양성다핵세포는광학현미경 (Olympus) 으로촬영하고집계하여생존세포수를구하였다. 4. 핵및액틴염색동조배양으로얻은성숙파골세포는 curcumin (5 μm) 과 RANKL (100 ng/ml) 을처리하고 12 시간배양하였다. 배양후, 세포는 10% formalin으로고정하고 0.1% Triton X-100으로세포막을투과한후핵과액틴염색을위해각각 DAPI 또는 rhodamine-conjugated phalloidin 용액으로 5분간세포를염색하고형광현미경 (Zeiss) 으로촬영하였다. 5. Western blot 분석성숙파골세포는 curcumin 및 RANKL로처리하고 lysis buffer (50 mm tris-cl, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, 1 mm sodium fluoride, 1 mm sodium vanadate, 1% deoxycholate, and protease inhibitors) 를이용하여용해하였다. 용해된세포는 14,000 rmp으로원심분리하여순수한단백질을얻었다. 단백질을정량화한후, 30 μg 단백질은 10% SDS-polyacrilamide gel에서전기영동한후, PVDF 막 (Amersham Biosciences) 으로단백질을옮겼다. 단백질을옮긴 PVDF 막은비특이단백질이붙는것을예방하기위해 5% non-fat dry milk로처리한후적절한 1차항체로처리하고 TBS-T 완충용액으로세척한후 2차항체를처리하였다. TBS-T 완충용액으로세척한 PVDF 막은 enhanced chemiluminescence 를이용하여단백질발현을관찰하였다. 6. Bone resorption 분석골수세포와조골세포의동조배양으로얻은성숙파골세포를 10% FBS와항생제가포함된 α-mem에부유하여 hydroxyapatite-coated 48-well plate에넣고 1시간배양하였다. 1 시간배양후, trypsin을처리하여조골세포를제거하고순수파골세포에 curcumin과 RANKL을처리하고 6시간배양하였다. 파골세포는 0.1% Triton X-100으로처리하여제거하고광학현미경으로촬영하였다. 골흡수영역은 Image Pro-plus program version, version. 4.0 (Media Cybernetics) 을이용하여정량화하였다. 7. 통계분석모든정량적인결과는평균 ± 표준편차로표시하였으며 Student's t-test로분석하여 p값이 0.05 이하인경우유의성이있는것으로간주하였다

김정중 김동주 이병기 김광진 이명수 이재훈 김헌수 이창훈 변승재 장성조 송정훈 오재민 이준석 김광미 전철홍 결과 1. 파골세포의생존에 curcumin의효과조혈모세포에서유래된파골세포는세포간의융합을통해다핵형파골세포로분화된다. 그러나다핵형파골세포는 RANKL과같은생존인자가없을때급격히세포사멸과정을겪는다. 따라서파골세포의생존은골흡수에중요한과정이다.

동조배양으로얻은파골세포에 RANKL을단독으로처리했을때와 curcumin을같이처리했을때 Akt와 ERK의인산화의변화는관찰되지않았다 (Fig 3A). 다음으로우리는세포사멸의생화학적특성중에하나인 caspase 활성에 curcumin의효과를검증하였다.

Effect of curcumin on osteoclast apoptosis. Mature osteoclasts from co-culture were plated on 48-well plates and cultured for 4 h.

3 김정중 김동주 이병기 김광진 이명수 이재훈 김헌수 이창훈 변승재 장성조 송정훈 오재민 이준석 김광미 전철홍 결과 1. 파골세포의생존에 curcumin의효과조혈모세포에서유래된파골세포는세포간의융합을통해다핵형파골세포로분화된다. 그러나다핵형파골세포는 RANKL과같은생존인자가없을때급격히세포사멸과정을겪는다. 따라서파골세포의생존은골흡수에중요한과정이다. 우리는파골세포의생존에 curcumin의효과를검증하였다. 순수파골세포는 curcumin과 RANKL을처리하여 24시간배양하였다. 대조군과비교하여 RANKL을처리한파골세포는생존하였다. 그러나 RANKL에 curcumin을같이처리한실험군에서는세포사멸로파골세포가급격히줄어들었다 (Fig. 1). 한 Akt와 ERK의활성에 curcumin의효과를검증하였다. 동조배양으로얻은파골세포에 RANKL을단독으로처리했을때와 curcumin을같이처리했을때 Akt와 ERK의인산화의변화는관찰되지않았다 (Fig 3A). 다음으로우리는세포사멸의생화학적특성중에하나인 caspase 활성에 curcumin의효과를검증하였다. 대조군에비하여 RANKL을처리한실험군에서는활성화된 caspase-9와 caspase-3의발현이감소되었지만 curcumin을동시에처리한실험군에서는활성화된 caspase-9과 caspase-3의발현이증가되는것을확인하였다 (Fig. 3A). 위의결과로 curcumin 이 caspase의활성을촉진하여파골세포의세포사멸을촉진한다할수있다. Fig. 2. Effect of curcumin on osteoclast apoptosis. Mature osteoclasts from co-culture were plated on 48-well plates and cultured for 4 h. Osteoblasts were removed by trypsin treatment, and purified osteoclasts were incubated for 12 h with RANKL (100 ng/ml) in the presence or absence of curcumin (5 μm). Cells were fixed with 3.7% formalin, permeabilized with 0.1% Triton X-100, stained for DAPI, and photographed under a fluorescence microscope. Fig. 1. Effect of curcumin on RANKL-mediated osteoclasts survival. (A) Mature osteoclasts from co-cultures of bone marrow cells and calvarial osteoblasts were seeded on 48-well plates and cultured for 4 h. Osteoblasts were removed by trypsin treatment, and purified osteoclasts were incubated for 24 h with RANKL (100 ng/ml) in the presence or absence of curcumin (5 μm). Cells were fixed with 3.7% formalin, permeabilized with 0.1% Triton X-100, and stained for TRAP. (B) TRAP-positive cells were counted as osteoclasts. Significant differences (*P<0.01)between the indicated groups are marked by an asterisk. 2. 세포사멸형태변화에 curcumin의효과세포사멸의특징으로핵이수축되고 DNA가파괴되는형태를나타낸다. 우리는파골세포의사멸에 curcumin의효과를 DAPI 염색으로 curcumin을처리한실험군핵의형태를확인하였다. 파골세포에 curcumin을전처리하고 RANKL을처리하여 RANKL을단독으로처리한군을 DAPI 염색으로핵의형태를관찰하였다. RANKL을처리한실험군은핵이많고형태가뚜렷했지만 RANKL에 curcumin을같이처리한실험군은핵의수가감소되었고모양도수축된것을관찰하였다 (Fig. 2). 이결과로 curcumin은전형적인세포사멸을유도한다고할수있다. 3. 세포생존신호전달물질의활성에 curcumin의효과 Lee 등은 Akt와 ERK 신호전달경로의활성이파골세포의생존에중요하다고보고하였다 17). 따라서우리는 RANKL에의 Fig. 3. Effect of curcumin on signal transduction induced by RANKL. Mature osteoclasts were obtained from co-cultures of bone marrow cells and osteoblasts. (A) Purified osteoclasts were pretreated with curcumin (5 μm) for 1 h and then stimulated with RANKL for 10 min. The cells were lysed in lysis buffer, and the cell lysates were subjected to western blotting with the indicated antibodies. (B) Purified osteoclasts were pretreated with curcumin (5 μm) for 1 h and then stimulated with RANKL for 6 min. Cell lysates were subjected to western blotting with the indicated antibodies. 4. Acin 형성에 curcumin 의효과 액틴의형성은파골세포의골흡수에필수적인과정이다 18)

먼저우리는파골세포의액틴고리형성에 curcumin의효과를검증하였다. 그림 4에서보는것과같이 RANKL을처리한실험군에서는파골세포의액틴고리형성이잘이루어진것을볼수있지만, curcumin을같이처리한실험군은파골세포의액틴고리형성이파괴되는것을볼수있었다 (Fig. 4). Fig. 4. Effect of curcumin on osteoclast actin ring formation.

4 먼저우리는파골세포의액틴고리형성에 curcumin의효과를검증하였다. 그림 4에서보는것과같이 RANKL을처리한실험군에서는파골세포의액틴고리형성이잘이루어진것을볼수있지만, curcumin을같이처리한실험군은파골세포의액틴고리형성이파괴되는것을볼수있었다 (Fig. 4). Fig. 4. Effect of curcumin on osteoclast actin ring formation. Mature osteoclasts from co-culture were plated on 48-well plates and cultured for 4 h. Osteoblasts were removed by trypsin treatment, and purified osteoclasts were incubated for 12 h with RANKL (100 ng/ml) in the presence or absence of curcumin (5 μm). Cells were fixed with 3.7% formalin, permeabilized with 0.1% Triton X-100, stained for rhodamine-conjugated phalloidin, and photographed under a fluorescence microscope. 5. 골흡수에 curcumin 의효과 골흡수의억제는골다공증과같은골질환의치료제로널리이용될수있으며 bisphosphonate 계열의골흡수억제제가골질환치료제로널리사용되고있다. 따라서동조배양으로얻은파골세포는 hydroxyapatite-coated plate에넣고 curcumin을처리하여골흡수에 curcumin의효과를검증하였다. RANKL에의한골흡수는증가되었지만 RANKL과 curcumin을같이처리했을때골흡수는현저히감소되었다 (Fig. 5A & B). Fig. 5. Effect of curcumin on bone resorption. Mature osteoclasts from co-culture were plated on hydroxyapatite- coated 48-well plates and further incubated for 1 h. After osteoblasts were removed, purified osteoclasts were incubated for 6 h with RANKL (500 ng/ml) in the presence or absence of curcumin (5 μm). (A) Cells were removed from the plates and photographed under a light microscope. (B) Pit area was quantified using Image Pro-plus program, version 4.0. Significant differences (*P<0.01)between the indicated groups are marked by an asterisk. 고찰 세포사멸은생물체의항성성유지에매우중요한과정으로세포사멸의불균형으로암, 자가면역질환, 골다공증과같은질환이유도된다 7,8). 성숙파골세포는생존기간이짧고 RANKL, TNF-α, IL-1과같은생존인자가없을때빠르게세포사멸과정을겪는다. 따라서파골세포의생존기간의증가는골흡수와밀접한관련이있어골대사에중요하다 19). 본연구에서우리는파골세포의세포사멸에강황에서추출한 curcumin의효과를검증하였다. Curcumin은 RANKL에의한파골세포의생존을억제하였다 (Fig. 1). 이결과로 curcumin의효과가세포사멸을유도하는것인지독성에의한세포괴사인지알수가없었다. 세포괴사는짧은시간안에세포막이파괴되지만, 세포사멸은생화학적, 형태학적특징으로알수있다. 생화학적특징으로는 DNA가파쇄되며 caspase가활성화되고형태학적특징은세포의수축및염식질의응축으로나타난다. 우리는 DAPI 염색으로 curcumin이세포사멸의형태학적특징인세포및핵의수축유도로파골세포의사멸을촉진한다는것을확인하였다 (Fig. 2). RANKL, TNF-α, IL-1과같은생존유도인자는 PI-3K/Akt 와 Ras/ERK 신호전달경로의활성을통하여파골세포의생존을유지한다 20). 최근 Akiyama 등은파골세포의사멸중에세포사멸유도인자인 Bim 단백질발현이증가되어파골세포의사멸에중요하다는것을 Bim 유전자가결여된세포에서확인하였으며, Bim의현은 Ras/ERK 경로의활성에의하여억제된다고보고하였다 21). 또한 Liu 등은 RANKL에의해유도되는 Akt의활성이파골세포의생존에중요하다고보고하였다 22). 이들의결과로파골세포의생존에 PI-3K/Akt와 Ras/ERK 신호전달경로의활성이파골세포의생존에중요하다고할수있다. 우리는 curcumin에의한파골세포사멸이생존유도경로인 PI-3K/Akt 와 Ras/ERK 신호전달경로의활성과관련있는지확인하였다. 비록 RANKL에의한 Akt와 ERK의활성은 curcumin에의해변화되지않았지만세포사멸에중요한 caspase의활성을촉진하였다 (Fig. 3A). 위의결과로 curcumin에의한 caspase의활성이 curcumin에의한세포사멸의원인으로사료된다. 파골세포는 vitronectin 수용체가골표면에 RGD (arginineglycine-arspartic acid) sequence를인지하여골에흡착된다. 이런파골세포는골흡수를하기위해서 "actin ring" 이라불리는액틴고리구조가형성되어골과단단히흡착하고, 흡착된파골세포는골흡수부분으로산 (H+) 과 lysosome 효소를분비하여골을흡수한다 3). 최근 calcitonin이파골세포의생존에는영향없이액틴구조를파괴하여골흡수를억제한다고보고되었다 23). 이결과는파골세포의액틴고리구조가골흡수에필수적인요소로간주된다. 본연구에서우리는 curcumin이 RANKL에의한액틴고리구조를파괴하고골흡수를억제한다는것을확인하였다 (Fig. 4 & 5). 전사인자 NF-kB는파골세포의분화및골흡수에중요한인자로 MMP-9, carbonic anhydrase II와같은단백질의발현을촉진한다 24,25). Curcumin은 NF-κB의억제제로잘알려져있다 26). 이들의결과로 curcumin에의한골흡수억제는

5 김정중 김동주 이병기 김광진 이명수 이재훈 김헌수 이창훈 변승재 장성조 송정훈 오재민 이준석 김광미 전철홍 NF-κB 신호전달경로의억제와관련있으리라사료된다. 결론으로, 우리는파골세포의생존에강황에서분리한 curcumin의효과를검증하였다. Curcumin은 caspase-9와 caspase-3의활성을유도하여파골세포사멸을촉진하였으며액틴고리구조를파괴하여파골세포의골흡수를억제하였다. 이들의결과로 curcumin은골다공증과같은골질환치료제개발에도움이될것이라사료된다. 감사의글 이논문은 2007년도원광대학교교비지원에의해수행되었습니다. 참고문헌 1. Rodan, G.A., Martin, T.J. Therapeutic approaches to bone diseases. Science 289: , Boyle, W.J., Simonet, W.S., Lacey, D.L. Osteoclast differentiation and activation. Nature 423: , Teitelbaum, S.L., Ross, F.P. Genetic regulation of osteoclast development and function. Nat Rev Genet 3: , Takayanagi, H. Osteoimmunology: shared mechanisms and crosstalk between the immune and bone systems. Nat Rev Immunol 7: , Thompson, C.B. Apoptosis in the pathogenesis and treatment of disease. Science 267: , Saudou, F., Finkbeiner, S., Devys, D., Greenberg, M.E. Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions. Cell 95: 55-66, Salvesen, G.S. and Dixit, V.M. Caspases: intracellular signaling by proteolysis. Cell 91: , Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S.M., Ahmad, M., Alnemri, E.S. and Wang, X. Cytochrome c and datp-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91: , Lindsten, T., Ross, A.J., King, A., Zong, W.X., Rathmell, J.C., Shiels, H.A. et al. The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. Mol Cell 6: , Jiang, X., Wang, X. Cytochrome c-mediated apoptosis. Annu Rev Biochem 73: , Strasser, A., O Connor, L. and Dixit, V.M. Apoptosis signaling. Annu Rev Biochem 69: , Wang, X. The expanding role of mitochondria in apoptosis. Genes Dev 15: , Wang, Z.B., Liu, Y.Q. and Cui, Y.F. Pathways to caspase activation. Cell Biol Int 29: , Goel, A., Kunnumakkara, A.B., Aggarwal, B.B. Curcumin as "Curecumin": From kitchen to clinic. Biochem Pharmacol 75: , Bharti, A.C., Takada, Y., Aggarwal, B.B. Curcumin (diferuloylmethane) inhibits receptor activator of NF-kappa B ligand-induced NF-kappa B activation in osteoclast precursors and suppresses osteoclastogenesis. J Immunol 172: , Kwak, H.B., Lee, S.W., Li, Y.J., Kim, Y.A., Han, S.Y., Jhon, G.J. et al. Inhibition of osteoclast differentiation and bone resorption by a novel lysophosphatidylcholine derivative. SCOH Biochem Pharmacol 67: , Lee, S.E., Chung, W.J., Kwak, H.B., Chung, C.H., Kwack, K.B., Lee, Z.H. and Kim, H.H. Tumor necrosis factor-alpha supports the survival of osteoclasts through the activation of Akt and ERK. J Biol Chem 276: , Vaananen, H.K., Zhao, H., Mulari, M. and Halleen, J.M. The cell biology of osteoclast function. J Cell Sci 113: , Tanaka, S., Miyazaki, T., Fukuda, A., Akiyama, T., Kadono, Y., Wakeyama, H., Kono, S., Hoshikawa, S., Nakamura, M., Ohshima, Y., Hikita, A., Nakamura, I. and Nakamura, K. Molecular mechanism of the life and death of the osteoclast. Ann N Y Acad Sci 1068: , Glantschnig, H., Fisher, J.E., Wesolowski, G., Rodan, G.A., Reszka, A.A. M-CSF, TNFalpha and RANK ligand promote osteoclast survival by signaling through mtor/s6 kinase. Cell death Differ 10: , Akiyama, T., Bouillet, P., Miyazaki, T., Kadono, Y., Chikuda, H., Chung, U.I., Fukuda, A., Hikita, A., Seto, H., Okada, T., Inaba, T., Sanjay, A., Baron, R., Kawaguchi, H., Oda, H., Nakamura, K., Strasser, A. and Tanaka, S. Regulation of osteoclast apoptosis by ubiquitylation of proapoptotic BH3-only Bcl-2 family member Bim. EMBO J 22: , Liu, W., Wang, S., Wei, S., Sun, L., Feng, X. Receptor activator of NF-kappaB (RANK) cytoplasmic motif, 369PFQEP373, plays a predominant role in osteoclast survival in part by activating Akt/PKB and its downstream effector AFX/FOXO4. J Biol Chem 280: , Karsdal, M.A., Henriksen, K., Arnold, M., Christiansen, C. Calcitonin a drug of the past or for the future? Physiologic inhibition of bone resorption while sustaining osteoclast numbers improves bone quality. BioDrugs 22(3): , Bond, M., Fabunmi, R.P., Baker, A.H. and Newby, A.C. Synergistic upregulation of metalloproteinase-9 by growth

6 factors and inflammatory cytokines: an absolute requirement for transcription factor NF-kappaB. FEBS Lett 435: 29-34, Kamolmatyakul, S., Chen, W., Yang, S., Abe, Y., Moroi, R., Ashique, A.M. and Li YP: IL-1alpha stimulates cathepsin K expression in osteoclasts via the tyrosine kinase-nf-kappab pathway. J Dent Res 83: , Singh, S., Aggarwal, B.B. Activation of transcription factor NF-kappaB is suppressed by curcumin (diferuloylmethane). J Biol Chem 270: 24995,

23(6)12최민규 hwp

23(6)12최민규 hwp 동의생리병리학회지제 23 권 6 호 Korean J. Oriental Physiology & Pathology 23(6):1299 1304, 2009 파골세포의골흡수에미치는녹용의억제효과 김윤경 최윤홍 1 송정훈 2 장성조 3 김현정 4 이창훈 4 안선호 4 이지은 4 김정중 1 최민규 1 * 원광대학교약학대학한약학과, 1 : 의과대학해부학교실, 2 : 성형외과학교실,