The Functional Analysis of MLKs-JNK Pathways in Osteoclast Formation SY Ji, et al ORIGINAL ARTICLE Vol. 19, No. 1, 2012 파골세포형성에있어서 MLKs-JNK 경로의역할규명 숙명여자대학교약학대학 지선영 정정 노아롱새미 문미란 임미정 * = Abstract = The Functional Analysis of MLKs-JNK Pathways in Osteoclast Formation Sun Young Ji, Zheng Ting, A Long Sae mi Noh, Miran Moon, Mijung Yim * College of Pharmacy, Sookmyung Women's University, Seoul, Korea Objectives: Osteoclasts (OCs) are bone-resorbing multinucleated cells derived from hematopoietic progenitors of the monocyte-macrophage lineage. OC precursors, such as bone marrow-derived macrophages (BMMs), are formed in the presence of macrophage colony-stimulating factor (M-CSF) and differentiate into OCs in response to M-CSF and receptor activator of nuclear factor κb ligand (RANKL). In this study, we investigated the role of mixed lineage kinases (MLKs)-c-Jun amino-terminal kinase (JNK) pathways in OC formation. Methods: We performed an OC formation assay and reverse transcription polymerase chain reaction (RT-PCR) analysis. Results: We first explored the role of JNK on osteoclst formation using mouse bone marrow (BM) culture system. We found that OC formation was impaired when the JNK inhibitor was added either in early or late stage, suggesting the requirement for JNK activation during OC formation. MLKs are serine/threonine kinases that regulate signaling by the JNK. Since the JNK activity is specifically required for osteoclastogenesis, we examined the messenger ribonucleic acid (mrna) levels of MLKs in BMs, BMMs and OCs by RT-PCR. Among MLKs, the level of MLK3 mrna expression is highest in BMs, BMMs and OCs. Moreover, we found that the mrna expression of MLK2 and MLK3 is increased with the differentiation of BMs to BMMs, and is sustained in OCs. Finally we investigated the role of MLK3 in OC differentiation using gene knock-down techniques. The silencing of MLK3 in BMMs partly attenuated RANKL-induced OC differentiation. Conclusions: These data suggest that JNK and MLK3 may positively regulate OC formation. [Korean Journal of Bone Metabolism, 19(1): 21-28, 2012] Key Words: JNK, MLKs, Osteoclast Received: March 26, 2012, Revised: April 21, 2012, Accepted: April 23, 2012 * Address for Correspondence: Mijung Yim, College of Pharmacy, Sookmyung Women's University, Cheongpa-ro 47-gil 100, Yongsan-gu, Seoul, 140-742, Korea. Tel: +82-2-710-9572, Fax: +82-2-710-9871, e-mail: myim@sookmyung.ac.kr 지선영과정정은본논문에대해동일하게기여하였습니다. CC This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/license/by-nc/3.0/). 21
서론현재우리나라는빠른속도로초고령화사회에진입하고있으며, 노인성질환도크게증가하고있다. 그중골다공증은골량이감소하고미세구조의이상으로인해결과적으로뼈의강도가약해져서부러지기쉬운상태가되는질환이다. 골다공증은노인의삶의질을급격히저하시키는문제점을지님에도불구하고발병율이매우높아그에따른대책이시급한실정이다. 뼈의항상성은새로운골기질의형성과오래된골의기질을용해시켜골흡수분해를담당하는파골세포 (osteoclast) 의적절한균형에의해유지된다. 1,2 조골및파골세포는밀접한관계를맺고있어, 파골세포의분화는조골세포에의해엄격하게조절된다. 1,2 즉, 조골세포는파골세포분화인자인 macrophage colony-stimulating factor (M-CSF, or CSF-1) 및 receptor activator of nuclear factor κb ligand (RANKL, osteoprotegerin ligand [OPGL], osteoclast differentiation factor [ODF], tumor necrosis factor-related activation induced cytokine [TRANCE]) 를통해파골세포의분화를조절함으로써체내골형성과골흡수의동적인평형을유지한다. 2-5 여러요인으로인해이균형이깨지면골의생성보다흡수가증가하여결국골손실 (bone loss) 이일어나게된다. 파골세포분화에관여하는다양한신호전달분자가알려져있다. 그중 c-jun amino-terminal kinase (JNK) 는파골세포의형성과유지에반드시필요한것으로보고되었다. 6-8 세포에대한다양한자극은 14종의 mitogen-activated protein kinase kinase kinase (MAP3K) 를활성화시키고, 이는 mitogenactivated protein kinase kinase (MKK) 4 또는 MKK7를인산화시켜최종적으로 JNK를활성시킨다. 파골전구세포에서 JNK의활성화를억제하면 RANKL 자극에의한파골세포의분화가저해되었으며, 이는파골세포분화의중요전사인자인 activator protein (AP)1과 nuclear factor of activated T-cells, cytoplasmic (NFATc)1을매개한것임이밝혀졌다. 6,7 또한 JNK는파골세포에서 AP1의구성성분인 c-jun의활성화를통해세포사를억제하여수명연장에관여하는것으로나타났다. 8,9 JNK를조절하는상위신호와파골세포와의연관성도일부보고되었다. 파골세포에 MAP3K의일종인 mitogen-activated protein kinase kinase kinase (MEKK) 1을과잉발현시키면 JNK의활성화가관찰되는동시에세포사가억제되었다. 9 또한파골전구세포에서 MKK7의억제는 JNK 의활성을감소시켰으며이에따라파골세포형성도억제되 었다. 10 이상의보고로미루어 JNK는파골세포의형성과유지에매우중요한역할을담당하고있는것으로생각되나, JNK를조절하는상위신호인 MAP3K와파골세포형성과의연관성에대해서는아직많은부분이알려져있지않다. 따라서본연구자는 JNK를활성화시키는 14종의 MAP3K 신호중 mixed lineage kinases (MLKs) 에주목하여파골세포형성에서 MLK의역할을규명하고자하였다. MLKs (MLK1, MLK2 및 MLK3) 는 serine/threonine kinase 로 MKK7을통해 JNK를활성화시킨다. MLKs는초기에신경세포의세포사를조절하는것이알려졌으며, 따라서신경질환에대한잠재적인약물타겟으로여겨지고있다. 11 실제로반합성 MLK 저해제 (CEP1347) 가파킨슨질환치료제로임상시험이진행중이다. 한편최근의연구는 MLKs가면역세포에서도중요한역할을담당할가능성이있음을시사하고있다. 12 Lipopolysaccharide (LPS) 를처리한단구세포에서 MLK의억제는 tumor necrosis factor-alpha (TNF-alpha) 의분비를감소시켰다. 13 또한 MLK 저해제는 LPS와 poly (I:C) 자극에의한수지상세포의분화와항원제시기능을저해하였으며, TNF-alpha, interleukin (IL)-10, IL-6와같은사이토카인의분비를감소시켰다. 14 본연구자는파골세포형성에있어서 MLKs의역할을마우스 bone marrow-derived macrophages (BMMs) 세포를사용하여검토하였기에보고하는바이다. 연구대상및방법 1. 마우스골수세포의배양 Imprinting control region (ICR) 마우스 (6-9주, 수컷 ) 를경추탈골한후 70% 에탄올로소독하였다. 경골부분의피부를절개하여부착근육을떼어냈다. 경골원심부를절단하고슬개골을탈골시켜경골을적출하였다. 뼈양끝을조금잘라한쪽끝에 25G의주사바늘을꽂고 alpha minimum essential medium (α-mem) 을흘려보내골수세포를시험관에모았다. 원심분리한후 α-mem에현탁하고 2배의 Gey's solution을가해적혈구를제거했다. 원심분리한후 10% fetal bovine serum (FBS) 가함유된 α-mem으로재현탁했다. 2. 파골세포의분화유도초기배양한골수세포를 M-CSF 10 ng/ml로하룻밤배양한후부유세포를 M-CSF 30 ng/ml로 3일간추가배양하여 BMM 세포를얻었다. BMM 세포를회수후 1 10 4 22
The Functional Analysis of MLKs-JNK Pathways in Osteoclast Formation SY Ji, et al Table 1. Primer sequences for RT-PCR Primer name MLK1 MLK1 anti MLK2 MLK2 anti MLK3 MLK3 anti CD11b CD11b anti F4/80 F4/80 anti CTR CTR anti CathepsinK CathepsinK anti β-actin β-actin anti Sequence 5'-CCTGGTAGATGGATACAA-3' 5'-CGGGTAGAGTTGCATTCA-3' 5'-GAGCCTTCTCCTTGTGTT-3' 5'-GCCCTCCATGTCCATGT-3' 5'-CGGTCCCGCATGGATGAA-3' 5'-CCTGGGCACCCATGTCTT-3' 5'-CTTAAAGCTCTTCTGGTCACAGCC-3' 5'-GTATTCTCCTTGCAGTTTTGGTGC-3' 5'-GAATCTTGGCCAAGAAGAGAC-3' 5'-GAATTCTCCTTGTATATCATCAGC-3' 5'-TTTCAAGAACCTTAGCTGCCAGAG-3' 5'-CAAGGCACGGACAATGTTGAGAAG-3' 5'-CTTCCAATACGTGCAGCAGA-3' 5'-ACGCACCAATATCTTGCACC-3' 5'-TGTGATGGTGGGAATGGGTCAG-3' 5'-TTTGATGTCACGCACGATTTCC-3' MLK, mixed lineage kinase; CTR, calcitonin receptor; RT-PCR, reverse transcription polymerase chain reaction cells/well에 RANKL 50 ng/ml과 M-CSF 30 ng/ml 존재하에서 4-6일간배양했다. 배양이끝난세포는 10% formalin 으로 10분간고정한후 ethanol-aceton (1:1) 로 1분간재고정하여 tartrate-resistant acid phosphatase (TRAP) 염색하였다. 현미경하에서관찰하여 3개이상의핵을가진 TRAP+ 세포를다핵파골세포로판정하였다. 3. Reverse transcription polymerase chain reaction (RT-PCR) 분석 Total ribonucleic acid (RNA) 추출은 easy-blue (intron Biotechnology, Sungnam, Korea) 를이용하였다. Complementary deoxyribonucleic acid (cdna) 는 1 mg의 total RNA 를 oligo(dt) primer, 10 mm deoxyribonucleotide triphosphate (dntp), 1 unit RNase inhibitor 그리고 4 unit Script reverse transcriptase (Fermentas, York, UK) 로 42 에서 60분처리하여합성한후, 70 에서 10분가열함으로써반응을중지시켰다. PCR은 Table 1에명시된프라이머를사용하여 94 3분간처리한후, 94 30초, 58 45초, 72 1분의과정을 32회또는 28회 (β-actin) 반복하였다. PCR 후, 증폭된 cdna는 1% agarose gel에서분리하였고 ethidium bromide 로염색하여 ultraviolet (UV) 상에서관찰하였다. 4. Small interfering RNA (sirna) 를이용한 MLK3 유전자발현저하마우스 MLK3 sirnas (Cat. no. SI01299893) 는 Qiagen GmbH (Hilden, Germany) 에서구입하였다. 대조군 sirna 로는 Allstars Negative Control sirna (Cat. no. 1027280) (Qiagen GmbH, Hilden, Germany) 를사용하였다. BMM 세포에 sirna (20 nm) 를 Lipofectamine TM 2000 CD (Cat. no. 12566-014; Invitrogen, Carlsbad, USA) 로도입시켰으며, 하루동안배양한후 RT-PCR 분석과파골세포형성실험에사용하였다. 5. 통계분석실험결과는평균 ± 표준편차로표기하였고, Student's t-test 로분석하여 P 값이 0.05 미만일때통계적으로유의하다고판단하였다. 결과 1. JNK 저해제에의한파골세포형성억제성숙파골세포는골수세포로부터유래하며파골전구세포를거쳐형성된다 (Fig. 1A). 즉, 마우스골수세포를 M-CSF 로처리하여파골전구세포인 BMMs가형성되는첫번째단계와 (1), 파골전구세포를 M-CSF와 RANKL로처리하여성숙파골세포로분화시키는두번째단계 (2) 를거쳐최종적으로파골세포가형성된다. 기존의많은연구를통해 JNK 23
A B 였다 (2+SP). 또한골수세포에 JNK 저해제를처리하여파골전구세포를형성한경우에도 RANKL에의한성숙파골세포형성이크게감소하였다 (1+SP). 이는 JNK 저해제가파골전구세포로의분화를억제한때문으로생각되며, 이상의결과로부터 JNK가성숙파골세포분화뿐아니라파골전구세포의분화에도중요한역할을담당하고있음을짐작할수있다. 2. Bone marrow (BM), BMM, osteoclast (OC) 에서 MLKs 의발현 Fig. 1. Effects of JNK inhibitor on osteoclatstogenesis. (A) Schematic design for treatment of JNK inhibitor. (B) Bone marrow cells were cultured with 10 ng/ml M-CSF overnight. Non-adherent cells were harvested and cultured with 30 ng/ml M-CSF in the absence or presence of SP for 3 days. Floating cells were removed and adherent cells were used as BMMs (1). BMMs were cultured with 30 ng/ml M-CSF and 200 ng/ml RANKL in the absence or presence of SP for 4 days (2). Cultured cells were fixed and stained for TRAP. TRAP+ multinucleated cells containing more than three nuclei were counted as osteoclasts. Data represent means ± SD of triplicate samples in a representative experiment. *P < 0.05 versus vehicle. (JNK, c-jun amino-terminal kinase; M-CSF, macrophage colony-stimulating factor; SP, SP600125 [a JNK inhibitor] BMM, bone marrow-derived macrophage; RANKL, receptor activator of nuclear factor κb ligand; TRAP, tartrateresistant acid phosphatase; MNC, multinucleated cells) 를매개하는신호전달경로가두번째단계인 RANKL 처리에의한성숙파골세포로의분화에필수적이라는것이밝혀졌다. 6-8 그러나파골세포분화의첫번째단계인파골전구세포의형성에있어서 JNK의역할은보고된바없다. 따라서이를알아보기위해 JNK 저해제인 SP600125 (SP) 를파골세포분화단계별로처리하여파골세포형성여부를조사하였다 (Fig. 1B). 구체적으로는마우스골수세포에 JNK 저해제를처리하여파골전구세포 (BMM) 를형성한후 RANKL 처리한군과 (1 +SP), 마우스골수세포를파골전구세포 (BMM) 로분화시킨후 RANKL과 JNK 저해제를처리한군 (2 +SP) 으로나누어성숙파골세포형성정도를비교하였다. 기존에보고된바와같이 JNK 저해제는파골전구세포 (BMM) 로부터성숙파골세포로의분화를완벽하게억제하 세포내에서 JNK를활성화시키는다양한상위신호전달경로가존재하며, 그중 MLKs는 MKK7을통해 JNK를활성화시키는것으로알려져있다. 11 파골세포분화에있어서 MLKs의역할을분석하기위해골수, 파골전구및성숙파골세포에서의 MLKs의발현을 RT-PCR 방법으로분석하였다 (Fig. 2). 먼저 MLK1, MLK2와 MLK3 mrna 발현을각각골수, 파골전구및성숙파골세포를사용하여조사하였다. 세종류의 MLK 중 MLK3가모든세포에서가장높이발현되고있음을알수있다. 반면 MLK1과 MLK2는모든세포에서상대적으로낮은발현량을보였다. 다음은파골세포분화단계에따른 MLKs 발현변화를 RT-PCR 방법으로분석하였다 (Fig. 3). 먼저골수세포와파골전구세포에서의 MLKs 발현비교에앞서, 파골전구세포의형성을마커유전자인 CD11b 와 F4/80의발현으로확인하였다. 골수세포에서와달리파골전구세포에서 CD11b 와 F4/80이높게발현되어, 파골전구세포가올바르게형성되었음을알수있다. 골수세포가파골전구세포로분화함에따라 MLK1의발현은크게변화하지않으나, MLK2와 MLK3 의발현은증가하였다. 골수세포에비해파골전구세포에서 MLK2은약 2배, MLK3는약 4배발현이증가하였다. 이는 MLK2와 MLK3가파골전구세포로의분화에관여할가능성을시사하는결과이다. 다음은파골전구세포와성숙파골세포에서 MLKs 발현을비교하였다 (Fig. 4). 성숙파골세포의형성은마커유전자인 calcitonin receptor (CTR) 와 Cathepsin K의발현증가로확인하였다. 성숙파골세포에서 MLK1, MLK2와 MLK3의발현은크게변화하지않는것으로나타났다. 즉, MLK1은파골전구세포에서와동일하게성숙파골세포에서발현이유도되지않았으며, 파골전구세포에서발현이증가한 MLK2와 MLK3는성숙파골세포에서도그발현이계속유지되었다. 이상의결과를통해 MLKs 중 MLK2 와 MLK3가파골전구세포및성숙파골세포의분화에관여 24
The Functional Analysis of MLKs-JNK Pathways in Osteoclast Formation SY Ji, et al Fig. 2. The mrna expression levels of MLKs in BMs, BMMs and OCs. Whole cell lysates were harvested from cultured cells. Total RNA were then isolated from the cells and cdna templates prepared. The mrna expression level was determined by RT-PCR using specific primers designed for MLK1, MLK2 and MLK3. Data represent means ± SD of three independent experiments. (mrna, messenger ribonucleic acid; MLK, mixed lineage kinase; BM, bone marrow; BMM, bone marrow-derived macrophage; OC, Osteoclast; cdna, complementary deoxyribonucleic acid; RT-PCR, reverse transcription polymerase chain reaction) CD11b / β-actin F4/80 / β-actin N.S. MLK1/ β-actin MLK2 / β-actin MLK3 / β-actin Fig. 3. Expression pattern of MLKs in BM and BMM. (A, B) Whole cell lysates were harvested from cultured cells and analyzed by RT-PCR. Data represent means ± SD of three independent experiments. *P < 0.05 versus BM. (N.S., non-significant; MLK, mixed lineage kinase; BM, bone marrow; BMM, bone marrow-derived macrophage; RT-PCR, reverse transcription polymerase chain reaction) 25
MLK1/ β-actin MLK2 / β-actin A B β-actin MLK3 / β-actin Cathepsin K / β-actin CTR / β-actin N.S. N.S. MLK3 / β-actin Fig. 4. Expression pattern of MLKs in BMM and OC. (A, B) Whole cell lysates were harvested from cultured cells and analyzed by RT-PCR. Data represent means ± SD of three independent experiments. *P < 0.05 versus BMM. (N.S., non-significant; CRT, calcitonin receptor, MLK, mixed lineage kinase; BMM, bone marrow-derived macrophage; OC, Osteoclast; RT-PCR, reverse transcription polymerase chain reaction) Fig. 5. Effects of MLK3 knock-down on osteoclatstogenesis. (A, B) BMMs were transfected with non-targeting sirna or sirna specific for MLK3. (A) Transfected BMMs were harvested from cultured cells and analyzed by RT-PCR. (B) Transfected BMMs were cultured for 4 days with 30 ng/ml M-CSF and 200 ng/ml RANKL. Cultured cells were fixed and stained for TRAP. TRAP-positive (+) multinucleated cells (MNCs) having more than 3 (n > 3) or 10 (n > 10) nuclei were counted. Data represent means ± SD of triplicate samples in a representative experiment. *P < 0.05 versus sint. scale bar = 200 μm. (sint, small interfering non-targeting; MLK, mixed lineage kinase; TRAP, tartrate-resistant acid phosphatase; MNC, multinucleated cells; sirna, small interfering ribonucleic acid; BMM, bone marrowderived macrophage; RT-PCR, reverse transcription polymerase chain reaction; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of nuclear factor κb ligand; TRAP, tartrate-resistant acid phosphatase) 26
The Functional Analysis of MLKs-JNK Pathways in Osteoclast Formation SY Ji, et al 할가능성이있다고판단하였다. 3. MLK3 유전자 Knock-down 에의한파골세포형성억제다음은 MLKs가파골세포형성에관여하는지의여부를조사하였다. MLKs 중 MLK3가골수세포에서파골전구세포로분화함에따라발현량이증가하고, 파골전구및성숙파골세포에서가장발현량이높았으므로 MLK3를대상으로실험을진행하였다. 파골세포형성에있어서 MLK3의기능을직접적으로규명하기위해 sirna를사용해 MLK3를 knock-down 시킨후파골세포형성의변화를관찰하였다 (Fig. 5). 먼저파골전구세포에 sirna를도입한후 MLK3의발현정도를 RT-PCR 로조사하였다. sirna 도입후 MLK3 mrna 발현이대조군에비해약 60% 감소한것을확인하였다 (Fig. 5A). 다음은 MLK3 sirna를도입한파골전구세포를 RANKL 처리하여성숙파골세포로분화시켰다 (Fig. 5B). MLK3 발현이감소한경우대조군에비해성숙파골세포형성이유의하게억제되었으며, 특히 10개이상의핵을가지는다핵파골세포로의분화가저해되는것으로나타났다. 이상의결과로 JNK의상위신호전달분자인 MLK3가성숙파골세포형성에일정부분관여하고있을가능성이제시되었다. 본연구자는 JNK 저해제를사용하여 JNK가성숙파골세포의형성뿐아니라파골전구세포의형성에도중요함을밝혔다. 따라서 JNK는파골전구세포및성숙파골세포의형성에중요하다고할수있다. 세포내 JNK의활성화를조절하는상위신호전달분자로 MLKs가알려져있다. 본연구자는 RT-PCR 분석법을통해골수세포에서파골전구세포로분화시 MLKs 중 MLK3 발현이가장많이증가하며, 성숙파골세포에서도그발현이지속되는것을확인했다. 또한 simlk3를사용한 knock-down 실험을통해 MLK3가파골세포형성에관여할가능성을제시하였다. 최근 MLK3 유전자를결손시킨 MLK3 녹아웃마우스는건강하고표면적으로특이사항이없는것으로보고된바있다. 15 이는 in vitro 실험을통해 MLK3의중요성을제시한다수의실험결과와는일치하지않는것이다. 이러한모순은 in vivo 상에서 MLK3의기능을타 MLKs가부분적으로보완하여발생한것으로생각되며, 추가연구를통해규명이필요하다. 본연구를통해파골세포형성을조절하는새로운기전이제시되었으며, 향후이를이용한골대사장애치료제등의개발도가능할것으로생각된다. 결론 고찰골다공증은고관절등의부위에골절을유발하여노인의삶의질을급격히저하시키는문제점을지님에도불구하고발병율이매우높은질환중의하나이다. 따라서골다공증치료제에대한수요도증가하여골다공증에대한 7개주요제약시장에서의의약품시장은 2016년 111억달러규모를형성할것으로전망한다. 지금까지개발된골다공증치료제중특히파골세포에작용하는골흡수억제제는비용이적게들며골절의위험을감소시키는데효과적인것으로나타나, 가장많이처방되고있다. 따라서새로운골다공증치료제의개발에있어서파골세포의분화기전을이해하는것은매우중요하다고할수있다. 파골세포의형성에있어서 JNK를매개한신호전달경로는필수적으로, JNK1 -/- 마우스유래의골수세포는파골세포로의분화가급격히감소하는것으로보고된바있다. 6 이는 JNK가파골세포분화인자인 RANKL의신호를매개하여성숙파골세포의형성을조절하기때문으로알려져왔다. JNK를매개한신호전달경로는파골전구세포및성숙파골세포의형성에중요하다. 또한 JNK의상위조절분자중하나인 MLK3는성숙파골세포형성에일부관여한다. 참고문헌 1. Takahashi N, Akatsu T, Udagawa N, et al. Osteoblastic cells are involved in osteoclast formation. Endocrinology 1988; 123:2600-2. 2. Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 1999;20:345-57. 3. Wong BR, Rho J, Arron J, et al. TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-jun N-terminal kinase in T cells. J Biol Chem 1997;272:25190-4. 4. Yasuda H, Shima N, Nakagawa N, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis- 27
inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A 1998;95:3597-602. 5. Lacey DL, Timms E, Tan HL, et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 1998;93:165-76. 6. David JP, Sabapathy K, Hoffmann O, Idarraga MH, Wagner EF. JNK1 modulates osteoclastogenesis through both c-jun phosphorylation-dependent and -independent mechanisms. J Cell Sci 2002;115:4317-25. 7. Chang EJ, Ha J, Huang H, et al. The JNK-dependent CaMK pathway restrains the reversion of committed cells during osteoclast differentiation. J Cell Sci 2008;121:2555-64. 8. Otero JE, Dai S, Foglia D, et al. Defective osteoclastogenesis by IKKbeta-null precursors is a result of receptor activator of NF-kappaB ligand (RANKL)-induced JNK-dependent apoptosis and impaired differentiation. J Biol Chem 2008; 283:24546-53. 9. Ikeda F, Matsubara T, Tsurukai T, Hata K, Nishimura R, Yoneda T. JNK/c-Jun signaling mediates an anti-apoptotic effect of RANKL in osteoclasts. J Bone Miner Res 2008;23: 907-14. 10. Yamamoto A, Miyazaki T, Kadono Y, et al. Possible involvement of IkappaB kinase 2 and MKK7 in osteoclastogenesis induced by receptor activator of nuclear factor kappab ligand. J Bone Miner Res 2002;17:612-21. 11. Gallo KA, Johnson GL. Mixed-lineage kinase control of JNK and p38 MAPK pathways. Nat Rev Mol Cell Biol 2002;3: 663-72. 12. Handley ME, Rasaiyaah J, Chain BM, Katz DR. Mixed lineage kinases (MLKs): a role in dendritic cells, inflammation and immunity? Int J Exp Pathol 2007;88:111-26. 13. Ciallella JR, Saporito M, Lund S, et al. CEP-11004, an inhibitor of the SAPK/JNK pathway, reduces TNF-alpha release from lipopolysaccharide-treated cells and mice. Eur J Pharmacol 2005;515:179-87. 14. Handley ME, Rasaiyaah J, Barnett J, et al. Expression and function of mixed lineage kinases in dendritic cells. Int Immunol 2007;19:923-33. 15. Brancho D, Ventura JJ, Jaeschke A, Doran B, Flavell RA, Davis RJ. Role of MLK3 in the regulation of mitogenactivated protein kinase signaling cascades. Mol Cell Biol 2005;25:3670-81. = 국문초록 = 연구목적 : 파골전구세포는 M-CSF 존재하에서조혈모세포로부터형성되며, M-CSF와 RANKL 존재하에서성숙파골세포로분화된다. 본실험에서는성숙파골세포형성에있어서 MLKs-JNK 경로의역할을알아보고자하였다. 연구방법 : 파골세포형성측정법과 RT-PCR 분석법을사용하였다. 결과 : 먼저파골세포형성에있어서 JNK의역할을조사한결과, JNK 저해제는파골전구세포와성숙파골세포의형성을모두억제하는것으로나타났다. JNK의활성은 MLK에의해조절된다는보고가있으므로 MLKs mrna의발현을 RT-PCR 방법으로조사하였다. MLKs 중, MLK3의발현이골수, 파골전구, 성숙파골세포에서가장높은것으로나타났다. 또한 MLK2와 MLK3의발현은골수에서파골전구세포로분화시증가하였으며, 성숙파골세포에서도발현이높게유지되었다. sirna를사용한 MLK3 유전자의 knock-down은파골세포분화를일부억제하였다. 결론 : JNK는파골전구세포와성숙파골세포로의분화에필수적이며, MLK3는파골세포분화에일부관여한다. 중심단어 : JNK, MLKs, 파골세포 28