대한내분비학회지 : 제 22 권제 6 호 2007 원저 인간부갑상선호르몬의골형성촉진작용과 Wnt/β-catenin 신호전달체계와의관련성규명 관동대학교의과대학내과학교실 1, 연세대학교의과대학내분비연구소 2, 연세대학교의과대학내과학교실 3 김세화 1 안연희 2 이유미 2,3 임승길 2,3 Identification and Validation of the Relationship of the Anabolic Effect of Parathyroid Hormone with the Wnt/β-catenin Canonical Pathway Se Hwa Kim 1, Juan Ji An 2, Yumie Rhee 2,3, Sung-Kil Lim 2,3 Department of Internal Medicine, Kwandong University College of Medicine 1, Institute of Endocrine Research ², Department of Internal Medicine 3, Yonsei University College of Medicine ABSTRACT Background: It has been well established that daily injections of low dose parathyroid hormone (PTH) increase bone mass in animals and humans. However, the precise mechanisms by which PTH exerts its anabolic action on bone are incompletely understood. The canonical Wnt-β-catenin signaling pathway has recently been demonstrated to have an important role in bone cell function. In the present study, we have examined the interaction between the PTH and Wnt signaling pathways in mouse osteoblastic MC3T3-E1 cells. Methods & Results: MC3T3-E1 cells were treated with 0.01 0.84 μm recombinant PTH. β-catenin expression was significantly increased after 30 minutes of exposure to PTH and reached a maximum 2.7 fold increase at 1 hr and expression then faded at 6 hrs. In addition, treatment with PTH increased nuclear accumulation of activated β-catenin; the ratio between the nuclear to cytoplasmic protein was more than three fold at 30 minutes and beyond. Moreover, PTH stimulated T-cell factor/lymphoid enhancer factor (TCF/LEF) reporter gene activity in MC3T3-E1 cells. Confocal microscopy revealed nuclear translocation of β-catenin by PTH as compared with a glycogen synthase kinase-3β (GSK-3β) inhibitor. Conclusion: These results suggest that the anabolic mechanism of PTH might be partially associated with the Wnt-canonical pathway. The appropriate target of another anabolic agent should be determined through further studies of this pathway. (J Kor Endocr Soc 22:411~418, 2007) ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ Key Words: β-catenin, Bone, Parathyroid hormone, TCF/LEF, Wnt-canonical pathway 1) 서 골다공증에대한치료는현재까지주로골흡수억제제가쓰이고있다. 그러나, 골흡수억제제는근본적인골형성을 접수일자 : 2007년 7월 3일통과일자 : 2007년 9월 22일책임저자 : 이유미, 연세대학교의과대학내과학교실 * 본논문은 2004년도한국학술진흥재단의지원에의하여연구되었음 (KRF-2004-003-E00152) 론 촉진시키지는않으므로그한계가뚜렷하다. 부갑상선호르몬은저용량으로매일투여시동물과사람에서모두골량을증가시키는것이입증되었고 [1], 최근미국식약청에서골다공증치료제로승인된유일한골형성촉진제이다. 부갑상선호르몬이골형성을증가시키는기전으로는조골세포활성증가, 조골전구세포들의분화촉진, lining cell의 recruitment, 그리고조골세포사멸의억제등이제시되고있으나아직까지도정확한기전은정립되지않았다 [2~4]. 한편, low-density lipoprotein receptor-related protein 5 (LRP5) 는조골세포에서 Wnt-β-catenin 시스템을통해서조 - 411 -
대한내분비학회지 : 제 22 권제 6 호 2007 Fig. 1. Effect of PTH on levels of β-catenin in mouse osteoblastic cells. β-catenin expression was significantly increased after 30 minutes of exposure to PTH (10-8 M) and maximized to 2.7 folds at 1 hr then faded at 6 hrs. 골세포의기능을증진시키는것으로잘알려져있다 [5,6]. 이 Wnt-β-catenin 신호전달체계에의해서촉진되는대표적인전사인자들이 cyclin D 등의세포증식과관련된인자들과세포고사억제를담당하는인자들이대부분이다. 따라서, 이번연구에서는부갑상선호르몬의골형성촉진의기전으로서 Wnt-β-catenin 시스템과의연관성을규명하여, 부갑상선호르몬의작용에대한하나의기전을제시하고자하였다. 대상및방법 1. 세포배양마우스조골세포주인 MC3T3-E1 세포를 α-minimal essential medium (α-mem), 10% fetal bovine serum (FBS), 및 1% penicillin/streptomycin을혼합한배지에서배양하였다. 배지는 3일에한번씩교환하였으며, 세포가 50~80% confluent 할때실험을진행하였다. 2. Western Blotting MC3T3-E1 세포에서부갑상선호르몬 [PTH(1-84)] 10-8 M을처리전, 후 15분, 30분, 1시간, 3시간, 및 6시간후에세포전체단백질을추출하였고, 세포질단백과핵내단백질을따로추출하였다. 추출된단백은 Bradford assay법으로농도를결정하였고, 98C에서 5분간가열하여 denature시켰다. 각각의검체를 SDS-PAGE (sodium dodecylsulfate -polyacrylamide gel electrophoresis) 로전기영동후 polyvinylidene difluoride (PVDF) membrane으로이동시키고항체와단백질간의비특이적인결합을차단하기위하 여 TBS-T (20 mm Tris-HCL, ph 7.4, 125 mm, 0.1% Tween 20) 용액에서 1시간반응시켰다. β-catenin (#610157, BD Biosciences, 92kDa), Active-β-catenin (#05-665, Upstate Biotechnology, 92kDa), β-actin (Santa Cruz, 43kDa) 에대한항체로 1시간동안반응시킨후 TBS-T 용액으로세척한후 anti-mouse 이차항체를첨가하여 1시간동안반응시켰다. TBS-T 용액으로세척후 ECL solution kit (Santa Cruz) 를이용해 X-ray 필름에감광시켜발현정도를측정하였다. 3. Confocal Laser Scanning Microscopy Gelatin 코팅을한슬라이드위에 MC3T3-El 세포를놓고 4% Para formaldehyde 로실온에서 10분간고정하였다. 고정된세포를인산완충용액 (phosphate buffered saline, PBS) 으로세척한후 10 mm NH 4Cl을 10분간처리하였다. 다시 PBS로세척후 0.1% Triton X 100을처리한후 1% BSA가함유된 PBS로블록시켰다. β-catenin: FITC (#6610155, BD Biosciences) 를 4C에서 16시간동안반응시킨후형광이표지된 2차항체를 1:100로희석하여암실에서실온상태로 1시간반응시켰다. SYTO 59 (Molecular Probes) 로대조염색을시행하였고공초점레이져현미경 (confocal microscopy, LSM Invert 410; Karl Zeiss) 으로관찰하였다. 4. TCF/LEF Luciferase 활성도평가 T cell factor/lymphoid enhancer factor (TCF/LEF) 유전자결합부위와 luciferase reporter 유전자를 fusion시킨 TOPflash - 412 -
- 김세화외 3인 : 인간부갑상선호르몬의골형성촉진작용과 Wnt/β-catenin 신호전달체계와의관련성규명 - Fig. 2. Effects of PTH on nuclear or cytoplasmic active β-catenin expression. PTH (10-8 M) increased nuclear accumulation of activated β-catenin; the ratio between nuclear to cytoplasmic protein were more than 3 folds at 30 minutes and thereafter reporter plasmid (Upstate biotechnology) 와 FOP luciferase reporter plasmid를사용하였다. MC3T3-E1 세포를 6-well plate에 1.5 10 5 개 /well로분주한후 24시간후에 Fugene TM 6 reagent (Roche Molecular Biochemicals, Indianapolis, IN) 를이용하여 TOPflash (1ug) 이나 FOP (1ug) 를 3시간동안 transfection 시켰다. 10시간후 0.1% FBS를함유한 α- MEM으로바꾼후 12시간동안 incubation시킨후부갑상선호르몬 PTH(1-84)] 10-8 M을 6시간동안처리하였다. Luciferase assay system kit (Promega, Madison, WI) 와 Luminometer (Dynatech MLX) 을이용하여 luciferase 활성도를측정하였다. 5. 통계학적분석 모든실험결과는평균 ± 표준편차로표시하였고, 통계분석은 SPSS 11.0 프로그램 (SPSS Inc, Chicago, IL) 을사용하였다. 실험결과의통계는경우에따라 Student s t-test와 ANOVA test를적용하여분석하였고 P < 0.05인경우를통계적으로유의성이있는것으로판정하였다. 결 1. 조골세포주에서부갑상선호르몬이 β-catenin 발현에미치는영향 조골세포주인 MC3T3-E1 세포를이용하여부갑상선호르몬을처치하여조골세포에서의 Wnt canonical 전달체계의가장중요한매개체인 β-catenin에어떠한영향을주는지정량적평가를하였다. 부갑상선호르몬 (PTH 10-8 M) 처리후 β-catenin의양이시간에따라의미있게증가함을관찰하였다. 부갑상선호르몬처리후 30분부터증가하기시작하여 1 과 시간째약 2.7배정도증가하였다가 6시간후에는기저농도로감소하였다 (Fig. 1). 이러한 β-catenin의양적증가가과연실제유전자전사인자의유도에영향을미쳤는지를확인하기위하여서활성화형태의비인산화된 β-catenin을세포질과핵단백질로분리하여분석을해보았다. 부갑상선호르몬 (PTH 10-8 M) 처리 30분후부터핵내에서활성화된 β-catenin이점차증가하였고, 세포질내의활성화형태의 β-catenin은감소하는경향을보였다. 핵단백 / 세포질단백의비를보면 30분이후부터 3배이상으로의미있게증가하였고 24시간동안지속됨을관찰하였다 (Fig. 2). 2. 부갑상선호르몬이 β-catenin 의세포내이동에미치는영향 부갑상선호르몬에반응하여증가된 β-catenin의세포내이동을관찰하기위해공초점레이져현미경을이용하여실험을진행하였다. 기저상태에서는 β-catenin이세포질과핵내에서모두약하게발현되었다. glycogen synthase kinase-3 (GSK-3) 저해제로써 Wnt-β-catenin 신호전달을활성화시키는리튬 (LiCl) 을양성대조군으로사용하였으며, LiCl에의해서 β-catenin 이세포질에존재하다가핵내로이동하여주로핵내에서만표현되었다. 부갑상선호르몬 (PTH 10-8 M) 투여 1시간후에는 β-catenin의발현이세포전체에서뚜렷하게증가하였고, 6시간후에는핵내에서만주로발현됨을관찰하였다 (Fig. 3). 이상의결과로부갑상선호르몬에의해서 β-catenin 발현이증가하고유전자활성의작용을하기위해핵안으로이동함을알수있었다. - 413 -
대한내분비학회지 : 제 22 권제 6 호 2007 Fig. 3. Effects of PTH on intracellular translocation of β-catenin in MC3T3-E1 cells. (A) β -catenin expression (green) was weak without PTH. PTH (10-8 M) treatment increases β -catenin expression in the cytoplasm as well as nuclei at 1 hr. β-catenin was mainly expressed in the nuclei after 6 hrs of PTH. LiCl (60 mm) increases β-catenin expression in the nuclei. (B) Hoechst staining of the same cells showing the nuclei (blue). (C) Confocal microscope images of the same field were taken and merged. 3. 부갑상선호르몬의 TCF/LEF Reporter 유전자활성도에대한효과 부갑상선호르몬에의해서증가되고활성화된 β-catenin이실제전사인자를조절하는지알아보기위해 TOPflash reporter 플라즈미드또는 FOPflash reporter 플라즈미드를이용하여조골세포주에 transfection시킨후 6시간후에 luciferase 활성도를측정하였다. LiCl을투여하였을때대조군에비해 luciferase 활성도가 4배증가하였고, 부갑상선호르몬을투여한경우 luciferase 활성도가 6배증가함을확인하였다 (Fig. 4). 고 본연구에서는인간부갑상선호르몬은조골세포주에서 찰 Wnt 신호전달체계의중요한구성요소인 β-catenin의발현, 특히활성화형태의발현을의미있게증가시켰으며, β -catenin 표적유전자인 TCF/LEF 유전자를활성화시킴을관찰하였다. 부갑상선호르몬은저용량으로매일투여시뼈에동화작용을나타내어서골양을증가시킨다는것은잘알려져있다. 현재까지의연구결과부갑상선호르몬은조골세포에세포사멸을억제할뿐아니라 [3] IGF-1및 TGF-와같은성장인자표현을증가시키고 [7,8] 조골세포의분화에필수적인 Runx2를증가시킴으로써작용할것으로생각되고있으나 [9] 명확한작용기전은아직까지밝혀지지않았다. 부갑상선호르몬은부갑상선호르몬수용체에결합한후 adenylyl cyclase와 camp증가를경유하여 protein kinase A (PKA) 를활성화시켜서작용하며 camp/pka 신호전달이조골세포에서의중 - 414 -
- 김세화외 3인 : 인간부갑상선호르몬의골형성촉진작용과 Wnt/β-catenin 신호전달체계와의관련성규명 - Fig. 4. Effects of PTH on the TCF/LEF reporter gene activity in mouse osteoblastic cells. PTH stimulates T-cell factor/ lymphoid enhancer factor (TCF/LEF) reporter gene activity in MC3T3-E1 cells. The changes in TCF/LEF reporter gene activity are expressed as fold change over control activity (no PTH treatment). 요한신호전달체계로알려져있고 [10], phospholipase C (PLC) 을경유하여 protein kinase C (PKC) 를활성화시키기도하며 [11] 일부다른경로가있을가능성도제기되고있다 [12]. Wnt 단백질들은 Frizzled 수용체와 LRP5 혹은 LRP6 수용체에결합해서 GSK3-β를억제함으로써 β-catenin을활성화시켜서 β-catenin가핵안으로이동하여 TCF/LEF와결합하여 Wnt 표적유전자를조절하게된다 [13,14]. 이러한 Wnt 신호전달은많은기관과조직의발달및유지에중요한역할을하지만, 특히뼈조직에서골량을조절하는데중요한역할을하는것으로알려져있다. 인간 LRP5 유전자의 loss-of-function 돌연변이가일어나면골밀도가낮고뼈가부러지기쉬운특징을나타내는 osteoporosis-pseudoglioma syndrome이발생하고 [15], 반대로 LRP5 유전자의다른돌연변이에서는 ( 예, G171V) 골밀도가매우높은특징을나타내고 LRP5와 Dickkopf-1 (DKK1) 의결합력이약해지는것이일부원인이될수있다고보고되었다 [16,17]. 마찬가지로 LRP5 결핍마우스에서도조골세포증식이감소하고골량이감소된특징을나타낸다 [5]. Wnt 신호전달이골형성을증가시키는경로는 Wnt/β-catenin 신호전달체계가중요한역할을하는데, 활성화된 β-catenin은 C3H10T1/2 세포에서조골세포로의분화를유도한다는것이보고되었고 [18], 동물모델에서의연구결과에서도 β-catenin이조골세포분화의초기단계에필요하며 β-catenin이결핍된경우중간엽줄기세포가조골세포로분화하지않고연골세포로의분화를촉진하는것으로보고되었다 [19,20]. 골형성에중요한부갑상선호르몬과 Wnt 경로사이에 cross-talk이있을가능성이충분히존재하며, 이전의연구에서 Kulkarni 등 [21] 은부갑상선을제거시킨랫트에서부갑상선호르몬투여후 Wnt 신호전달체계와연관이있는여러 구성요소중 LRP6와 frizzled-1 (FZD-1) 표현은증가하고, LRP5와 DKK-1은감소함을보고하였다. 또한부갑상선호르몬이랫트조골세포주인 UMR-106 세포에서 β-catenin을증가시키고 TBE6-luciferase reporter 유전자활성도를증가시킴을보고하여부갑상선호르몬의골형성을촉진시키는기전에 Wnt 신호전달이연관되어있을가능성을제시하였다. 본연구에서는부갑상선호르몬을처리하였을때 β-catenin 발현이 30분부터빠르게증가하여 1시간째약 3배로증가하였다가 6시간후에는기저농도로감소하였다. 그러나 Kulkarni 등의연구에서는동일농도의부갑상선호르몬에의해 β-catenin이 20~50% 정도로약하게증가하였고 24시간동안지속되었다는것이본연구에서와약간의차이를보였다. 이들결과의차이는실험에사용한세포와부갑상선호르몬의차이 (PTH 1-84 vs. PTH 1-38) 가영향을미쳤을가능성이있다. 본연구에서는전사인자를조절하는작용을나타내는활성화된형태의 β-catenin을핵내와세포질로구분하여각각측정하였는데활성화형태의 β-catenin이 24시간동안핵내에서뚜렷하게증가해있음을보여다른보고에서와비슷한결과를나타낸다고생각하였다. 최근 Tobimatsu 등 [22] 에의하면부갑상선호르몬이마우스조골세포에서 Smad3를통하여 β-catenin을증가시킴을보고하여 Wnt-β-catenin 신호전달경로가부갑상선호르몬의세포사멸억제기전에관여함을제시하였다. 이들저자들은부갑상선호르몬을처리하였을때 β-catenin 단백질발현이증가하고불활성화형태의인산화된 β-catenin의양이감소함을보고하였다. 그러나본연구에서는다른연구에서와달리실제로부갑상선호르몬자극에의한 β-catenin의세포내이동을입증하였는데, 자극이없을때에는세포전체에서매우약하게발현되었던 β - 415 -
대한내분비학회지 : 제 22 권제 6 호 2007 -catenin이부갑상선호르몬투여 1시간후에전반적으로증가되었고, 6시간후에는주로핵안에서뚜렷하게발현하는것을직접관찰하였다. 이것은부갑상선호르몬에의해실제로 β-catenin이작용을하기위해활성화된형태로핵내로이동함을의미한다고하겠다. 세포사멸은태생기근골격계발달과성숙, 골전환, 골절치유및골의퇴행성변화동안매우중요한역할을하는것으로알려져있다. 조골세포증식, 분화, 세포사멸간의균형이조골세포전체규모를결정한다 [23]. Hunter 등 [24] 은인간조골세포주에서 β-catenin의 proteolytic cleavage가 caspase-3 활성화와연관되어있으면서조골세포사멸을유도함을보고하여 β-catenin 신호의활성화가조골세포생존을촉진한다고하였다. 최근의다른보고에서는 Wnt-βcatenin 신호전달이 uncommitted 조골전구세포와분화된조골세포의세포사멸을억제한다고하였다 [25]. 이상의여러보고들을종합하여볼때명확한기전은아직까지밝혀지진않았지만 β-catenin이조골세포에서부갑상선호르몬의세포사멸억제의기전에중요할것으로생각된다. 본연구에서는부갑상선호르몬에의한세포사멸억제에 β -catenin이어떤역할을하는지에대해서직접적으로규명하지는못했지만, 여러보고들을종합하여볼때부갑상선호르몬에의해서활성화된 β-catenin이증가하고표적유전자인 TCF/LEF 유전자를활성화시켰다는것은위와같은맥락에서일치하는결과라고하겠다. 본연구에서는마우스조골세포인 MC3T3-E1 세포에서만실험을진행하였고다른세포주를포함한일차배양한조골세포등에서는실험을하지못하였으나, 다른연구에서랫트조골세포주에서비슷한결과를보고하여부갑상선호르몬에의한 β-catenin 발현의증가및표적유전자활성화등의결과가 MC3T3-E1 세포에만특이적으로보이는사실은아닐것으로사료된다. 결론적으로부갑상선호르몬은마우스조골세포에서 Wnt 신호전달체계의중요한구성요소인 β-catenin 발현을증가시켰고, 증가된 β-catenin은핵내로이동하여 TCF/LEF 유전자를활성화시킴을입증하였다. 따라서부갑상선호르몬의골형성촉진이 Wnt-β-catenin 신호전달체계와연관성이있음을제시한다고하겠다. 요약연구배경 : 부갑상선호르몬을저용량으로매일투여시골량을증가시킨다는것은잘알려져있으나아직까지골형성촉진효과에대한기전은명확하게밝혀져있지않다. 본연구에서는부갑상선호르몬과 Wnt/β-catenin 체계와의연관성을규명하여부갑상선호르몬작용에대한하나의기전을제시하고자하였다. 방법 : 조골세포주인 MC3T3-E1 세포를이용하여부갑상선호르몬 (PTH 10-8 M) 을처리한후활성화형태의 β -catenin 발현을세포질과핵내로구분하여조사하였고, 공초점레이져현미경을이용하여부갑상선호르몬처리에의한 β-catenin의세포내위치이동을관찰하였다. 또한부갑상선호르몬이 TCF/LEF reporter 유전자활성도에미치는영향에대해조사하였다. 결과 : 부갑상선호르몬 [(PTH (1-84)] 10-8 M을처리한후 β-catenin의양이 30분부터증가하기시작하여 1시간째약 3배정도증가하였다가 6시간후에는기저농도로감소하였다. 활성화형태의 β-catenin발현을세포질과핵으로분리하여분석하였는데핵내 β-catenin이부갑상선호르몬처리 30분후부터의미있게증가하여 24시간까지지속함을관찰하였다. 또한기저상태에서는 β-catenin이세포전체에서약하게발현되었던것이부갑상선호르몬투여 1시간후에는전체적으로뚜렷하게증가하고 6시간후에는주로핵안에서뚜렷하게발현됨을관찰하였다. 또한부갑상선호르몬에의해활성화된 β-catenin이실제전사인자를조절하는지 luciferase 분석을통해실험하였는데부갑상선호르몬은 TCF/LEF reporter 유전자활성도를 6배정도증가시킴을확인하였다. 결론 : 부갑상선호르몬은마우스조골세포에서 Wnt 신호전달의중요한구성요소인 β-catenin 발현, 특히활성화형태의 β-catenin을증가시켰고증가된 β-catenin은핵내로이동하여 TCF/LEF 유전자를활성화시킴을입증하였다. 따라서부갑상선호르몬의골형성촉진작용에일부 Wnt-βcatenin 신호전달체계가연관되어있음을제시한다고하겠다. 참고문헌 1. Dempster DW, Cosman F, Parisien M, Shen V, Lindsay R: Anabolic actions of parathyroid hormone on bone. Endocr Rev 14:690-709, 1993 2. Dobnig H, Turner RT: Evidence that intermittent treatment with parathyroid hormone increases bone formation in adult rats by activation of bone lining cells. Endocrinology 136:3632-3638, 1995 3. Jilka RL, Weinstein RS, Bellido T, Roberson P, Parfitt AM, Manolagas SC: Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone. J Clin Invest 104:439-446, 1999 4. Onyia JE, Bidwell J, Herring J, Hullman J, Hock JM: In vivo, human parathyroid hormone fragment (hpth 1-34) transiently stimulates immediate early response gene expression, but not proliferation, in trabecular bone cells of young rats. Bone 17:479-484, 1995-416 -
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