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1 대한정형외과연구학회지제 10 권제 2 호 J. of Korean Orthopaedic Research Society Volume 10, Number 2, October, 2007 관절연골세포에서인테그린과 SMAD 신호경로사이의신호교차반응규명 연세대학교의과대학정형외과학교실 *, 연세대학교 BK-21 의과학사업단 한수봉 * 박민성 * 김윤희 * 이진우 * = Abstract = Cross-talk between Integrin and SMAD Signal Pathway in Articular Chondrocyte Soo Bong Hahn, M.D.*, Min Sung Park, M.S.*, Yun Hee Kim, Ph.D.*, Jin Woo Lee, M.D., Ph.D.* Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, South Korea* Brain Korea 21 Project for Medical Science, Yonsei University, Seoul, South Korea Purpose: Identifying the signal cross-talk between integrin signaling cascade and TGF- β1 signaling cascade in articular chondrocytes. Materials and Methods: To analyze integrin or TGF-β1 mediated signaling pathways from extracellular stimuli, type II collagen was coated on the cell culture plate and TGF-β1 was added to cell culture media. Chondrocytes were cultured in the conditioned media with each or both stimuli. Altered activation of signaling proteins detected with western blot technique. Results: More rapid attachment of cells was observed in the type II collagen coated group than non-coated group. The phosphorylated SMAD 2 and 3 were expressed in the type II collagen coated group and synergistically up-regulated phosphorylation in the co-treated group. The phosphorylated FAK at tyrosine 925 was activated by TGF- β1 treatment and synergistically up-regulated by both stimuli. But there was no meaningfully changed phosphorylation of extracellular signal regulated protein kinase (ERK) 1/2 and p38, as known downstream molecules of FAK cascade. Conclusion: This result means that SMAD 2, SMAD 3 and tyrosine 925 of FAK are involved in this signal cross-talking in articular chondrocytes. Key Words: Articular chondrocyte, Signal cross-talk, Type II collagen, TGF-β1, FAK, SMAD 2/3 통신저자 : 이진우서울특별시서대문구신촌동 134 번지연세대학교의과대학정형외과학교실 TEL: 02) FAX: 02) ljwos@yuhs.ac 본연구는연세대학교의과대학 2004년도교수연구비 ( 과제번호 : ) 에의해수행되었음. 65

2 대한정형외과연구학회지제 10 권제 2 호 2007 년 서론제 2형교원질은관절연골의가장큰비중을차지하는기질성분이다. 연골세포들은연골의분자적성질과물리적특징들을결정하는세포외기질을끊임없이생산하고재구성하며, 세포와교원질의상호작용은세포대사에크게영향을준다 8,18). 제 2형교원질과관절연골세포의결합에인테그린 (integrin) β1 이관여하며, 이는제 2형교원질과의상호작용을통해관절연골세포의증식, 분화와생존등에관련된세포외부신호를전달해주는역할을한다 1,11,13). 또한 tyrosine kinase의일종인 focal adhesion kinase (FAK) 등과같은세포막부위의 focal adhesion complex를이루는단백들은인테그린으로부터받는세포증식, 분화, 생존을조절하는세포외부신호를전달한다 5,7). 연골세포의인테그린 β1 이골아세포의특이적표현형인제 1형교원질과결합하면인테그린β1 하위 tyrosine kinase를활성화되고관절연골의기질분해효소인 matrix metalloproteinase-13 (MMP-13) 의발현이증가되며 21), 또한관절연골세포의인테그린 α2β1 이제 1형교원질이아닌제 2형교원질에결합했을때, 세포증식이촉진되고관절연골의주요기질성분인제 2 형교원질과단백다당의합성이증가한다 11,18). 따라서정상적인관절연골세포와관절연골의유지또는재생을위해제 2형교원질과연골세포의상호작용이매우중요하다는것을알수있다. TGF-β1 은관절연골세포의기질형성을촉진하는대표적인성장인자로알려져있다 16,17,18). 세포외부에존재하는 TGF-β1 은 TGF-β수용체에결합하여 SMAD 2와 3를인산화함으로써다양한세포기능을조절한다 28). TGF-β1 은관절연골세포의성장과분화를조절하며, 세포외기질생성을촉진한다고알려져있다 17,18,30). 수년전까지다양한신호전달경로는수용체를활성화시키는분자에의해유전자의전사를조절하는일차원적인경로로써생각되어왔으나, 현재다양한신호경로사이에서복잡한상호작용을통해신호교차반응 (signal cross-talk) 이일어남이알려졌다 4). 예를들면, 제 2형교원질에부착된연골세포의인테그린 β1 신호전달경로와 insulinlike growth factor-i (IGF-I) 사이에서신호교차반응이일어나며, 연골세포의분화를조절한다고보고된바있다 26). 연골세포의증식및분화를조절하는대표적인성장인자인 TGF-β1 과다른성장인자의신호교차반응도알려졌다. 연골세포에서 TGF-β1 이 transforming growth factorbeta-activated kinase-1 (TAK-1) 를통해 mitogen activated protein kinase (MAPK) 를활성화하여제 2형교원질의합성이증가된다는보고와 19), TGF-β1 과 IGF binding protein- 3 사이의신호교차반응이일어나며 15), SMAD 2 및 3와 MAPK 사이의전사인자수준에서신호교차반응이일어나며 aggrecan의발현이증가된다고알려졌다 32). 최근에는 TGF-β1 과제 2형교원질의신호전달경로간의교차반응이일어나며, 이로인한제 2형교원질의발현에대해시너지효과가확인되었으나 24), 이에대한자세한신호교차반응기전은알려지지않았다. 따라서본연구는관절연골세포에서제 2형교원질에의해활성화된인테그린 β1 경로가 TGFβ1 경로를활성화한다는가설하에, 두신호경로의활성을분석함으로써, 제 2형교원질매개인테그린β1 과 TGF-β1 신호경로의교차반응기전에관여하는신호전달인자를밝히고자하였다. 대상및방법 1. 관절연골세포의분리돼지의슬관절로부터연골을채취한후, 1% antibiotic-antimycotic 용액이첨가된 4 의 Dulbecco s modified eagle s medium-high glucose (DMEM-HG, GIBCO-BRL, Grand Island, NY, U.S.A.) 로세척하고연골조직을효소용액 (DMEM-HG9.3 ml/ 연골 1 g, collagenase Ⅱ 10 mg/ 연골 1 g, hyaluronidase 6 mg/ 연골 1 g, 102 mm CaCl ml/ 연골 1 g) 에넣고 12 시간동안녹였다. 연골조직이녹아있는용액을원심분리하고 10% DMEM-HG 로세척한후, 기본배양액 (10% 우태아혈청과 1% antibiotic-antimycotic 용액이첨가된 DMEM-HG) 에서 7일간배양하였다. 66

3 한수봉외 관절연골세포에서인테그린과 SMAD 신호경로사이의신호교차반응규명 2. 세포의배양분리된관절연골세포는기본배양액 (10% 우태아혈청과 1% antibiotic-antimycotic 용액이첨가된 DMEM-HG) 을첨가하여 7일간배양하였다. 각세포는실험전 12시간동안 serum starvation을실시한후, 실험에사용하였고, 0.5% 우태아혈청과 1X insulin-transferrinselenium (ITS, GIBCO-BRL), 1% antibiotic-antimycotic 용액이첨가된 DMEM-HG에배양하며실험을수행하였다. 3. 제 2형교원질의코팅세포배양용기에 PBS (GIBCO-BRL) 에 1 μg/ ml 의농도로희석한제 2형교원질용액 (Chondrex, Redmond, WA, U.S.A.) 을넣고 4 에서 3일간코팅하고제 2형교원질용액을제거하고차가운 PBS 로한번세척한후 30 분간건조하여실험에사용하였다. 4. 실험군의분류실험조건에따라연골세포를세포배양용기에서배양한군 (Group A), 제 2형교원질 (1 μg/ml) 이코팅된세포배양용기에서배양한군 (Group B), 세포배양용기의배양액에 TGF-β1 (10 ng/ml, R&D Systems, Inc., Minneapolis, U.S.A.) 처럼처리한군 (Group C), 제 2형교원질 (1 μg/ml) 이코팅된세포배양용기에 TGF-β1 (10 ng/ml) 을처리하여배양한군 (Group D) 으로분류하였다. 액만취해서 -70 에보관하였다. 추출된단백질의정량은 Bradford 방법에따라실시하였다. 6. Western Blot 분석세포추출액은 SDS-PAGE를이용하여단백질을분리한후, western blotter (Bio-RAD) 를이용하여 PVDF membrane (Amersham Pharmacia, Piscataway, NJ, U.S.A.) 으로옮겼다. Membrane을 5% 의무지방우유가함유된 1X TBST (100 mm Tris (ph7.5), 1.5 M NaCl, 0.5% tween-20) 용액으로 blocking하고, 1X TBST로세척한후, 일차항체를 1% 무지방우유를포함한용액에서부착시켰다. 일차항체반응후, membrane을 1X TBST를이용하여세척하고, 이차항체를반응시킨후, 1X TBST를이용하여세척하고 ECL western blotting detection kit (Amersham Pharmacia) 을사용하여발광을유도한후, ECL hyper film (Amersham Pharmacia) 을사용하여현상하였다. Western blot에사용된일차항체는 phospho-smad 2, SMAD 2, phospho-smad 3, SMAD 3, phospho-fak Y397, phospho-fak Y925, FAK, phospho-erk1/2, ERK1/2, phospho-p38, p38, GAPDH (Santa Cruz, Santa Cruz, CA, U.S.A.) 이며, Western blot analysis를통해얻은결과는 TINA pixel analyzer version 2.10 (Raytest Isotopenme gerate GmbH, Straubenhardt, Germany) 을이용하여정량화하였고, 각단백질의발현정도는 GAPDH로표준화하였다. 5. 단백질추출 결 과 세포배양용기에부착된세포를 PBS 로 2회세척하고 passive lysis buffer (Promega, Madison, WI, U.S.A.), 500X protease inhibitor (Sigma, St. Louis, MO, U.S.A.), 100X phosphatase inhibitor Ⅰ와 Ⅱ (Sigma) 를세포배양용기에첨가하여 cell scraper를이용하여세포를모은후단백질을추출하였다. 단백질추출액을원심분리한후, 상층 1. 제 2형교원질에부착된연골세포형태관찰세포부착 1시간째세포형태를현미경상으로관찰한결과, 제 2형교원질에서배양된군 (Group B, D) 이코팅되지않은군 (Group A, C) 에비해빠른세포부착과넓게퍼진세포형태를관찰하였으며, 이러한연골세포부착은성장인자보다세포외기질즉, 제 2형교원질에의해영향을받는 67

(A) Non-treated group, (B) Type II collagen coated group, (C) TGF-β1 treated group, (D) Type II collagen and TGF-β1 co-treated group. D Fig. 2.

4 대한정형외과연구학회지제 10 권제 2 호 2007 년 A B C Fig. 1. Microscopic morphological features of attached articular chondrocytes. Articular chondrocytes were seeded on type II collagen (1 μg/ml) coated or non-coated plates after serum starvation for 12 hours and 1 hour later, we observed with an optical microscopy ( 200). (A) Non-treated group, (B) Type II collagen coated group, (C) TGF-β1 treated group, (D) Type II collagen and TGF-β1 co-treated group. D Fig. 2. SMAD 2 and SMAD 3 phosphorylation by the type II collagen. The whole cell lysates were obtained at 1, 2, 4, 12, 24, 48, 96 hours after stimulation and detected by western blot. SMAD 2 and 3 were phosphorylated by type II collagen (1 μg/ml, Right) more quickly and stronger than non-coated group (Left) were detected with western blot analysis. 68

한수봉외 관절연골세포에서인테그린과 SMAD 신호경로사이의신호교차반응규명 것으로확인되었다 (Fig. 1). 2. 제 2형교원질에의한 SMAD 2 및 SMAD 3의인산화분석 SMAD 2의인산화가부착후 24 시간까지꾸준히증가하였으나, 48시간이후에감소되었다. 특히, 대조군에비해제 2형교원질에서배양된세포에서세포부착후 1시간부터빠르게증가하였다.

5 한수봉외 관절연골세포에서인테그린과 SMAD 신호경로사이의신호교차반응규명 것으로확인되었다 (Fig. 1). 2. 제 2형교원질에의한 SMAD 2 및 SMAD 3의인산화분석 SMAD 2의인산화가부착후 24 시간까지꾸준히증가하였으나, 48시간이후에감소되었다. 특히, 대조군에비해제 2형교원질에서배양된세포에서세포부착후 1시간부터빠르게증가하였다. SMAD 3의인산화는제 2군에서 1시간부터 4시간까지확연히증가하였으며, 12시간이후급격하게감소하였다. 따라서세포부착초기에있어서 SMAD 2와 SMAD 3이서로다르게관여하는것으로보이며, 또한제 2형교원질에의해 TGF-β1 신호전달물질인 SMAD 2 및 SMAD 3 이활성화됨을확인하였다 (Fig. 2). 3. 제 2형교원질및 TGF-β1 에의한 SMAD 2 및 SMAD 3의인산화분석 TGF-β1 또는제 2형교원질의각자극에의해 SMAD 2의활성이시간이지남에따라감소하였으나, 두자극을함께가한군 (Group D) 에서 48 시간부터 96 시간까지지속되는것으로나타났다 (Fig. 3A). SMAD 2와유사하게, Group B와 Group C에서 SMAD 3의활성이시간이지남에따라감소하였으나, Group D에서 48 시간부터 96 시간까지지속되는활성을확인하였다 (Fig. 3B). 따라서제 2형교원질과 TGF-β1 을동시에처리하였을경우, 일어나는신호교차반응에 SMAD 2와 SMAD 3이관여하는것으로보인다. 4. FAK의인산화분석 인테그린의하위신호전달단백인 FAK 의 A B Fig. 3. Synergic effect on phosphorylation of SMAD 2 and 3 by combination of type II collagen and TGF-β1. After stimulation, unattached cells were washed out with PBS and attached cells were lysed directly with lysis buffer at 12, 24, 48, 96 hours. Phospho-SMAD 2 (A) and phospho-smad 3 (B) were detected using western blotting. Phosphorylation of these two molecules in type II collagen (Group B) or TGF-β1 (Group C) stimulated group was time dependently decreased after 24 hours. Until 96 hours, however, phosphorylation of those was maintained by co-stimulation (Group D). This means that there was synergic effect by signal cross-talk between integrin and TGF-β1 pathways. 69

6 대한정형외과연구학회지제 10 권제 2 호 2007 년 tyrosine 397번 (FAK Y397) 잔기의인산화를분석한결과, 모든군에서시간이지남에따라 FAK Y397 의인산화가증가하였으나, 두자극을모두가했을때에인산화양상에유의한차이를보이지않았다 (Fig. 4A). 그러나, FAK의인산화 부위인 tyrosine 925번 (FAK Y925) 을확인한결과, 대조군에비해제 2형교원질과 TGF-β1 을가한군그리고두자극을함께가한군에서 FAK Y925 의활성이증가되었으며, 또한두자극을함께가했을때활성이 96 시간까지지속되는것 A B Fig. 4. Synergistically activated tyrosine 925 residue of FAK. Chondrocytes were seeded in coated or non-coated plates containing TGF-β1 or not. Whole cell lysates were obtained at 1, 2, 4, 12, 24, 48, 96 hours after stimulation and western blotting was performed with phosphorylation site (tyrosine 397 (A) or 925 residue (B)) specific antibodies of FAK. There was synergic effect on phospho-fak at tyrosine 925 residue. 70

한수봉외 관절연골세포에서인테그린과 SMAD 신호경로사이의신호교차반응규명 을확인하였다 (Fig. 4B). 따라서 TGF-β1 과제 2형교원질에의해 FAK 의 tyrosine 397번이아닌 tyrosine 925번인산화에시너지효과를보였으며, 이것이 SMAD 2와 3에서보인시너지효과와관련이있을것으로추측된다. 5.

7 한수봉외 관절연골세포에서인테그린과 SMAD 신호경로사이의신호교차반응규명 을확인하였다 (Fig. 4B). 따라서 TGF-β1 과제 2형교원질에의해 FAK 의 tyrosine 397번이아닌 tyrosine 925번인산화에시너지효과를보였으며, 이것이 SMAD 2와 3에서보인시너지효과와관련이있을것으로추측된다. 5. MAPK의인산화분석 자극을함께가한군에서또한증가되지않았다 (Fig. 5A). p38의인산화를확인한결과, 각군의차이가없었으며, 두자극을동시에처리한군에서도유의한변화양상을확인할수없었다 (Fig. 5B). 따라서제 2형교원질및 TGF-β1 의신호교차반응은위의 MAPK 보다상위단계에서일어날것으로사료된다. 위에서보인시너지효과가 FAK 의하위경로인 MAPK에서도일어나는지살펴보았다. ERK1/2의인산화를확인한결과, 모든군에서인산화양상의유의한차이를볼수없었으며두 고찰관절연골은한번손상되면재생되지않는단점이있다 17). 통증, 활동의제약, 체형변형등을유 A B Fig. 5. Phosphorylation of ERK and p38. Cells were seeded in type II collagen coated or non-coated plates containing TGF-β1 or not. Whole cell lysates were obtained at 1, 2, 4, 12, 24, 48, 96 hours after stimulation and western blot analysis was performed with phospho-specific ERK1/2 (A) and p38 (B) MAPKs. 71

8 대한정형외과연구학회지제 10 권제 2 호 2007 년 발하는가장흔한관절질환인골관절염이발생된연골은연골기능이크게손상된상태로, 연골기질의변성이일어나고, matrix metalloproteinases (MMPs) 가분비되어단백다당을분해시키고균열 (fibrillation) 이발생하며, 결국에는연골조직이파괴된다 2,3). 세포의생물학적기능은세포내에서발현되는유전자의발현을조절하는다양한세포신호들의복잡한전달과정이관여함으로써조절된다 4). 따라서정상연골의기능을유지하고회복하기위해서는연골세포내신호전달과정의이해가필수적이다. 세포부착단백의일종인인테그린은세포외부로부터연골세포내부로성장, 생존, 분화, 이동등의관련된신호를전달하는매개체의역할을한다 1,6,9,22). 현재 TGF-β1 에의한연골세포의제 2형교원질의발현이증가한다는보고와 16,17,19,30), 발현된제 2형교원질및그외의기질성분들이연골유지에주요역할을한다고알려져있다 5,8,11,18). 또한연골세포에서두종류의성장인자가신호교차반응을통해연골세포의대사를촉진한다고한다 26,32). 따라서본연구는관절에서분리한연골세포에연골의주요구성성분인제 2형교원질과대표적인연골성장인자인 TGF-β1 의자극을가하였을때또는두가지자극을동시에가했을때변화하는세포내신호전달단백의활성을살펴보았다. TGF-β1 의신호전달단백인 SMAD 2와 SMAD 3이제 2형교원질자극이가해진군에서빠르게활성화되었음이확인되었다. 즉, 제 2형교원질로인해활성화된인테그린신호전달경로가 SMAD 2와 SMAD 3의신호전달경로와상호작용을한다는것을의미한다. SMAD 2와 SMAD 3이제 2형교원질과 TGF-β1 에의해활성화되었으나자극을가한후 48 시간이지나자활성이감소되었다. 하지만두가지자극이함께가해졌을때에활성이 96 시간까지지속되었고, 약 4배증가된것으로보아, 두자극의교차반응에의한시너지효과가있는것을확인하였다. 이결과는제 2형교원질로부터매개된인테그린하위신호전달계와 TGF-β1 하위 SMAD 2와 SMAD 3의신호전달경로에서신호교차반응이일어난다는것을보여준다. 인테그린 β1 의세포내부위에부착하는 FAK 는 인테그린의신호전달과정의중심적인역할을한다. 하지만현재까지연골세포특성의유지에크게영향을주는것으로알려져있는 TGF-β1 과관련하여인테그린과 FAK 에의해전개되는세포내신호전달과정의자세한기전에대해서밝힌연구는매우미흡한상태이다 7,12). 본연구결과에서, FAK 의 tyrosine 397번째잔기의인산화는시간이지남에따라증가되는것으로나타났으나, 모든군에서차이가없었으며, 시너지효과또한확인할수없었다. 한편, FAK의 tyrosine 925번째아미노산잔기의인산화가제 2형교원질에의해서빠르게증가되었으며, TGF-β1 에의해서도활성을보였다. 특히두자극을함께가한군에서빠르게활성화되어 96 시간까지강한활성이지속되는것으로확인되었다. 따라서 TGF-β1 과인테그린 β1 신호교차반응에 FAK 의 tyrosine 925번부위가관여할것으로사료된다. 관절연골세포에서 TGF-β1 과제 2형교원질의공동작용에의해 DNA 의합성과 aggrecan의 mrna 발현증가에시너지효과가있다는보고가있다 16,17). 따라서본연구에서보인 TGF-β1 과제 2형교원질에의한 FAK의 tyrosine 925 잔기및 SMAD 2와 3 의활성에대한시너지효과가위의보고와관련하여연골세포의특성을유지하고, 연골기질의합성에중요한역할을할수있을것으로사료된다. 세포와교원질의상호작용에의해인테그린으로전달된세포외부자극은인테그린 β1 의 cytoplasmic domain에결합된 FAK와 Src, paxillin, talin, p130 CAS, cavaolin 등의보조인자들을통해세포내로신호전달이이루어지고증식, 분화, 생존등과관련된세포대사가조절된다. FAK tyrosine 397의활성화는 FAK와 Src 의상호작용에의해 tyrosine 576, tyrosine 577, tyrosine 861 그리고 tyrosine 925번잔기등의인산화로이어진다. 특히 FAK의 tyrosine 925 잔기의인산화가일어나게되면 FAK 로전달된신호는 MAP kinase의활성에영향을준다고도알려져있다 7,12,14,25). MAP kinase의활성화기전은 Src, p130 CAS, Crk 등이관여하는 FAK 에의존적인신호경로와 Shc, Fyn, caveolin-1 등이관여하는 FAK 에비의존적인신호경로의두가지경로를통해 MAPK가활성화되는두가지경로 72

9 한수봉외 관절연골세포에서인테그린과 SMAD 신호경로사이의신호교차반응규명 Fig. 6. A diagram of signal cross-talk between integrin and TGF-β1 cascades. We suggested in this study that there was the signal cross-talk between type II collagen triggered integrin signal cascade and TGF-β1 signal pathway. Also, the tyrosine 925 residue and SMAD 2 and SMAD 3 were related on this signal cross-talk. 로나눌수있다 10,23,31). 최근연구에따르면, IGF 와 fibroblast growth factor (FGF) 등의성장인자들에의해 MAP kinase가활성화되고연골세포의대사가조절된다고한다 20,27). 한편, Starkman 등 29) 은연골세포에서 IGF 에의한단백다당합성이 MAPK 활성에비의존적으로증가된다고보고하였다. 본연구에서는 MAPK의일종인 p38 과 ERK 1/2가 TGF-β1 또는제 2형교원질에의해유의하게활성이증가되거나두자극에의한시너지효과를보이지않았다. MAPK 의활성에는수많은신호전달인자들이관여하며, 세포증식, 분화및생존등을조절하는신호전달경로또한매우복잡하게구성되어있다. 따라서 FAK 의하위경로로써 p38 과 ERK 1/2에비의존적인경로가관여할것으로추측된다. 본연구에서 TGF-β1 에의한신호와제2형교원질자극에의한인테그린신호가 FAK 의 tyrosine 925와 SMAD 2 및 SMAD 3의활성에대해시너지효과를보였으며, 이단백들이 TGF-β 1과제 2형교원질의신호교차반응에중요한역할을할것으로사료된다 (Fig. 6). 결 론 연골세포의대사에중요한역할을하는 TGF-β 1과제 2형교원질의신호경로간에일어나는신호교차반응에 SMAD 2와 SMAD 3, 그리고 FAK tyrosine 925가중요한역할을하며, 이신호교차반응이연골기질생성에영향을줄것으로보인다. 따라서본연구가관절연골세포의대사조절기전을이해하는데에도움이될것으로기대되며, 또한외상또는관절염이유발된연골조직의회복에적용되어질수있는기본적인이론및기전에관한이해뿐만아니라, 손상된관절의치료및재생, 조직공학등에관한임상적응용에필요한이론적인이해에도움을줄수있을것으로사료된다. REFERENCES 01) Albelda SM and Buck CA: Integrins and other cell adhesion molecules. FASEB J, 4: , ) Buckwalter JA: Articular cartilage injuries. Clin Orthop Relat Res, 402: 21-37, ) Burrage PS, Mix KS and Brinckerhoff CE: Matrix metalloproteinases: role in arthritis. Front Biosci, 11: , ) Dumont JE, Pecasse F and Maenhaut C: Crosstalk and specificity in signalling. Are we crosstalking our selves into general confusion? Cell Signal, 13: , ) Geiger B, Bershadsky A, Pankov R and Yamada KM: Transmembrane crosstalk between the extra cellular matrix-cytoskeleton crosstalk. Nat Rev Mol Cell Biol, 2: , ) Giancotti FG and Ruoslahti E: Integrin signaling. Science, 13, 285: , ) Guan JL: Focal adhesion kinase in integrin signaling. Matrix Biol, 16: , ) Hynes RO: Integrins: Bidirectional, Allosteric Signaling Machines. Cell, 110: , ) Hynes RO: Related Integrins: versatility, modulation, and signaling in cell adhesion. Cell, 69: 11-25, ) Lee JW and Juliano R: Mitogenic signal transduction by integrin- and growth factor receptor- 73

10 대한정형외과연구학회지제 10 권제 2 호 2007 년 mediated pathways. Mol Cells, 17: , ) Lee JW, Qi WN and Scully SP: The involvement of beta1 integrin in the modulation by collagen of chondrocyte-response to transforming growth factor-beta1. J Orthop Res, 20: 66-75, ) Li W, Duzgun A, Sumpio BE and Basson MD: Integrin and FAK-mediated MAPK activation is required for cyclic strain mitogenic effects in Caco-2 cells. Am J Physiol Gastrointest Liver Physiol, 280: G75-87, ) Loeser RF: Integrins and cell signaling in chondrocytes. Biorheology, 39: , ) Mitra SK, Mikolon D, Molina JE, et al.: Intrinsic FAK activity and Y925 phosphorylation facilitate an angiogenic switch in tumors. Oncogene, 28, 25: , ) O Rear L, Longobardi L, Torello M, et al.: Signaling cross-talk between IGF-binding protein-3 and transforming growth factor-βin mesenchymal chondroprogenitor cell growth. J Mol Endocrinol, 34(3): , ) Qi WN and Scully SP: Effect of type II collagen in chondrocyte response to TGF-beta 1 regulation. Exp Cell Res, 25, 241: , ) Qi WN and Scully SP: Extracellular collagen modulates the regulation of chondrocytes by transforming growth factor-beta 1. J Orthop Res, 15: , ) Qi WN and Scully SP: Type II collagen modulates the composition of extracellular matrix synthesized by articular chondrocytes. J Orthop Res, 21: , ) Qiao B, Padilla SR and Benya PD: Transforming growth factor (TGF)-beta-activated kinase 1 mimics and mediates TGF-beta-induced stimulation of type II collagen synthesis in chondrocytes independent of Col2a1 transcription and Smad3 signaling. J Biol Chem, 280: , ) Raucci A, Laplantine E, Mansukhani A and Basilico C: Activation of the ERK1/2 and p38 mitogen-activated protein kinase pathways mediates fibroblast growth factor-induced growth arrest of chondrocytes. J Biol Chem, 279: , ) Ronzière MC, Aubert-Foucher E, Gouttenoire J, et al.: Integrin alpha1beta1 mediates collagen induction of MMP-13 expression in MC615 chondrocytes. Biochim Biophys Acta, 1746: 55-64, ) Salter DM, Hughes DE, Simpson R and Gardner DL: Integrin expression by human articular chondrocytes. Br J Rheumatol, 31: , ) Schlaepfer DD, Jones KC and Hunter T: Multiple Grb2-mediated integrin-stimulated signaling pathways to ERK2/mitogen-activated protein kinase: summation of both c-src- and focal adhesion kinase-initiated tyrosine phosphorylation events. Mol Cell Biol, 18: , ) Schneiderbauer MM, Dutton CM and Scully SP: Signaling cross-talk between TGF-β1 and ECM signals in chondrocytic cells. Cell Signal, 16: , ) Schoenwaelder SM and Burridge K: Bidirectional signaling between the cytoskeleton and integrins. Curr Opin Cell Biol, 11: , ) Shakibaei M, John T, De Souza P, Rahmanzadeh R and Merker HJ: Signal transduction by beta1 integrin receptors in human chondrocytes in vitro: collaboration with the insulin-like growth factor-i receptor. Biochem J, 342: , ) Shakibaei M, Seifarth C, John T, Rahmanzadeh M and Mobasheri A: Igf-I extends the chondrogenic potential of human articular chondrocytes in vitro: Molecular association between Sox9 and Erk1/2. Biochem Pharmacol, 72: , ) Shi Y and Massague J: Mechanisms of TGFbeta signaling from cell membrane to the nucleus. Cell, 113: ,

11 한수봉외 관절연골세포에서인테그린과 SMAD 신호경로사이의신호교차반응규명 29) Starkman BG, Cravero JD, Delcarlo M and Loeser RF: IGF-I stimulation of proteoglycan synthesis by chondrocytes requires activation of the PI 3-kinase pathway but not ERK MAPK. Biochem J, 389: , ) Verrecchia F and Mauviel A: Transforming growth factor-beta signaling through the Smad pathway: role in extracellular matrix gene expression and regulation. J Invest Dermatol, 118: , ) Wary KK, Mariotti A, Zurzolo C and Giancotti FG: A requirement for caveolin-1 and associated kinase Fyn in integrin signaling and anchorage-dependent cell growth. Cell, 94: , ) Watanabe H, de Caestecker MP and Yamada Y: Transcriptional cross-talk between Smad, ERK1/2, and p38 mitogen-activated protein kinase pathways regulates transforming growth factor-beta-induced aggrecan gene expression in chondrogenic ATDC5 cells. J Biol Chem, 276: ,

2016 학년도약학대학면접문제해설 문제 2 아래의질문에 3-4분이내로답하시오. 표피성장인자수용체 (epidermal growth factor receptor, EGFR) 는수용체티로신인산화효소군 (receptor tyrosine kinases, RTKs) 의일종으로서세

2016 학년도약학대학면접문제해설 문제 2 아래의질문에 3-4분이내로답하시오. 표피성장인자수용체 (epidermal growth factor receptor, EGFR) 는수용체티로신인산화효소군 (receptor tyrosine kinases, RTKs) 의일종으로서세 본문제에대한지적소유권은동국대학교에있습니다. 본교의서면허락없이무단으로출판, 게재, 사용할수없습니다. 문제 2 2016 학년도약학대학면접문제 아래의질문에 3-4 분이내로답하시오. 표피성장인자수용체 (epidermal growth factor receptor, EGFR) 는수용체티로신 인산화효소군 (receptor tyrosine kinases, RTKs) 의일종으로서세포의생존과증식