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1 사람폐섬유아세포의전환성장인자 -β1 에의한 fibronectin 분비와 α-smooth muscle actin 표현에있어서활성산소족의역할 순천향대학교현암신장연구소 1, 이화여자대학교약학대학, 순천향대학교의과대학내과학교실 3 하헌주 1,, 유미라 1, 어수택 1,3, 박춘식 1,3, 이희발 1,3 Role of Reactive Oxygen Species in Transforming Growth Factor-β1- inuduced Fibronectin Secretion and α-smooth Muscle Actin Expression in Human Lung Fibroblasts Hunjoo Ha 1,, Mi-Ra Yu 1, Soo-taek Uh, M.D. 1,3, Choon Sik Park, M.D. 1,3, and Hi Bahl Lee, M.D. 1,3 Hyonam Kidney Laboratory, Soon Chun Hyang University 1, Ewha Womans University College of Pharmacy, and Department of Internal Medicine, Soon Chun Hyang University College of Medicine 3 Background : The transforming growth factor-β1 (TGF-β1) plays a key role in lung fibrosis. However, the mole cular mechanisms involved in TGF-β1-induced lung fibrosis are unclear. TGF-β1 is the key inducer of myofibroblast transdifferentiation via de novo synthesis of α-smooth muscle actin (α-sma). Since TGF-β1 signals through reactive oxygen species (ROS) and ROS have been shown to induce accumulation of extracellular matrix (ECM) in various tissues, this study examined if ROS play a role in TGF-β1-induced fibronectin secretion and α-sma expression in human lung fibroblasts, MRC-5 cells. Methods : Growth arrested and synchronized MRC-5 cells were stimulated with TGF-β1 (.-1 ng/ml) in the presence or absence of N-acetylcysteine (NAC) or diphenyleneiodonium (DPI) for up to 9 hours. Dichlorofluorescein (DCF)-sensitive cellular ROS were measured by FACScan and secreted fibronectin and cellular α-sma by Western blot analysis. Results : TGF-β1 increased the level of fibronectin secretion and α-sma expression in MRC-5 cells in a dosedependent manner. Both NAC ( and 3 mm) and DPI (1 and 5 μm) significantly inhibited TGF-β1-induced fibronectin and α-sma upregulation. The TGF-β1-induced cellular ROS level was also significantly reduced by NAC and DPI. Conclusions : The results suggest that NADPH oxidase-dependent ROS play an important role in TGF-β1-induced fibronectin secretion and α-sma expression in MRC-5 cells, which leads to myofibroblast transdifferentiation and progressive lung fibrosis. (Tuberc Respir Dis 5; 58:7-75) Key words : Pulmonary fibrosis, Transforming growth factor-beta1, Reactive oxygen species, α-smooth muscle actin, Fibroblast, Myofibroblast, Fibronectin, N-acetylcysteine, Diphenyleneiodonium 서 세포외기질 (extracellular matrix: ECM) 의과다한축적으로정의되는조직섬유화는다양한원인에의한폐질환에서도관찰되는공통적인병리소견이다 1. 본연구는학술진흥재단중점연구소육성과제 ( F) 지원에의하여수행되었음. Address for correspondence : Hi Bahl Lee, M.D. Hoynam Kidney Laboratory Soon Chun Hyang University 57 Hannam-dong, Yongsan-gu Seoul 1-73 Phone : Fax : hblee@hkl.ac.kr Received : Dec. 1. Accepted : Feb.. 5 론 α-smooth muscle actin (α-sma) 의신생합성 (de novo synthesis) 은폐섬유아세포 (lung fibroblast) 를근섬유아세포 (myofibroblast) 표현형으로변화시키고이근섬유아세포의표현은폐섬유화가진행되는부위에서상향조절되고지속적으로표현 1-5 되기때문에폐섬유화의발생과진행에주역할을할것으로추정되고있다. Zhang 등은 bleomycin에의한폐섬유화에서폐섬유아세포양 (fibroblast-like) 세포에서 α-sma 의표현과함께 collagen I 과전환성장인자-β1 (trans forming growth factor-β1: TGF-β1) 이표현되고 7 세포수축을유도할 8 뿐아니라단핵구화학주성단백-1 (monocyte chemoattractant protein-1: MCP-1) 의표현을증가시킴을보고하였다 9. 폐의근섬유아세포는 7

2 HJ Ha, et al.: ROS and lung fibrosis 기관지또는혈관외막 (perivascular adventitium) 에존재하던섬유아세포의표현형이변화된것으로생각된다. α-sma 의표현이근섬유아세포로의변화를특징짓고있기때문에폐섬유아세포에서의 α-sma의표현조절기전을이해하면폐섬유화의발생과진행을억제할수있는새로운치료법을제시할수있을것이다. TGF-β1 은정상폐조직의기관지상피세포와섬유아세포에서생산되는주된 TGF-β 아형이다 1. TGF-β 1은 ECM 단백을생산하는유전자를상향조절하여그합성을증가시키고, plasminogen activator inhibi tor-1 (PAI-1) 의표현을상향조절하여 plasmin이나 collagenase 등 ECM 분해효소의활성을감소시킴으로써 ECM 축적을매개한다 11,1. TGF-β1은상피세포 -중간엽세포이행 (epithelial-mesenchymal transition: EMT) 을유도하는주된인자로이과정에서 α-sma 의신생합성을유도하고상피세포의표현형을근섬유아세포로변화시킨다 13. 또 TGF-β1 에의한 α-sma 의표현은폐섬유아세포를근섬유아세포로변화시키는중요한지표이다 1. TGF-β1 에의한세포활성화에는 Smad 경로 15 와 mitogen-activated protein kinases (MAPK) 경로가관여함이잘알려져있다 그러나최근의연구결과로활성산소족 (reactive oxygen species: ROS) 도 TGF-β1 의세포내신호전달자로관여한다는것이알려져있다 19-. Junn 등 은폐섬유아세포에서 TGFβ1에의한 IL-의상향조절에 ROS가관여한다고보고한바있다. 본연구진은신장사구체혈관간세포에서 ROS가 TGF-β1 에의한 PAI-1 상향조절에관여하고 TGF-β1 에의한 ROS 증가에는 NADPH oxidase 가관여함을보고하였다,7. 또외부에서투여한과산화수소 (H O ) 가신장세포의 fibronectin 단백생산을상향조절할뿐아니라 PAI-1 을상향조절함으로써 ECM 분해를억제하여 ECM 축적을유발하고,7 α-sma 를상향조절함 8 을보고한바있다. Hagiwara 등 9 은항산화제 N-acetylcysteine (NAC) 이 bleomycin에의한폐섬유화를억제하는것을보고하여 ROS가폐섬유화에관여함을시사하였다. 본연구에서는조직섬유화의기전을이해하고섬유화를예방또는억제하 는전략을수립하기위한연구의일환으로폐섬유아세포에서 TGF-β1 에의한 fibronectin 분비와 α-sma 표현상향조절에 ROS가관여하는지를검색하였다. 이를위하여 MRC-5 세포를이용하여 dichlorofluore scein (DCF) 에민감한세포내 ROS를 FACScan으로측정하였고, TGF-β1 에의한 fibronectin 분비와 α- SMA 표현상향조절에미치는항산화제의효과를관찰하였다. 대상및방법실험에사용한재료는따로제시되지않은한 Sigma Chemical Company (St. Louis, MO, USA) 와 Nalge Nunc International (Naper Ville, IL, USA) 에서구입하였다. 1. 폐섬유아세포주배양이미성상이확인되어실험에사용되고있는폐섬유아세포주 (MRC-5) 를 American Type Culture Collection (ATCC: Rockville, MD, USA) 에서구입하여 5% 우태아혈청이포함된 5. mm 포도당함유 Dulbecco's modified Eagle's medium (DMEM) 배지에서배양하였다. 세포가배양용기를가득채우면 trypsin/edta (.5%/.1%) 로처리하여계대배양하였고실험을위하여 well plate (1x1 5 cells/well) 에서배양하였다.. 실험조건세포가배양용기의대부분을채우면우태아혈청을포함하지않는배지로교체하여 시간배양함으로써세포성장을정지시키고성장주기를동일화하였다. 이후실험목적에알맞게혈청이배제된배지를대조군으로하고, 실험군은.-1 ng/ml TGF-β1 (R&D, Mpls., MN, USA) 을첨가한군으로구성하여 9시간까지배양하였다. 항산화제인 N-acetylcysteine (NAC: 5-3 mm) 이나 NADPH oxidase 억제제인 diphenyleneiodonium (DPI:.1-5 μm) 은 TGF-β1 투여 1 시간전부터실험이종료될때까지지속적으로 8

3 Tuberculosis and Respiratory Diseases Vol. 58. No. 3, Mar, 5 투여하였다. 배양액은 8시간에동일한실험조건의배양액으로교환함으로써포도당을비롯한영양공급을유지하였다. 3. 세포독성분석 NAC와 DPI에의하여손상을입은세포로부터배지내로유리되는 LDH를측정함으로써세포독성을정량하였다 3. 세포독성은실험이완료될때까지배지로유리된 LDH 양과분석시점에서 1% Triton X-1으로처치하여인위적으로세포들을모두파괴시킨후측정한세포내 LDH를합한총 LDH 양중배지로유리된 LDH 양의백분율 (%) 로표시하였다. 시료의 LDH는 pyruvic acid, 환원형태의 β-nicotin amide adenosine dinucleotide (NADH) 와반응시켜 SOFTMAX PRO multiple reader (Molecular Devices, Sunnyvale, CA, USA) 를이용하여 3 nm에서의흡광도를측정하였다. % 세포손상 = 1 x 손상된세포에서배지로유리된 LDH/( 배지로유리된 LDH+ 세포내 LDH). Western Blot 분석실험이완료된후배양상층액을취하여원심분리한후 aliquot하여분석할때까지 7 에서보관하였다. 세포용해액 (lysis buffer: 5mM Tris-HCl PH 7.5, 1mM NaCl, mm EDTA, 1mM dithiothritol,.1mm PMSF, leupeptin, aprotimin, 1% Triton X- 1) 으로세포를용해한후 Bio-Rad 분석시약 (Bio- Rad Laboratories, Hercules, CA, USA) 으로단백량을측정하였다. 세포단백량 ( μg) 으로보정된배양액 ( 농축이필요하지않음 ) 또는세포용해액에 reducing sample buffer를넣고 95 에서 5분간끓였다. Fibr onectin 단백과 α-sma 단백측정을위해서각각 5% 와 1% polyacrylamide gel에서전기영동하여단백질을분리하고완충액을이용하여 nitrocellulose 흡착지에전이하였다. 흡착지를 blocking 용액 (5% skim milk 및.1% Tween 이포함된 TBS) 에넣고 1시간동안 반응을시켰다. Peroxidase-conjugated rabbit antihuman fibronectin (DAKO, Glostrup, Denmark) 을이용하여 시간동안반응을시킨후 washing 용액 (.1% Tween 을포함한 TBS) 을이용하여 15분간한번과 5분씩세번을반복하여반응하지않은항체를제거하고 ECL solution kit (Amerham Pharmacia Biotech, England) 를이용하여 X-ray film을이용하여감광을시켰다. α-sma 단백은 1차항체로 anti-mouse α-sma antibody (1:) 와 차항체인 anti-mouse IgG (Santa Cruz Biotechnology, Inc, Snata Cruz, CA, USA) 를이용하여각각 시간동안반응시켰다. 감광된필름을 scan 하여 density 를구하여대조군과비교하였다. α-sma 단백은 anti-mouse β-actin antibody (1:1,) 로보정하였다. 5. 세포내 DCF-sensitive ROS 측정 DCF에민감한세포내 ROS를정량하기위하여 FACS (Becton Dickinson Immunocytometry System, Mountain View, CA, USA) 를이용하였다. 실험이완료되면 5-(and-)-chloromethyl-',7'-dichloro-dihydr ofluorescein diacetate (CM-H DCFDA: Molecular Probes Inc., Eugene, OR, USA) 5 μm을첨가하여세포배양기에서 분간배양하였다. PBS로씻어준후 1, 세포에함유된산화된,7 -dichlorofluorescin (DCFH) 의형광을 88 nm excitation과 nm emission에서측정하였다. TGF-β1에의한세포내 ROS의측정은예비실험결과에따라 TGF-β1 자극후 3 분에측정하였다.. 자료분석모든자료는 평균값 + 표준오차 로기술하였다. 각조건에서실험군간의변화양상을검토하기위하여분산분석 (analysis of variance: ANOVA) 을이용하였고, F 값이의의가있을때에는각군간의차이를 Fisher s least significant difference 방법으로비교하였다. P 값이.5 이하일때를통계적으로유의한차이로정의하였다. 9

4 HJ Ha, et al.: ROS and lung fibrosis 결과 1. TGF-β1이 MRC-5 세포의 fibronectin 분비와 α-sma 표현에미치는영향 TGF-β1 을첨가하고 8시간 (Figure 1A) 또는 9시간 (Figure 1B) 후배양액으로분비된 fibronectin을 Western blot 분석으로측정하였다. TGF-β1 은 MRC-5 세포의 fibronectin 분비를용량의존적으로증가시켰다. 혈청배제 DMEM 에서배양한 MRC-5 세포는 α- SMA 단백을미량표현하였으며 (Figures 1C와 D), TGF-β1 자극후 8시간 (Figure 1C) 또는 9시간 (Figure 1D) 후 α-sma 단백표현은 TGF-β1 에의하여용량의존적으로증가하였다.. N-acaetylcystein (NAC) 또는 diphenylene iodonium (DPI) 이 TGF-β1 에의한 fibronectin 분비와 α-sma 표현에미치는영향 MRC-5 세포의 TGF-β1 에의한 fibronectin 분비와 α-sma 단백표현은 ng/ml 이상에서 8 시간과 9 시간모두에서대조군과비교하여통계적으로의미있게증가하였으므로항산화제 NAC와 NADPH oxidase 억제제 DPI 의영향을관찰하기위하여 ng/ml 의 TGF- A. FN B. FN C. D. α-sma α-sma β-actin β-actin Figure 1. Effect of TGF-β1 on fibronectin secretion (A, B) and α-sma expression (C, D) in MRC-5 cells. After incubating the quiescent MRC-5 cells with increasing TGF-β1 concentrations for 8 (A, C) or 9 (B, D) hours, aliquots of the conditioned media (A, B) or cell lysate (C, D) containing the same concentrations of proteins were electrophoresed under reducing conditions, and Western blots were performed as described in the text. The upper panel shows a representative Western blot and the lower panel represents the relative change as the mean ± SE of 3 experiments. P<.5 compared with the control 7

5 Tuberculosis and Respiratory Diseases Vol. 58. No. 3, Mar, 5 β1으로 8 시간동안자극하였다. NAC는 과 3 mm 에서 TGF-β1 에의한 fibronectin 분비를의미있게억제하였고 (Figure A), 3 mm에서 α-sma 표현을의미있게억제하였다 (Figure B). DPI는 1과 5 μm에서 TGF-β1 에의한 fibronectin 분비 (Figure 3A) 를, 5 μm 에서 α-sma 표현 (Figure 3B) 을의미있게억제하였다. NAC는 3 mm까지그리고 DPI는 1 μm까지 MRC- 5세포의 LDH 분비에영향을주지않았다 (Figure ). A. B. α-sma FN NAC, mm NAC, mm β-actin NAC, mm NAC, mm Figure. Effect of N-acetyl cysteine (NAC) on the TGF-β1-induced fibronectin secretion (A) and α-sma expression (B) in MRC-5 cells. After incubating the quiescent MRC-5 cells for 8 hours under TGF-β1 in the presence or absence of NAC, aliquots of the conditioned media (A) or the cell lysate (B) containing the same concentrations of proteins were electrophoresed under reducing conditions, and Western blots were performed as described in the text. Values are expressed as the mean ± SE of experiments. P<.5 compared with the control, +P<.5 compared with the TGF-β1 control A. B. α-sma FN β-actin DPI, μm DPI, μm DPI, μm DPI, μm Figure 3. Effect of diphenyleniodonium (DPI) on TGF-β1-induced fibronectin secretion (A) and α-sma expression (B) in MRC-5 cells. After incubating the quiescent MRC-5 cells for 8 hours under TGF-β1 in the presence or absence of DPI, aliquots of the conditioned media (A) or the cell lysate (B) containing the same concentrations of proteins were electrophoresed under reducing conditions, and Western blots were performed as described in the text. Values are expressed as the mean ± SE of experiments. P<.5 compared with the control, +P<.5 compared with the TGF-β1 control 71

6 HJ Ha, et al.: ROS and lung fibrosis A. B. (%) (%) mm μm NAC DPI Figure. Effect of N-acetyl cystein (NAC) (A) or dipheyleniodonium (DPI) (B) on LDH release by MRC-5 cells. After incubating the quiescent MRC-5 cells for 8 hours with NAC (A) or DPI (B), the level of LDH released was measured as described in the text. NAC: N-acetylcysteine, DPI: diphenyleniodonium. The values are expressed as a mean ± SE of 3 experiments 3. TGF-β1 이 MRC-5 세포의세포내 ROS에미치는영향 MRC-5 세포내의 DCF에민감한 ROS는 TGF-β1 투여후 3분에최대에도달하였다 ( 자료제시않음 ). TGF-β1 에의하여세포내 ROS는의미있게증가하였고, NAC와 DPI는 TGF-β1 에의한세포내 ROS 증가를의미있게억제하였다 (Figure 5). 고찰 본연구의결과는사람폐섬유아세포인 MRC-5 세 Figure 5. Effect of N-acetyl cystein (NAC) or diphey leniodonium (DPI) on TGF-β1-induced DCF-sensitive cellular ROS in MRC-5 cells. After incubating the quiescent MRC-5 cells for 3 minutes under TGF-β1, the level of DCF-sensitive cellular ROS were measured as described in the text. Control: culture media without TGF-β1, TGF-β1: ng/ml TGF-β1 without inhibitors, NAC: N-acetylcysteine 5 mm, DPI: diphenyleniodonium.5μm. Values are ex pressed as the mean ± SE of experiments. P<.5 compared with the control, +P<.5 compared with the TGF-β1 control. 포에서 TGF-β1 이세포내 ROS를증가시키는동시에 fibronectin의분비와 α-sma 표현을증가시키고항산화제인 NAC와 NADPH oxidase 억제제인 DPI가 TGF-β1 에의한세포내 ROS의증가억제와동시에 fibronecin 분비와 α-sma 표현의상향조절을억제함을관찰함으로써 NADPH oxidase에의하여생산된 ROS가 TGF-β1 에의한폐섬유화의발생과진행에관여함을증명하였다. Hagiwara 등 9 은 NAC가 ble omycin에의한폐섬유화를억제하는것을관찰하여 ROS가폐섬유화에관여함을시사하였다. TGF-β1에의한 fibronectin 분비상향조절은신장사구체혈관간세포 31 와세뇨관상피세포 8 에서보고되었고, TGF-β1 에의한 α-sma 표현의상향조절은신장세뇨관상피세포 13,15,8 와폐섬유아세포 1 에서보고되었다. 본연구의결과는 TGF-β1 이폐섬유아세포에서 collagen I 뿐아니라 fibronectin의분비도상향조절함으로써 ECM 축적에관여하고근섬유아세포 7

7 Tuberculosis and Respiratory Diseases Vol. 58. No. 3, Mar, 5 로의표현형변화를유도하는 α-sma 표현을상향조절함으로써폐섬유화의발생과진행에중요한역할을함을확인하였다. TGF-β1 이 ROS 를증가시킴은잘알려져있다. MRC- 5 폐섬유아세포에서 TGF-β1이세포내 ROS를증가시킨본연구의결과는사람의폐섬유아세포인 HRF, 신장의사구체혈관간세포,7 및세뇨관상피세포 8 의결과와일치한다. 그러나폐섬유아세포에서세포내 ROS를증가시키는성장인자인혈소판유래성장인자 (platelet-derived growth factor: PDGF) 나섬유아세포성장인자 (fibroblast growth factor: FGF) 와달리 TGF-β1 은세포외로의 ROS 분비를증가시키고 3 ECM 단백의 tyrosine 잔기를산화시켜교차결합을증가시킴으로써폐섬유화에관여할수있음이보고 33 된바있다. 본연구의목표는신호전달자로서의세포내 ROS의역할검색임으로세포내 ROS를측정하였다. MRC-5 세포에서 NADPH oxidase 억제제인 DPI가 TGF-β1에의한세포내 ROS, fibronectin 분비및 α-sma 표현의상향조절을억제함은 TGF-β 1에의한세포내 ROS 증가에는 NADPH oxidase가관여함을시사한다. NADPH oxidase는흰쥐의간세포, 신장사구체혈관간세포,7 및세뇨관상피세포 7,8 에서도 TGF-β1에의한세포내 ROS 생산에주된역할을한다. 그러나세뇨관상피세포 8 에서는 mitochondria 의전자전달계도 TGF-β1 에의한 ROS 생산에관여함으로 MRC-5 세포에서도 mitochondria 의전자전달계가관여하는지를검색하여야할것이다. TGF-β1에의한세포활성화에는 Smad 경로 15, MAPK 경로 1-18 뿐아니라 ROS가세포내신호전달자로관여한다 19-. MRC-5 세포에서 TGF-β1 에의한 fibronectin 분비와 α-sma 표현증가에 ROS가관여함은신장세포의결과 -8 와일치한다. 흰쥐폐에서분리한섬유아세포에서 TGF-β1 에의한 α-sma 표현증가에 Smad 3 단백활성화가관여한다 1 고보고된바있다. TGF-β1에의한세포내신호전달에 Smad 경로와 MAPK 경로사이의 cross-talk 3,35 이있다는것과 ROS가 MAPK 의상위신호전달자임 7 은알려져있다. 폐섬유아세포에서 ROS에의한 fibronectin 분 비와 α-sma 표현의상향조절에관여하는정확한신호전달계는추후연구에서규명되어야할것이다. 결론적으로, 본연구는폐섬유아세포에서 TGF-β1 에의한 α-sma의표현과 fibronectin 분비의증가에 ROS가관여하는것을증명함으로써 TGF-β1 에의한폐섬유화의발생과진행에있어서 ROS가중요한역할을담당한다는증거를제시하였다. 요약연구배경 : 전환성장인자-β1(transforming growth factor-β1: TGF-β1) 은폐섬유화를매개하는주된인자이지만 TGF-β1 에의한폐섬유화의발생과진행기전의이해는아직불완전하다. TGF-β1 은다양한세포에서활성산소족 (reactive oxygen species: ROS) 을통하여세포내신호를전달하고 α-smooth muscle actin (α- SMA) 의신생합성을통하여상피세포와폐섬유아세포를근섬유아세포표현형으로의변화를유도하는주된인자이다. ROS는또다양한세포에서세포외기질 (extracellular matrix: ECM) 축적을유발하는것이알려져있음으로본연구에서는폐섬유아세포인 MRC-5 세포에서 TGF-β1 이 ROS 를매개하여 fibronectin 분비와 α-sma 표현의증가에관여하는지를검색하였다. 방법 : 성장이동일화된 MRC-5 세포를 TGF-β1 (.-1 ng/ml) 으로 9 시간까지자극하였고, 필요에따라항산화제인 N-acetylcysteine (NAC) 이나 NADPH oxidase 억제제인 diphenyleniodonium (DPI) 을 TGF-β1 투여 1 시간전부터전처리하였다. Dichlorofluorescein (DCF) 에민감한세포내 ROS는 FACS 로, 분비된 fibronectin과세포의 α-sma 표현은 Western blot 분석으로측정하였다. 결과 : TGF-β1은용량의존적으로 fibronectin 분비와 α- SMA 표현을상향조절하였다. NAC와 DPI는 TGF-β1 에의한 fibronectin 분비증가와 α-sma 상향조절을유의하게억제하였다. TGF-β1 에의한세포내 ROS의 73

8 HJ Ha, et al.: ROS and lung fibrosis 증가도 NAC나 DPI에의하여유의하게억제되었다. 결론 : 본연구의결과는폐섬유아세포에서 NADPH oxidase 에의하여생산된 ROS가 TGF-β1 에의한 fibronectin 분비와 α-sma 표현을상향조절함으로써폐섬유화의발생과진행에관여할수있음을증명하였다. 참고문헌 1. Phan SH. The myofibroblast in pulmonary fibrosis. Chest ;1:8S-9S.. Adler KB, Low RB, Leslie KO, Mitchell J, Evans JN. Contratile cells in normal and fibrotic lung. Lab Invest 1989;: Mitchell J, Woodcock-Mitchell J, Reynolds S, Low R, Leslie K, Adler K, et al. α-smooth muscle actin in parenchymal cells of bleomycin-injected rat lung. Lab Invest 1989;:3-5.. Kuhn C, McDonald JA. The role of myofibroblast in idiopathic pulmonary fibrosis: ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis. Am J Pathol 1991;138: Pache JC, Christakos PG, Gannon DE, Mitchell JJ, Low RB, Leslie KO. Myofibroblasts in diffuse alveolar damage of the lung. Mod Pathol 1998;11:1-7.. Zhang K, Rekhter MD, Gordon D, Phan SH. Myo fibroblasts and their role in lung collagen gene ex pression during pulmonary fibrosis: a combined im munohistochemical and in situ hybridization study. Am J Pathol 199;15: Zhang K, Flanders KC, Phan SH. Cellular localization of transforming growth factor β expression in bleo mycin-induced pulmonary fibrosis. Am J Pathol 1995; 17: Zhang HY, Gharaee-Kermani M, Zhang K, Karmiol S, Phan SH. Lung fibroblast α-smooth muscle actin expression and contractile phenotype in bleomycininduced pulmonary fibrosis. Am J Pathol 199;18: Zhang K, Gharaee-Kermani M, Jones ML Warren JS, Phan SH. Lung monocyte chemoattractant protein-1 gene expression in bleomycin-induced pulmonary fibrosis. J Immunol 199;153: Coker RK, Laurent GJ, Shahzeidi S, Hernandez- Rodriguez NA, Pantelidis P, du Bois RM, et al. Diverse cellular TGF-β1 and TGF-β3 gene expression in normal human and murine lung. Eur Respir J 199;9: Eickelberg O, Kohler E, Reichenberger F, Bertschin S, Woodtli T, Erne P, et al. Extracellular matrix deposition by primary human lung fibroblasts in response to TGF-β1 and TGF-β3. Am J Physiol 1999; 7:L Border W, Noble NA. Transforming growth factorbeta in tissue fibrosis. N Engl J Med 199;331: Yang J, Liu Y. Dissection of key events in tubular epithelial to myofibroblast transition and its implications in renal interstitial fibrosis. Am J Pathol 1;159: Hu B, Wu Z, Phan SH. Smad3 mediates transforming growth factor-β-induced α-smooth muscle actin ex pression. Am J Respir Cell Mol Biol 3;9: Li JH, Zhu HJ, Huang XR, Lai KN, Johnson RJ, Lam HY. Smad7 inhibits fibrotic effect of TGF-β on renal tubular epithelial cells by blocking smad activation. J Am Soc Nephrol ;13: Hartsough MT, Mulder KM. Transforming growth factor beta activation of pmapk in proliferating cultures of epithelial cells. J Biol Chem 1995;7: Zavadil J, Bitzer M, Liang D, Yang YC, Massimi A, Kneitz S, et al. Genetic programs of epithelial cell plasticity directed by transforming growth factor-beta. Proc Natl Acad Sci U S A 1;98: Ellenrieder V, Hendler SF, Boeck W, Seufferlein T, Menke A, Ruhland C, et al. Transforming growth factor-β1 treatment leads to an epithelial-mesenchymal transdifferentiation of pancreatic cancer cells requiring extracellular signal-regulated kinase activation. Cancer Res 1;1: Janda E, Lehmann K, Killisch I, Jechlinger M, Herzig M, Downward J, et al. Ras and TGF-β cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathway. J Cell Biol ;15: Lafon C, Mathieu C, Guerrin M, Pierre O, Vidal S, Valette A. Transforming growth factor-β1-induced apoptosis in human ovarian carcinoma cells: protection by the antioxidant N-acetylcysteine and bcl-. Cell Growth Differ 199;7: Hong YH, Peng HB, la Fata V, Liao JK. Hydrogen peroxide-mediated transcriptional induction of ma crophage colony-stimulation factor by TGF-β1. J Immunol 1997;159: Junn E, Lee KN, Ju HR, Han SH, Im JY, Kang HS, et al. Requirement of hydrogen peroxide generation in TGF-β1 signal transduction in human lung fibr oblast cells: involvement of hydrogen peroxide and Ca + in TGF-β1-induced IL- expression. J Immunol 7

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Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee University College of Medicine & Hospital E mail : ycell2@yahoo.co.kr Abstract