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담배연기에의한 Muc5ac 유전자발현에관여하는세포내신호전달경로로서의 ERK1/2 와 p38 MAPK 가톨릭대학교의과대학내과학교실, 가톨릭대학교의과대학성모병원호흡기질환연구실 1 김용현, 윤형규, 김치홍, 안중현, 권순석, 김영균, 김관형, 문화식, 박성학, 송정섭, 조경숙 1 Muc5ac Gene Expression Induced by Cigarette Smoke is Mediated Via a Pathway Involving ERK1/2 and p38 MAPK Yong Hyun Kim, M.D., Hyoung Kyu Yoon, M.D., Chi Hong Kim, M.D., Joong Hyun Ahn, M.D., Soon Seog Kwon, M.D., Young Kyoon Kim, M.D., Kwan Hyoung Kim, M.D., Hwa Sik Moon, M.D., Sung Hak Park, M.D., Jeong Sup Song, M.D., Kyung Sook Cho 1 Division of Pulmonology, Department of Internal Medicine and 1 Institute of Respiratory Disease, St. Mary s Hospital, College of Medicine, the Catholic University of Korea, Seoul, Korea Object : Cigarette smoking is a major cause of mucus hypersecretion, which is a pathophysiological feature of many inflammatory airway diseases. Mucins, which are an important part of the airway mucus, are synthesized from the Muc gene in airway epithelial cells. However, the signaling pathways for cigarette smoke-induced mucin synthesis are unknown. The aim of this study was to determine the signal pathway for smoking induced Muc5ac gene expression. Methods : A549 cells were cultured and transiently transfected with the Muc5ac promoter fragment. These cells were stimulated with 5% cigarette smoke extract (CSE) alone or with CSE after a pretreatment with various signal transduction pathway inhibitors (AG1478, PD98059 and SB203580). The Muc5ac promoter activity was examined using the luciferase reporter system, and the level of phosphorylated EGFR, ERK1/2, p38 MAPK and JNK were all examined using Western blot analysis. Muc5ac mrna expression was also examined using reverse transcriptase polymerase chain reactions (RT-PCR). Results : 1. The peak level of luciferase activity of the Muc5ac promoter was observed at 5% concentration and after 3 hours of incubation with the CSE. The level of EGFR phosphorylation and the luciferase activity of the transfected cells caused by the CSE were significantly suppressed by AG1478 or PD98059 (P<0.01). 2. CSE phosphorylated ERK1/2 or p38 MAPK but not JNK. The Muc5ac mrna expression level was increased by the CSE but that was suppressed by PD98059 or AG1478. 3. The CSE-induced phosphorylation of ERK1/2 was blocked by PD98059 and that of p38 MAPK was blocked by either PD98059 or SB203580. Either PD98059 or SB203580 suppressed the luciferase activity of the transfected cells (P<0.0001). Conclusion : The Muc5ac mrna expression level was increased by the CSE. The increased CSE-induced transcriptional activity was mediated via EGF receptor activation, which led to ERK1/2 and p38 MAPK phosphorylation. (Tuberc Respir Dis 2005; 58: 590-599) Key words : Cigarette smoke, MUC5AC, MAPK, Signal transduction 서 흡연은오래전부터만성폐쇄성폐질환 (Chronic ob 론 Address for correspondence : Jeong Sup Song, M.D. 150-713 Division of Pulmonology, Department of Internal Medicine and Institute of Respiratory Disease, St. Mary s Hospital, College of Medicine, the Catholic University of Korea, Seoul, Korea Phone : 02-3779-1146 Fax : 02-780-3132 E-mail : jssong@catholic.ac.kr Received : Feb. 21. 2005 Accepted : May. 19. 2005 structive lung disease) 과죽상경화증의주요위험인자 1-4 로잘알려져있다. 또흡연은만성폐쇄성폐질환환자에서뿐만아니라건강한사람에서도기도내점액분비를증가시킨다. 특히만성폐쇄성폐질환의주요임상양상인점액의과다분비는이질환의중요한병태생리학적소견이며과도하게분비된점액은하부기도의폐쇄를일으켜호흡곤란과나아가호흡부전을야기하는중요한원인이된다 5. 기도점액은기도상피를덮고있으며수분의증발을막고병균이나화학적자극물질들로부터상피를보호하는역할을하는데, 590

Tuberculosis and Respiratory Diseases Vol. 58. No. 6, Jun. 2005 이는대기도상피에존재하는배상세포와점막하분비선에존재하는점액세포와장액세포에서분비된다 6-8. 점액은 95% 의물과소량의지방, 단백질및특정유전자에의해만들어지는당단백질등으로구성되어있으며 6 어떤이유에의해과다분비가일어나면문제를일으킨다. 점액의과다분비는기도내배상세포의증식및화생 (metaplasia) 때문이다 9. 점액의과다분비를일으키는원인은매우다양한데이중한가지가흡연이다. 담배연기내에는대략 4000 여가지의다양한물질들이기체혹은결정의형태로존재하며 nitrogen oxide, hydrogen peroxide, hydrogen cyanide, acrolein 등의다양한산화제를포함하고있다 10. 이들산화제는만성폐쇄성폐질환, 기관지확장증, 낭성섬유증과같은기도내염증질환의병태생리에중요한역할을한다고알려져있다. 기도내의점액분비를유발하고조절시키는기전은아직까지정확하게알려져있지않지만점액의주성분인점액당단백질을합성하는데관여하는기도상피세포내의점액유전자, 즉 Muc 유전자가있음이알려져있다. 현재까지인체에서는 15종류의 Muc 유전자가존재한다 11. 그중에서도 Muc5ac 유전자에의해합성되는당단백은정상인과기관지천식환자에서기도분비액의주된성분으로알려져있고 12 기도내배상세포증식및화생과가장관련성이있다고보고되었다 13. 최근이 Muc 유전자의발현과정에대한여러연구에서유전자를활성화시키는세포내신호전달과정에표피성장인자 (epidermal growth factor) 수용체가관여함이밝혀졌다 14,15. 하지만, 표피성장인자수용체활성화이후의세포내신호전달과정에대해서는확실하게규명이되어있지않고일부의연구들에서이신호전달과정에 mitogen-activated protein kinase (MAPK) 가관련되어있음을최근보고하였다 16-18. 저자들은흡연에의노출이점액유전자의발현을활성화시키고이유전자의발현에어떠한 MAPK가신호전달과정에관여하는지알아보고자기도상피세포의특성을가지고있는폐선암세포주인 A549세포를담배연기추출물에노출시켜실험하였다. 방법 1. 세포배양사람의폐선암세포주인A549 세포 (KCLB, 한국세포주은행, Korea) 를 10% 우태아혈청 (Glbco-BRL, California, USA), penicillin (Glbco-BRL) 100 U/mL, streptomycin (Glbco-BRL) 100 μg /ml, HEPES 640 (Sigma, St. Louis, USA) 25 mmol 배지에서 37, 5% CO 2 의배양기에서배양하였다. 2. 담배연기추출용액 (cigarette smoke extract solution) 25 ml의 RPMI1640 medium (10% 우태아혈청, HEPES 50 mmol without penicillin and streptomy cin) 내로 one puff/min 의비율로 5분동안담배연기를모았다. ph 7.4가되도록적정을하고, 0.22 μm filter unit (Millipore, Bedford) 을사용하여여과를하였다. 이때만들어진담배연기추출용액은사용하기 30분전에만들어썼다. 또한이용액은동일한배지로 5% 혹은 10% 로희석하여세포에처리하였다. 3. Transient transfection과세포자극 A549 세포를 6-well 조직배양용기 (Nunc, Roskilde, Denmark) 에 1 10 5 세포 /ml 의양으로 2mL씩접종후 48시간동안배양하여, 용기의 80% 정도채워자랐을때 Muc5ac 의 transfection 실험을실시하였다. Trans fection을수행할 PGL3-Muc5ac-3752pro luciferase reporter plasmid와 PGL3-basic vector는실험을수행하기전에각각 200 ng/ μl로정량화하였고, 대조군으로사용할 β-galactosidase의양은 100 ng/ μl로정량화하였다. Transfection 전에세포는혈청이없는배지로세척을하고각용기당 800μl의혈청이없는배지를넣어미리적응을시켰다. 한튜브에는혈청이없는배지 300μl, PGL3-Muc5ac-3752pro luciferase reporter plasmid 5μl, plus reagent (Gibco-BRL) 5μl, β-galactosidase 3μl를혼합하여준비하고다른한튜 591

YH Kim, et al.: The signal transduction pathway for the Muc5ac gene expression 브에는혈청이없는배지 300μl, LIPOFECTAMINETM reagent (Gibco-BRL) 4μl를넣은후각각의튜브를상온에서 15분간반응시킨후, 두개의튜브를잘섞어서상온에서 15분간반응을시켜복합체를형성하도록하였다. 준비된복합체를미리적응시킨배양용기에 200μl씩골고루넣은후, 20% FBS를 1 ml을배양용기에첨가하여 24시간동안배양하였다. Trans fection한세포주를혈청이없는배지에 24시간배양한후, 5% 담배연기추출용액과 mitogen-activated protein kinase kinase (MAPKK) 의억제제인 PD98059 40 mmol (Sigma), epidermal growth factor receptor (EGFR) kinase의억제제인 AG1478 (Calbiochem, Bad Soden, Germany) 50 μmol, p38 MAPK 억제제인 SB203580 (Calbiochem) 50 μmol을넣어 30분간전처리한후, 다시 5% 담배연기추출용액으로 3시간자극하였다. phosphate buffer solution (PBS) 으로 2번세척하고 reporter lysis buffer (Promega, Madison, USA) 를넣고상온에서 20 분간반응시켜 12,000 rpm에서 15 초간원심분리한후, 세포단백질을얻었다. 4. Luciferase receptor assay 얻어진세포단백질을 20μl으로정량한후 lucife rase 활성화정도를측정하기위해서세포추출단백질에 100μl의 luciferase assay substrate (Promega) 를넣어반응시킨후, luminometer (Turner Designs, Turku, Finland) 를이용하여활성화정도를측정하였다. 또한각각의활성도는 β-galactosidase를이용하여보정하였다. 5. 역전사효소연쇄중합반응 (Reverse transcription polymerase chain reaction) A549 세포가배양용기의 80% 정도채워자랐을때, transient transfection 실험과동일한조건으로 Tri- ZolTM reagent (Invitrogen, California, USA) 500 μl로상온에서 5분간반응시키고제조자가제시한방법으로 RNA를추출하였다. 추출한 RNA는 spectropho tometer (Bio-RAD, California, USA) 를이용해 1 μg / μl 로정량화하였고, 여기에서 1 μl를채취하여역전사효소 (SUPERSCRIPT Ⅲ, Invitrogen) 0.5μl로 BioNeer Accupower PCR premix (BioNeer, Seoul, Korea) 를이용하여역전사효소연쇄중합반응을수행하였다. 활성화정도는 GAPDH 를사용하여보정하였으며사용한 Muc5ac 및 GAPDH 의 primer 염기서열은다음과같다. Muc5ac ;(F) 5 TCCGGCCTCATCTTCTCC 3 (R) 5 ACTTGGGCACTGGTGCTG 3 GAPDH ; (F) 5 ACCACAGTCCATGCCATCAC 3 (R) 5 TCCACCACCCTGTTGCTGTA 3 6. Western blot A549 세포를위에서언급한방법으로처리하여배양하고 Bradford assay (Bio-RAD) 방법으로정량하여세포단백질을준비하였다. 준비된세포단백질 30 μg을 10% SDS-polyacrylamide gel에서 120V에서약 1시간 30분간전기영동을하였다. 이어 PVDF mem brane (Bio-RAD) 으로 4, 250mA에서 1시간 30분간전기영동하여단백질을전이시키고차단용액으로차단시켰다. Phosphorylated EGFR (P-EGFR/EGFR), phosphorylated extracellular signal-related kinase (P-ERK/ERK), phosphorylated p38 MAPK (P-p38 MAPK/p38 MAPK), phosphorylated c-jun N-ter minal kinase (P-JNK/JNK) 에대한일차항체 (rabbit anti-human, Santacruz, California & Cell signaling, Beverly, MA, USA) 로 4 에서밤새반응시키고세척후이차항체 (anti-rabbit IgG HRP-linked antibody 또는 anti-mouse IgG HRP-linked antibody, Cell si gnaling) 로 1시간 30분동안반응시켰다. 세척후, ECLTM reagent (Amersham-Pharmacia Biotech, Seoul, Korea) 를이용하여제조자가제시한방법으로반응시킨후, X-ray 필름에감광시키고자동현상기를이용하여현상하였다. 7. 통계방법통계적검정은 SPSS 프로그램을사용하여 one-way 592

Tuberculosis and Respiratory Diseases Vol. 58. No. 6, Jun. 2005 ANOVA와 independent sample T-test 를사용하였으며 P 값이 0.05 이하인경우를통계적유의성이있는것으로하였다. 결과 1. 담배연기추출물자극에의한 Muc5ac 유전자전사활성도의증가및표피성장인자수용체의인산화 자극시가장높은활성도를보였다 ( 그림 2). Western blot을이용하여표피성장인자수용체의인산화, 즉활성화된수용체를본결과대조군에비해담배연기추출물자극에의해수용체의인산화가증가되었고이는수용체인산화를억제하는 AG1478에의해억제되었다 ( 그림 3). 담배연기추출물의농도를달리하여 A549 세포를자극하였을때 5% 담배연기추출용액일때가장높은 luciferase 활성도를보여이농도에서 Muc5ac pro moter 의전사가가장증가됨을알수있어이후세포자극시 5% 담배연기추출용액을사용하였다 ( 그림 1). 자극시간에따른활성도는일반적으로자극시간이길수록활성도가더증가하는경향을보였으며 3시간 Figure 2. Effect of incubation time on the CSE-induced Muc5ac transcriptional activity. Transcriptional activity of Muc5ac promoter was the highest when cells were exposed to 5% CSE for three hours (PGL3-basic: PGL3-basic vector without Muc5ac promoter region, * P<0.01). Figure 1. Effect of cigarette smoke extract solution (CSE) concentration on the transcriptional activity of Muc5ac promoter. MUC5AC reporter plasmid was transfected into A549 cells and transfected cells were treated with CSE for one hour. The transcriptional activity of Muc5ac promoter was the highest when the transfected cells were exposed to 5% CSE. Luciferase activity was expressed as a relative value to a PGL3-basic value (PGL3-basic: PGL3-basic vector without Muc5ac promoter region, CTL: control/muc5ac promoter transfected, CSE: cigarette smoke extract solution, * P<0.01). Figure 3. Western blot analysis of epidermal growth factor receptor (EGFR) and phosphorylated epidermal growth factor receptor (P-EGFR). Western blot analy sis was performed under conditions as described in methods. A549 cells were incubated with 5% CSE for three hours. EGFR phosphorylation was increased by CSE and decreased by EGFR tyrosine kinase inhibitor, AG1478 or mitogen-activated protein kinase kinase inhibitor, PD98059 pretreatment (M: marker, CTL: control/ MUC5AC promoter transfected, 5% CSE: 5% cigarette smoke extract solution) 593

YH Kim, et al.: The signal transduction pathway for the Muc5ac gene expression 2. 세포내신호전달경로로써 MAPK와 Muc5ac 유전자의전사발현 Muc5ac 유전자의전사과정에서세포내신호를전달하는데표피성장인자수용체가관여하며그하위신호전달과정에 MAPK, 특히 ERK1/2 가관계있음이알려져있어저자들은표피성장인자수용체의 tyr osine kinase 억제제인 AG1478, MAPK kinase 억제제인 PD98059를사용하여 Muc5ac 유전자의전사가억제되는지 luciferase 활성도를통하여관찰하였다. 담배연기추출물자극시증가된 Muc5ac 유전자의전사는이들억제제에의하여모두억제되었다 ( 그림 4). Western blot을이용하여 3가지 MAPK 의활성화된형태, 즉인산화된 kinase 활성도를본결과담배연기추출물의자극에서인산화된 ERK1/2 와 p38 MAPK 의활성화는관찰되었으나 JNK의활성화는볼수없었다. 또표피성장인자수용체의인산화를억제하는 AG1478에의해 ERK 1/2와 p38 MAPK 활성도는예상대로억제되었고 MAPK kinase 억제제인 PD98059 처리시 ERK 1/2와 p38 MAPK 활성도역시감소하였다 ( 그림 5). 이를다시확인하기위하여역전사효소중 합연쇄반응을이용하여 Muc5ac mrna 발현을관찰해보니담배연기추출물자극시 Muc5ac mrna 발현이증가되었고 AG1478과 PD98059에의해 Muc5ac mrna 발현은억제되었다 ( 그림 6). PGL3-basic + - - - - MUC5AC - + + + + 5% CSE - - + + + PD98059 - - - + - AG1478 - - - - + Figure 4. The effect of cell signal transduction inhibi tors on luciferase activity. Mitogen-activated protein kinase kinase inhibitor, PD98059 and EGFR tyrosine kinase inhibitor, AG1478 suppressed the CSE-induced luciferase activity (PGL3-basic: PGL3-basic vector with out Muc5ac promoter region, MUC5AC: Muc5ac pro moter transfected, 5% CSE: 5% cigarette smoke extract solution, * P<0.01). Figure 5. Effect of CSE and signal transduction in hibitors on the phosphorylation of ERK, p38 MAPK and JNK. Western blot analysis was performed to as sess the phosphorylation of mitogen-activated protein kinases under same conditions above described. CSE phosphorylated both ERKs and p38 MAPK but not JNK. Phosphorylations of ERKs and p38 MAPK were inhibited by PD98059 or AG1478 (M: marker, CTL: control, 5% CSE: 5% cigarette smoke extract solution, MAPK: mitogen-activated protein kinase, P-ERK: phosphorylated extracellular signal-related kinase, P-JNK: phosphorylated c-jun N-terminal kinase). 594

Tuberculosis and Respiratory Diseases Vol. 58. No. 6, Jun. 2005 Figure 6. Reverse transcription polymerase chain re action (RT-PCR) analysis of Muc5ac mrna expression in A549 cells. PCR reaction was run on a 1% agarose gel and the band density was expressed as a relative value using densitometry measurement. CSE increased the Muc5ac mrna expression. Pretreatment of PD98059 or AG1478 suppressed the Muc5ac expression near to the control levels (CTL: control, 5% CSE: 5% cigar ette smoke extract solution). Figure 7. Western blot analysis of ERK and p38 MAPK. PD98059 inhibited phosphorylation of both ERKs and p38 MAPK. SB203580 also inhibited p38 MAPK phosphorylation as much as to the level of control and CSE treatment group (CTL: control, CSE: 5% cigarette smoke extract solution). 3. SB203580 처리시세포내신호전달경로와 Muc5ac 유전자발현 담배연기추출물에의한 Muc5ac 유전자발현에관여하는 MAPK 중본실험에서 ERK1/2 와 p38 MAPK 의인산화가확인되어 p38 MAPK의선택적억제제인 SB203580으로처리한후 p38 MAPK 인산화와 Muc5ac promoter 활성도를관찰하였다. PD98059와 SB203580 은예상대로 p38 MAPK 인산화를억제하였으며 ( 그림 7) SB203580은PD98059에비하여훨씬강하게 lu ciferase 활성도를감소시켰다 ( 그림 8). 고찰기도내기류제한과반복적인기도염증으로요약되는만성폐쇄성폐질환에서기도내점액분비의증가는 Figure 8. The effect of cell signal pathway inhibitors on luciferase activity. PD98059 and SB203580 signi ficantly suppressed CSE-induced luciferase activity, respectively (PGL3-basic: PGL3-basic vector without Muc5ac promoter region, CSE: 5% cigarette smoke extract solution, * P< 0.0001). 특징적인임상양상이다. 만성폐쇄성질환의원인은여러가지가알려져있으나그중흡연이가장중요한원인이며이는기도내점막의염증을일으키고기도내점액분비증가에기여한다 2,19. 기도점액의주성분은점액소 (mucin) 라는당단백질복합체로써구조의뼈대를이루는여러단백질이각각의특이유전자에 595

YH Kim, et al.: The signal transduction pathway for the Muc5ac gene expression 의해만들어짐이최근밝혀져있는데현재까지모두 15개의점액유전자 (Muc gene) 가알려져있다. 점액소는막결합성점액소와분비형점액소로나누며이중폐혹은기도내에존재하는분비형점액소는 Muc2, Muc5ac, Muc5B, Muc7 그리고 Muc8이고 11 이중 Muc5ac 가흡연에의해분비가유도됨이알려졌다 20. 하지만, 이러한점액유전자가어떠한세포내신호전달과정을통하여점액분비를증가시키는지에대해서는명확하게밝혀진바가없으나최근호중구의 ela stase, 흡연, 금속, 세균감염, 산화제의자극등에의하여표피성장인자수용체가활성화되고이어점액유전자의전사가일어난다는사실이알려졌다 17,21-23. 본실험에서도담배연기로처리하였을때 A549 세포의 Muc5ac mrna 발현이증가되었고표피성장인자수용체의활성화를동반하며표피성장인자수용체인산화를억제하는 AG1478 처리시 Muc5ac 유전자의발현이억제되어유전자발현에표피성장인자수용체가관여함을확인할수있었다. 표피성장인자수용체가자극에의해활성화되어세포내신호를전달하는데는 ERK1/2, JNK1/2/3, p38 MAPK 혹은 protein ki nase C를매개하는것이알려져있는데 Muc2 혹은 Muc5ac 유전자의발현에표피성장인자수용체의활성이먼저일어나고이어 ERK 신호전달체계를경유하여 NF-κB 나 SP1같은전사인자가 DNA에결합하여유전자의발현을증가시킨다는보고들이있다 16,18. 세포내신호전달에관여하는신호전달체계중 MAPK 군은 proline-targeted serine-threonine kinase에속하며외부자극을핵에전달하는역할을하는데포유동물에서는최소 4가지종류의 MAPK, 즉 ERK1/2, JNK1/2/3, p38 MAPK, ERK5가존재한다 24. 본연구자는담배연기에의한 Muc5ac 유전자의발현에표피성장인자수용체의활성화를확인하고그이하단계에서의신호전달과정에여러 MAPK 중어떤종류가관여하는지확인하였다. 담배연기자극시에 ERK1/2 와 p38 MAPK 의인산화는관찰하였으나 JNK 인산화는관찰되지않았고 MAPK 를활성화시키는 MAPK kinase (MAPKK) 억제제인 PD98059와 p38 MAPK 의선택적억제제인 SB203580으로처리시각각 ERK1/2 와 p38 MAPK 의인산화가억제되고 Muc5ac mrna 발 현이감소되었다. 이상의결과로보아담배연기에의한 Muc5ac 유전자의발현증가를일으키는세포내신호전달에는 MAPK 중 ERK1/2 와 p38 MAPK 가관여하고 JNK는관계없음을유추할수있다. 담배연기가표피성장인자수용체를활성화시키는기전은아직명확하지않다. 표피성장인자수용체의특이리간드인표피성장인자와 transforming growth factor 는직접수용체에결합하여표피성장인자수용체인산화를유도하지만 14 이외에도외부에서과산화수소와같은산화제로자극하거나활성화된호중구로자극시수용체-리간드결합과관계없는표피성장인자수용체인산화경로가보고되고있다 23. 활성화호중구역시많은종류의염증매개물을생성하여 DNA 의산화손상을일으킨다고알려져있어다양한자극에의해생성된산화물질들이리간드결합과는무관하게직접적으로표피성장인자수용체를활성화시키고 MAPK 경로를통하여점액분비를증가시킨다고추측된다. 담배연기내에도다양한산화제들이존재하므로이들산화제에의해표피성장인자수용체활성화가일어난다고생각할수있다. 하지만흡연은생체에서기도내염증을유발하여기도내세포로부터다양한염증매개물들을분비하게하는데특히호중구의동원을일으키는 IL-8과같은시토카인분비를자극함으로써먼저호중구가동원되고이차적으로산화자극에의해수용체의활성화가이루어질가능성을배제할수는없다. 그러나본실험은호중구의동원이불가능한폐암세포주에서이루어졌으며호중구의자극혹은외부에서의산화제처리에의한점액유전자발현을본이전의보고 23 와같이신호전달과정에 ERK1/2 경로는관여하나 p38 kinase 경로는무관하다고결론을내렸다. 연구자의실험결과담배연기자극으로나타난신호전달경로에는 ERK1/2 와 p38 MAPK 가모두관여하는결과를보여표피성장인자수용체인산화는호중구동원의이차적인결과이거나단순히담배연기내에포함된산화물질에만의한것이라단정할수는없다. 담배연기내에들어있는대표적물질인 acrolein 으로기도상피세포를직접자극하였을때 Muc5ac mrna 의발현증가가보고되었으며 25,26 이의기전으로 acrolein 이세포내 glutathione 을 596

Tuberculosis and Respiratory Diseases Vol. 58. No. 6, Jun. 2005 소모시켜 reactive oxygen species 를생성하여유전자발현을증가시킨다는보고도있다 4,27. 또 acrolein 은생체에서염증반응을일으켜호중구, 대식세포및단핵구의활성화를촉진하고이들세포들은다양한염증매개물을분비하는데이들염증매개물로기도상피세포를자극하였을때도역시 Muc5ac mrna의발현이증가되고단백질합성이이루어지는것으로보아 acrolein 은직접적으로 Muc5ac 유전자를활성화시키고동시에염증반응을매개하여간접적으로도 Muc5ac 유전자를자극하는것으로생각된다. 이처럼다른신호전달경로를가진자극원이담배연기에존재하거나산화물질의종류에따라각각다른신호전달경로를가질가능성이있다. 담배연기내에 4,000여가지물질들이존재하고이들의생물학적기능이나영향이완전히규명되지않았기에이러한물질들이직접적으로표피성장인자수용체를활성화시키고세포내신호를전달하는과정에서이미알려진산화자극과는다른세포내신호경로, 즉 p38 MAPK 를경유한다고생각할수있다. 요약하면담배연기추출물에의하여 A549 세포의표피성장인자수용체가활성화되고이어 MAPKK를통하여활성화되는 MAPK 중 ERK1/2 와 p38 MAPK 의경로를경유하여세포내신호가전달되어 Muc5ac 유전자의 mrna 발현을촉진하여점액분비증가가일어난다고생각한다. 요약연구배경 : 만성폐쇄성폐질환에서나타나는기도점액의과다분비는이질환의중요한병리학적소견이며호흡곤란등환자의증상을악화시키는요인중의하나이다. 기도점액을구성하는여러성분중 Muc 유전자에의해만들어지는당단백이흡연에의해생성이증가하는데이에관여하는세포내신호전달과정에대하여확실히밝혀진바가없다. 저자는 Muc 유전자중인체의기도에가장많이분비되는 Muc5ac 점액생성을담당하는 Muc5ac 유전자의발현이흡연에의하여증가하는데관여하는세포내신호전달과정을알아보고자하였다. 재료및방법 : 사람폐선암세포주인 A549 세포를배양하여 Muc5ac 유전자의 promotor를 luciferase reporter plasmid 를사용하여세포내에 transfection시키고 5% 담배연기추출물로자극하여배양하였다. 또세포내신호전달에관여하는표피성장인자수용체 kinase의억제제인 AG1478, mitogen-activated protein kinase kinase (MAPKK) 억제제인 PD98059, p38 mitogen-activated protein kinase 억제제인 SB203580으로각각전처치후역시 5% 담배연기추출물로자극배양하였다. 배양된세포에서단백질을추출하여 luciferase 분석을통하여 Muc5ac promoter 활성도를측정하고 Western blot을이용하여표피성장인자수용체와 mitogen-act ivated protein kinase (MAPK) 인 extracellular signalrelated kinase (ERK)1/2, p38 MAPK, c-jun N-terminal kinase (JNK) 의발현을확인하였다. 또세포에서 RNA 를추출한후 Muc5ac primer를이용하여역전사효소중합연쇄반응을수행하여 Muc5ac mrna 발현을관찰하였다. 결과 : 1. Muc5ac promoter를삽입한 A549 세포를 5% 담배연기추출물로자극하였을때의의있게 luciferase 활성도가증가하였고 (P<0.001) 자극하는시간이 3시간이었을때 luciferase 활성도가최고치를보였다 (P<0.01). 또담배연기추출물자극은표피성장인자수용체를인산화시켰으며인산화는 AG1478과 PD98059 에의하여억제되었다. 2. AG1478 혹은 PD98059로전처치후 5% 담배연기추출물로자극한경우 5% 담배연기추출물단독으로자극한것에비하여유의하게 lucifearse 활성도가억제되었고 (P<0.01) 세가지종류의 MAPK 중 ERK1/2 와 p38 MAPK 의인산화는관찰되었으나 JNK의인산화는관찰되지않았다. 역전사효소중합연쇄반응을이용하여관찰한 Muc5ac mrna 발현은담배연기추출물에의해증가되었고 PD98059와 AG1478에의하여역시억제되었다. 3. 담배연기추출물에의하여인산화된 ERK1/2 는 PD98059에의하여인산화가감소하였고 p38 MAPK 의인산화는 PD98059와 SB203580에의하여감소하 597

YH Kim, et al.: The signal transduction pathway for the Muc5ac gene expression 였으며이두가지억제제는모두 luciferase 활성도를유의하게억제시켰다 (P<0.0001). 결론 : 담배연기추출물은 Muc5ac 유전자의발현을증가시켜기도내점액분비를증가시키며이는표피성장인자수용체를매개로 ERK1/2 와 p38 MAPK 를경유하여세포내신호전달이이루어진다고생각된다. 따라서점액유전자활성화를매개하는신호전달과정을차단하는약제나방법이개발된다면과도한점액분비를치료할수있을것으로생각한다. 참고문헌 1. Hogg JC, Macklem PT, Thurlbeck WM. Site and nature of airway obstruction in chronic obstructive lung disease. N Engl J Med 1968;278:1355-60. 2. Higgins M. Epidemiology of COPD: state of the art. Chest 1984;85:3S-8S. 3. Holbrook JH, Grundy SM, Hennekens CH, Kannel WB, Strong JP. Cigarette smoking and cardiovascular diseases. Circulation 1984;70:1114A-7A. 4. Grafstrom RC, Dypbukt JM, Willey JC, Sundqvist K, Edman C, Atzori L, et al. Pathobiological effects of acrolein in cultured human bronchial epithelial cells. Cancer Res 1988;48:1717-21. 5. Dunnill MS. The pathology of asthma, with special reference to changes in the bronchial mucosa. J Clin Pathol 1960;13:27-33. 6. Widdicombe JG. Airway mucus. Eur Respir J 1989; 2:107-15. 7. Rogers DF. Airway goblet cells: responsive and ada ptable front-line defenders. Eur Respir J 1994;7: 1690-706. 8. Jeffery PK, Li D. Airway mucosa: secretory cells, mucus and mucin genes. Eur Respir J 1997;10:1655-62. 9. Coles SJ, Levine LR, Reid L. Hypersecretion of mucus glycoproteins in rat airways induced by tobacco smoke. Am J Pathol 1979;94:459-71. 10. Hoffmann D, Wynder EL. Chemical constituents and bioactivity of tobacco smoke. IARC Sci Publ 1986: 145-65. 11. Leikauf GD, Borchers MT, Prows DR, Simpson LG. Mucin apoprotein expression in COPD. Chest 2002;121: 166S-82S. 12. Hovenberg HW, Davies JR, Herrmann A, Linden CJ, Carlstedt I. Muc5ac, but not MUC2, is a prominent mucin in respiratory secretions. Glycoconj J 1996;13: 839-47. 13. Zuhdi Alimam M, Piazza FM, Selby DM, Letwin N, Huang L, Rose MC. Muc-5/5ac mucin messenger RNA and protein expression is a marker of goblet cell metaplasia in murine airways. Am J Respir Cell Mol Biol 2000;22:253-60. 14. Takeyama K, Dabbagh K, Lee HM, Agusti C, La usier JA, Ueki IF, et al. Epidermal growth factor system regulates mucin production in airways. Proc Natl Acad Sci U S A 1999;96:3081-6. 15. Nadel JA. Role of epidermal growth factor receptor activation in regulating mucin synthesis. Respir Res 2001;2:85-9. 16. Li JD, Feng W, Gallup M, Kim JH, Gum J, Kim Y, et al. Activation of NF-kappaB via a Src-dependent Ras-MAPK-pp90rsk pathway is required for Pse udomonas aeruginosa-induced mucin overproduction in epithelial cells. Proc Natl Acad Sci U S A 1998; 95:5718-23. 17. Kohri K, Ueki IF, Shim JJ, Burgel PR, Oh YM, Tam DC, et al. Pseudomonas aeruginosa induces Muc5ac production via epidermal growth factor receptor. Eur Respir J 2002;20:1263-70. 18. Perrais M, Pigny P, Copin MC, Aubert JP, van Se uningen I. Induction of MUC2 and Muc5ac mucins by factors of the epidermal growth factor (EGF) family is mediated by EGF receptor/ras/raf/extracellular signal-regulated kinase cascade and Sp1. J Biol Chem 2002;277:32258-67. 19. Roth MD, Arora A, Barsky SH, Kleerup EC, Simmons M, Tashkin DP. Airway inflammation in young ma rijuana and tobacco smokers. Am J Respir Crit Care Med 1998;157:928-37. 20. Takeyama K, Jung B, Shim JJ, Burgel PR, Dao- Pick T, Ueki IF, et al. Activation of epidermal growth factor receptors is responsible for mucin synthesis induced by cigarette smoke. Am J Physiol Lung Cell Mol Physiol 2001;280:L165-72. 21. Voynow JA, Young LR, Wang Y, Horger T, Rose MC, Fischer BM. Neutrophil elastase increases Muc5ac mrna and protein expression in respiratory epithelial cells. Am J Physiol 1999;276:L835-43. 22. Wu W, Graves LM, Jaspers I, Devlin RB, Reed W, Samet JM. Activation of the EGF receptor signaling pathway in human airway epithelial cells exposed to metals. Am J Physiol 1999;277:L924-31. 23. Takeyama K, Dabbagh K, Jeong Shim J, Dao-Pick T, Ueki IF, Nadel JA. Oxidative stress causes mucin synthesis via transactivation of epidermal growth fa ctor receptor: role of neutrophils. J Immunol 2000;164: 598

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