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1 J. Exp. Biomed. Sci. 2012, 18(2): 104~111 pissn : Original Article Upregulation of MMP is Mediated by MEK1 Activation During Differentiation of Monocyte into Macrophage Jaewon Lim 1,3, Yoonjung Cho 1,3, Dong Hyun Lee 1, Byung Chul Jung 1, Han Sol Kang 1, Tack-Joong Kim 2, Ki-Jong Rhee 1, Tae Ue Kim 1, and Yoon Suk Kim 1, 1 Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju , Korea 2 Division of Biological Science and Technology, College of Science and Technology, Yonsei University, Wonju , Korea Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases which degrade extracellular matrix (ECM) during embryogenesis, wound healing, and tissue remodeling. Dysregulation of MMP activity is also associated with various pathological inflammatory conditions. In this study, we examined the expression pattern of MMPs during PMA-induced differentiation of THP-1 monocytic cells into macrophages. We found that MMP1, MMP8, MMP3, MMP10, MMP12, MMP19, MMP9, and MMP7 were upregulated during differentiation whereas MMP2 remained unchanged. Expression of MMPs increased in a time-dependent manner; MMP1, MMP8, MMP3, MMP10, and MMP12 increased beginning at 60 hr post PMA treatment whereas MMP19, MMP9, and MMP7 increased beginning at 24 hr post PMA treatment. To identify signal transduction pathways involved in PMA-induced upregulation of MMPs, we treated PMA-differentiated THP-1 cells with specific inhibitors for PKC, MEK1, NF-κB, PI3K, p38 MAPK and PLC. We found that inhibition of the MEK1 pathway blocked PMA-induced upregulation of all MMPs to varying degrees except for MMP-2. In addition, expression of select MMPs was inhibited by PI3K, p38 MAPK and PLC inhibitors. In conclusion, we show that of the MMPs examined, most MMPs were up-regulated during differentiation of monocyte into macrophage via the MEK1 pathway. These results provide basic information for studying MMPs expression during macrophage differentiation. Key Words: Matrix metalloproteinase (MMP), Phorbol-12-myristate-13-acetate (PMA), Macrophage, MEK1 서 Matrix metalloproteinases (MMPs) 는아연-의존적단백분해효소로서 collagen, proteoglycan, gelatin, elastin 등다양한세포외기질 (extracellular matrix, ECM) 을분해하며, * Received: 30 May, 2012 / Revised: 7 June, 2012 Accepted: 8 June, These authors contributed equally to this work. Corresponding author: Yoon Suk Kim. Department of Biomedical Laboratory Science, College of Health Science, Yonsei University, Wonju , Korea. Tel: , yoonsukkim@yonsei.ac.kr Corresponding author: Tae Ue Kim. Department of Biomedical Laboratory Science, College of Health Science, Yonsei University, Wonju , Korea. Tel: , kimtu@yonsei.ac.kr C The Korean Society for Biomedical Laboratory Sciences. All rights reserved. 론 배발생, 기관형성또는상처치유등에관여하는것으로보고되고있다 (Brinckerhoff et al., 2002; Deryugina and Quigley, 2006; Egeblad and Werb, 2002; Pollette et al., 2004). 이러한 MMPs가비정상적으로과발현또는활성화될경우결합조직등을파괴시켜다양한염증성질환을야기하는것으로알려져있다 (Brinckerhoff and Matriain, 2002). MMPs는각종질환발달에중요한역할을하는것으로알려져있는데암의발달및전이, 관절염등의발달에 MMPs가관여된것으로보고되고있고, 동맥경화증 (atherosclerosis) 의발달에도밀접한연관이있는것으로알려져있다 (Lendon et al., 1991; Moreno et al., 1994; Vihinen and Kahari, 2002; Westermarck and Kahari, 1999). MMPs는세포외기질분해기능과더불어세포의증식 (proliferation) 및이동 (migration), 세포사멸 (apoptosis) 과도연관되어있으며면역세포에서각종사이토카인

2 (cytokine) 과그수용체 (receptor) 또는세포증식인자 (growth factor) 를활성화또는비활성화시키는데관여하여염증성반응조절에중요한역할을하는것으로알려져있다 (Hojilla et al., 2003; Parks et al., 2004). 현재까지, 사람에게서발현하는 MMPs는 23종이보고되고있으며, 기질 (substrate) 에대한특이도및단백질을구성하는 domain 구조에따라총 6개의아형그룹 (subclass group) 으로분류된다. 6개의아형그룹은각각 Collagenases (MMP-1, -8, -13), gelatinases (MMP-2, -9), stromelysins (MMP-3, -10), matrilysins (MMP-7, -26), 세포막결합 MMPs (MT-MMPs) 그리고기타 MMPs (MMP-12, -19, -20, -27) 으로구성된다. (Fanjul-Ferna ndez et al., 2010; Nagase et al., 2006; Santiago et al., 2010; Ugalde et al., 2010). MMPs를발현하는세포들은다양한세포증식인자, 사이토카인, 케모카인 (chemokines) 및세포외기질과의직접적인접촉등에의하여감작되어 MMPs를발현하며주로 mrna 전사 (transcription) 단계에서 MMPs 발현이조절된다 (Andrea et al., 2010). 이렇게발현된 MMPs는 pro-peptide domain과아연이온이촉매부위 (catalytic site) 에결합하여기질과의결합을억제하며비활성화된상태로유지되다가다양한활성유도신호에의해 pro-peptide domain이제거되면서활성부위 (active site) 가기질과결합하게되어기질을분해할수있게된다 (Nagase et al., 2006; Page-McCaw et al., 2007; Stamenkovic, 2003). 이렇게활성화된 MMPs는활성억제가필요할경우세포에존재하는 tissue inhibitor of metalloproteinases (TIMPs) 라명명되는억제단백질에의해그활성이억제된다 (Schafers et al., 2010). 이러한 MMPs는내피세포 (endothelial cell), 평활근세포 (smooth muscle cell) 뿐아니라단핵구 (monocyte), 대식세포 (macrophage) 를포함하는다양한면역세포 (immune cell) 에서발현된다 (Schafers et al., 2010). 최근연구결과에의하면동맥경화증등을포함한각종염증성질환의발달에 MMPs가관련되어있으며, 특히단핵구및대식세포에서발현하는 MMPs가이러한염증성질환발달과깊게연관되어있는것으로생각된다 (Andrew, 2008). 인간단핵구에서 MMP-8, -11, 17, -23, -25 등의일부 MMPs 발현이보고되어있으며 (Bar-Or et al., 2003), 세포의체외배양시간에따라또는세포외기질을구성하는성분에의한자극이나다른세포에의한직, 간접적인자극등에의하여단핵구내일부 MMPs 발현이변화하는것으로알려져있다 (Amorino and Hoover, 1998; Matias- Roman et al., 2005). 이러한 MMPs의발현은단핵구가분화되어형성되는대식세포에서도관찰되는데 MMPs의발현양상은단핵구와차이가있는것으로보고되고있다 (Andrew, 2008). 인간단핵구에 glanulocyte/macrophagecolony stimulating factor (GM-CSF) 를처리하여대식세포로분화시킬경우 MMP-1, -2, -7, -9, -11, -12, -14의발현이증가하는반면, MMP-8은변화가없는것으로알려져있다 (Ardans et al., 2002; Bar-Or et al., 2003; Feinberg et al., 2000; Schonbeck et al., 1999; Welgus et al., 1990; Whatling et al., 2004). THP-1이나 U937과같은단핵구유사세포주 (monocyte-like cell lines) 에 phorbol myristate acetate (PMA) 를처리하게되면유사대식세포 (macrophage-like cells) 로분화되는데이때 MMPs 발현양상에대한일부보고는있으나 (Andrew, 2008; Amorino and Hoover, 1998) 체계적으로연구된바는없는실정이다. 본연구에서는 THP-1 세포를모델세포로이용하여 PMA 처리에의한단핵구에서대식세포로분화시세포막결합 MMPs를제외한나머지아형그룹에속하는 MMPs 발현양상을확인하고, 각각의 MMPs 발현에관여하는신호전달체계 (signal transduction pathway) 를규명하고자하였다. 대부분의 MMPs 발현이단핵구의대식세포로분화시증가하였지만증가양상및관련신호전달체계는아형그룹별로차이를보였다. 본연구는단핵구및대식세포에서발현되는 MMPs와각종염증성질환에대한관계연구에기초자료를제시할것으로기대된다. 재료및방법신호전달단백질억제제 Classical PKC (RO ), MEK1 (PD 98059), PI3-K (LY ), p38 MAPK (SB ) 및 craf-1 kinase (GW 5074) 에대한특이적억제제를 Calbiochem (Darmstadt, Germany) 에서구매하였다. NF-κB (BAY ) 에대한특이적억제제를 Enzo life science (New York, NY, USA) 에서구매하였다. PLC (U 73122) 에대한특이적억제제를 Cayman (Ann Arbor, MI, USA) 에서구매하였다. Dimethyl sulfoxide (DMSO) 는 Sigma-Aldrich (St. Louis, MO, USA) 에서구매하여사용하였다. 세포배양및처리 10% heat-inactivated fetal bovine serum (FBS), 2 mm L-glutamine, 100 U/ml penicillin 및 100 μg/ml streptomycin

3 이포함된 RPMI 1640 배지를이용하여인간단핵구백혈병세포주인 THP-1 세포를 5% CO 2 존재하에 37 세포배양기에서배양하였다. THP-1 세포를대식세포로분화시키기위하여 6-well plate에각 well 당 개의세포를심고 200 nm PMA를처리하였다. PMA에대한농도의존적 MMPs 발현변화를살펴보기위하여 PMA 를 72시간동안각각 0, 1, 2, 4, 10, 20, 40 및 100 nm 처리하였다. PMA에대한시간의존적 MMPs 발현변화를살펴보기위하여 200 nm의 PMA를각각 0, 6, 12, 24, 36, 48, 60 및 72시간동안처리하였다. 억제제처리에따른 MMPs 발현변화를확인하기위하여 48시간동안 200 nm의 PMA를처리한뒤, 대조군과비교하여각각 RO nm, PD μm, BAY μm, LY μm, SB μm, GW μm, U ng/ml를 200 nm PMA와같이 24시간동안처리하였다. RNA 분리및 semi-quantitative reverse transcriptase PCR (semi-qrt-pcr) THP-1 세포및 PMA에의해유도분화된 (PMAdifferentiated) THP-1 세포를 1 ml phosphate buffered saline (PBS) 로 2회세척후 1 ml의 Trizol (Invitrogen, Grand Island, NY, USA) 을처리한뒤 Invitrogen 사에서제공한 protocol 의지침에따라 RNA를분리하였다. cdna를합성하기위하여 2 μg의 total RNA를정량한뒤 random hexamer 0.25 μg, Murine Molony Leukemia Virus Reverse Transcriptase (MMLV-RT; Invitrogen) 200 unit을가하여 25 10분, 37 50분및 70 15분동안반응시켰다. 역전사된 (reversetranscribed) cdna를희석한뒤 20 μl PCR 증폭 (PCR amplification) 에이용하였다. PCR 증폭에는 Prime Taq premix PCR kit (Genet Bio, Chungnam, Korea) 를이용하였고 MMP-1, -8, -3, -10, -12, -19, -2, -9, -7 (MMP-1, 5'- GAT GGG AGG CAA GTT GAA AA -3' 및 5'- CCA GGT CCA TCA AAA GGA GA -3'; MMP-8, 5'- GGA AAC CCC AAG TGG GAA CG -3' 및 5'- AGT CCA TAG ATG GCC TGA ATG C -3'; MMP-3, 5'- GGC CAG GGA TTA ATG GAG AT -3' 및 5'- GCT GAC AGC ATC AAA GGA CA -3'; MMP-10, 5'-GTG GAG TTC CTG ACG TTG GT -3' 및 5'- TGC CTG ATG CAT CTT CTG TC -3'; MMP-12, 5'- ACA CCT GAC ATG AAC CGT GA -3' 및 5'- AGC AGA GAG GCG AAA TGT GT -3'; MMP-19, 5'- GGG TCC TGT TCT TCC TAC AT -3' 및 5'- CAA TCC TGC AGT ACT GGT CT -3'; MMP-2, 5'- TTT CCA TTC CGC TTC CAG GGC AC -3' 및 5'- TCG CAC ACC ACA TCT TTC CGT CAC T -3'; MMP-9, 5'- TGG AGA GTC GAA ATC TCT GG -3' 및 5'- ACC AAA CTG GAT GAC GAT GT -3'; MMP-7, 5'- AAC TCC CGC GTC ATA GAA ATA ATG -3' 및 5'- ACC CAA ACA ATG GCC AAG TTC ATG -3') 에대한 MMPs 특이적프라이머 (primer) 를사용하였다. PCR 증폭은 thermocycler (Applied Biosystems, New York, NY, USA) 를통해 25~40 cycles 동안수행하였다. MMPs 발현을비교확인하기위하여 glyceraldehyde 3-phosphate dehydrogenase (GAP-DH) 를내부기준 (internal control) 로사용하였다. 증폭된 PCR 산물은 0.5 μg/ml ethidium bromide (Et-Br) 가첨가된 2.0% (w/v) agarose gels에서전기영동을하였으며 100 bp DNA ladder marker (Intron, Gyeonggi, Korea) 를기준으로증폭된 PCR 산물을확인하였다. Gel-Doc (Bio-Rad, Hercules, CA, USA) 을이용하여 Gel-image를확인한뒤 Image Lab (version 2.0, Bio-Rad) 을통하여 GAP-DH에대한상대적 MMPs 발현정도를정량확인하였다. 결과 PMA 처리에의한단핵구의대식세포분화과정에서 MMPs 발현증가 PMA는단핵구나단핵구유사세포주에처리시에대식세포로의분화를유도하는것으로알려져있으며이때일부 MMPs의세포외분비 (secretion) 가촉진되는것이보고된바있다 (Ardans et al., 2002; Bar-Or et al., 2003; Feinberg et al., 2000; Watanabe et al., 1993; Welgus et al., 1990; Whatling et al., 2004). 본연구에서는 PMA 처리에의한단핵구의대식세포분화과정에서 MMPs의발현양상을확인하기위해 THP-1 세포에 PMA를농도의존적으로처리한후각 MMPs의 mrna 양을 RT-PCR 로확인하였다. MMP-2는 PMA 처리이전에도발현되어있었다 (Fig. 1). 그러나 MMP-2를제외한 collagenases (MMP-1, -8), stromelysins (MMP-3, -10), 기타 MMPs (MMP-12, -19), matrilysins (MMP-7), 그리고 gelatinases 중 MMP-9은 PMA 처리전에는 mrna 수준에서발현이관찰되지않다가 PMA 처리에의해대식세포로분화되면발현되는것을확인하였다 (Fig. 1). 확인한 MMP 중에 MMP-10은다른 MMP에비해고농도의 PMA 처리시에발현이증가하는것이관찰되었다 (Fig. 1). 특이적으로 MMP-2는저농도 (1~4 nm) 의 PMA 처리시에는증가하다고농도 (20~

A Fig. 1. Dose-dependent expression of MMPs during PMAinduced differentiation of THP-1 cells.

cdna was prepared from extracted total RNA and subjected to PCR to amplify MMPs.

Data are expressed as the mean ± SD and are presented as the expression levels of MMPs mrna relative to GAP-DH mrna.

Data are from three independent experiments. B 상 MMP 중 MMP-2를제외한다른 MMPs는 PMA 처리에의해 THP-1 세포가단핵구에서대식세포로분화할때발현이감작되는것을확인하였다.

Collagenases 아형그룹에속한 MMP-1 과 MMP-8, stromelysins 아형그룹에속하는 MMP-3과 MMP-10은각각 PMA 처리에따른발현시간대가유사한것을확인할수있다 (Fig. 2).

또한 Collagenases (MMP-1, -8), stromelysins (MMP-3, -10), 기타 MMP 중 MMP-12는 PMA 처리후 60시간경과후에발현이관찰된반면 MMP-19, MMP-9, MMP-7 등은 PMA 처리후 24

4 A Fig. 1. Dose-dependent expression of MMPs during PMAinduced differentiation of THP-1 cells. (A) THP-1 cells were treated with indicated concentrations of PMA (0, 1, 2, 4, 10, 20, 40 or 100 nm) for 72 hrs. cdna was prepared from extracted total RNA and subjected to PCR to amplify MMPs. The PCR products were analyzed on a 2% agarose gel and GAP-DH was used as an internal control. (B) Densitometric analysis. Data are expressed as the mean ± SD and are presented as the expression levels of MMPs mrna relative to GAP-DH mrna. The mrna expression level of MMPs relative to GAP-DH in cells treated with 100 nm PMA was set as 100. Data are from three independent experiments. B 상 MMP 중 MMP-2를제외한다른 MMPs는 PMA 처리에의해 THP-1 세포가단핵구에서대식세포로분화할때발현이감작되는것을확인하였다. 단핵구에서대식세포로의분화과정에서 PMA 처리시간의존적으로 MMPs 발현증가 다음으로 PMA를시간의존적으로처리하여각 MMPs 의발현양상을확인한결과 MMP-2를제외한다른 MMPs는 PMA 처리시간의존적으로발현이증가함을확인하였다 (Fig. 2). Collagenases 아형그룹에속한 MMP-1 과 MMP-8, stromelysins 아형그룹에속하는 MMP-3과 MMP-10은각각 PMA 처리에따른발현시간대가유사한것을확인할수있다 (Fig. 2). MMP-2는 PMA 처리후 24시간까지일시적으로증가하다가그이후에는시간이경과함에따라그발현량이감소하여 PMA 처리 74시간경과후에는 PMA 미처리시수준의발현량으로돌아오는것을관찰할수있었다 (Fig. 2). 또한 Collagenases (MMP-1, -8), stromelysins (MMP-3, -10), 기타 MMP 중 MMP-12는 PMA 처리후 60시간경과후에발현이관찰된반면 MMP-19, MMP-9, MMP-7 등은 PMA 처리후 24 시간경과후부터발현이관찰되었다 (Fig. 2). 본연구를통해실험대상 MMP 중 MMP-2를제외한다른 MMPs 는 PMA 처리에의해 THP-1 세포가단핵구에서대식세포로분화할때시간의존적으로발현이감작되며 MMP 발현의 PMA 시간의존성은아형그룹간에차이가있다는사실을확인하였다. PMA 처리에의한단핵구에서대식세포로의분화과정에서 Protein kinase C (PKC) 비의존적으로 MMPs 발현증가 nm) PMA 처리시에는 PMA 미처리시와비슷한발현수준을보이는것이관찰되었다. 본연구를통해실험대 THP-1 세포를대식세포로분화시키는데사용되는 PMA 는 PKC를활성화하는물질 (activator) 로알려져있다 (Van Leeuwen et al., 1994). 따라서 PMA 처리에따른 MMPs 발현증가에 PKC가관여하는지확인하였다. THP

5 A B Fig. 3. PKC is not involved in PMA-induced increase of MMPs expression. THP-1 cells were differentiated with PMA (200 nm) for 48 hrs and then treated with 50 nm RO (inhibitor specific for PKC) for another 24 hrs. cdna was synthesized and PCR performed using MMP-specific primers. The PCR products were resolved on a 2% agarose gel. GAP-DH was used as an internal control. 과를통해 THP-1 세포의대식세포로의분화과정에서 PMA에의한 MMPs 발현증가를매개하는신호전달체계에 PKC는관여하지않는다는것을확인할수있었다. Fig. 2. Time-dependent expression of MMPs during PMAinduced differentiation of THP-1 cells. (A) THP-1 cells were differentiated with PMA (200 nm) for the indicated times (0, 6, 12, 24, 36, 48, 60 or 72 hrs). Total RNA was extracted and cdna was prepared. PCR analysis was performed using MMP-specific primers. The PCR products were resolved on a 2% agarose gel. GAP-DH was used as an internal control. (B) Densitometric analysis. Data are expressed as the mean ± SD and are presented as the expression levels of MMPs mrna relative to GAP-DH mrna. The mrna expression level of MMPs relative to GAP- DH in THP-1 cells treated with 200 nm PMA for 72 hrs was set as 100. Data are from three independent experiments. 1 세포에 200 nm의 PMA를처리하여 48시간동안배양한뒤 classical PKC 억제제인 RO 를처리하고 24시간배양한후 MMPs 발현변화를확인하였다. 예상과다르게 PKC 활성을억제할경우에도 PMA에의한 MMPs 발현에는아무변화가없었다 (Fig. 3). 본연구결 PMA 처리에의한단핵구분화과정에서 MEK-1 의존적신호전달과정을통해 MMPs 발현증가 PMA에의한 THP-1 세포의대식세포로의분화시야기되는 MMPs의발현증가와연관된신호전달체계단백질을규명하기위하여각종신호전달물질에대한억제제를이용하여실험을진행하였다. THP-1 세포에 200 nm 의 PMA를 48시간동안처리한뒤각종신호전달물질특이적억제제를처리하고 24시간배양후 MMPs 발현양상을 RT-PCR 방법으로확인하였다. craf-1에대한억제제인 GW 5074와 PLC 억제제인 U 73122를처리시에는 PMA에의한 MMPs의발현증가에변화가없는것을확인할수있었다 (Fig. 4). 하지만 collagenases (MMP-1, -8), stromelysins (MMP-3, -8), 그리고기타 MMPs 아형그룹중 MMP-12와같은경우, MEK1에대한억제제인 PD 98059를처리하면 PMA에의한발현증가가대부분억제되는것을확인할수있었다. 그리고 MMP-19, MMP-9,

A Fig. 4. PMA-induced upregulation of most MMPs is mediated by the MEK-1 pathway.

5 μm), or U 73122 (50 ng/ml) for another 24 hrs. cdna was prepared from total RNA extracts and subjected to PCR to amplify MMPs. The PCR products were resolved on a 2% agarose gel.

The mrna expression level of MMPs relative to GAP-DH mrna in PMA-treated cells without treatment with inhibitors was set as 100. The data represent results from three independent experiments.

collagenases (MMP-1, -8) 의경우, NF-κB 특이억제제인 BAY 11-7085 처리시 PMA 에의한 MMPs 발현이일부감소하는것이관찰되었고 stromelysins (MMP-3, -10) 은 p38 MAPK 특이억제제인 SB 203580 처리시 PMA 처리에의한 MMP 증가가억제됨을확인하였다 (Fig. 4).

6 A Fig. 4. PMA-induced upregulation of most MMPs is mediated by the MEK-1 pathway. (A) THP-1 cells were incubated with PMA (200 nm) for 48 hrs and then treated with inhibitors PD (50 μm), BAY (10 μm), Ly (10 μm), SB (20 μm), GW 5074 (0.5 μm), or U (50 ng/ml) for another 24 hrs. cdna was prepared from total RNA extracts and subjected to PCR to amplify MMPs. The PCR products were resolved on a 2% agarose gel. GAP-DH was used as an internal control. (B) Densitometric analysis. Data are expressed as the mean ± SD and are presented as the expression levels of MMPs mrna relative to GAP-DH mrna. The mrna expression level of MMPs relative to GAP-DH mrna in PMA-treated cells without treatment with inhibitors was set as 100. The data represent results from three independent experiments. B MMP-7의경우에도 PD 처리시 PMA 처리에의한발현이일부억제되는것을확인하였다 (Fig. 4). 이러한결과를통하여 MMP-2를제외한 MMPs은 PMA 처리에의한발현증가가 MEK1 관련신호전달체계를통해이루어짐을확인할수있었다. collagenases (MMP-1, -8) 의경우, NF-κB 특이억제제인 BAY 처리시 PMA 에의한 MMPs 발현이일부감소하는것이관찰되었고 stromelysins (MMP-3, -10) 은 p38 MAPK 특이억제제인 SB 처리시 PMA 처리에의한 MMP 증가가억제됨을확인하였다 (Fig. 4). 또한 MMP-8과 MMP-12는 PI3-K 특이억제제인 LY 처리시 PMA에의한발현이억제되는경향을확인하였다 (Fig. 4). 이러한결과는 PMA의자극에의한 MMPs 발현이 MEK1 관련신호전달체계이외에 NF-κB, p38 MAPK, PI3-K 등의신호전달물질에의해매개됨을보여주며또한관련된신호전달물질이일부 MMPs 아형그룹에서는공통적으로관여함을보여주고있다. 고 찰 말초혈액내단핵구에도일부 MMPs가낮은수준으로발현되며 lipopolysaccharide (LPS) 나염증성사이토카인 (inflammatory cytokine) 에의한자극에의해서일부 MMPs 발현양상이변하는것이보고되고있다 (Andrew, 2008). 이와더불어기저막 (basement membrane) 등의세포외기질과접촉하면일부 MMPs 발현및분비가더욱활성화되며이렇게분비된활성 MMPs에의한세포외기질분해가단핵구의조직내이동에중요한역할을할것으로추측되고있다. 또한조직으로이동한단핵구가대식세포로분화되면서일부 MMPs의발현양상이변한다는보고도있다. 본실험에서는 THP-1 세포에 PMA를처리

7 하여단핵구를대식세포로분화시키는과정에서세포막결합 MMP 아형그룹을제외한대부분의 MMP 발현양상이어떻게변하는지확인하였다. PMA 처리에의해단핵구에서대식세포로분화함에따라 MMP-2를제외한대부분의 MMPs 발현이증가하는것을확인할수있었다 (Fig. 1, 2). collagenases와 stromelysins 등의아형그룹에같이속해있는 MMPs는 PMA 처리시간에따른발현증가양상이비슷한패턴을보이는것을확인할수있었다 (Fig. 2). 또한 PMA 처리시조기에발현증가를보이는 MMP (MMP-19, 9, 7) 와더시간이경과해야발현이증가하는 MMP (MMP-1, 8, 3, 10, 12) 등을확인할수있었다. 이러한결과를기초로 MMPs 활성시에단계적으로 MMPs가활성화되어먼저활성화된 MMPs가다른 MMPs를연쇄적으로활성화시켜세포외기질의분해를촉진시킬가능성을추론할수있다. MMP-2가다른 MMPs와는달리단핵구상태에서도지속적으로발현 (Fig. 1, 2) 되는것또한이러한가능성을설명할수있는이유로사료된다. 이전의보고에의하면 MMP-2는불활성화상태의 MMP-1, -2, -13 등을활성화시키는것으로알려져있는데 (Magnus et al., 2010) 단핵구에발현되어있는 MMP-2가대식세포로의분화시먼저발현된 MMP-19, 9, 7을활성화하고이렇게활성화된 MMP가나중에발현되는 MMP-1, 8, 3, 10, 12 등을활성화할가능성도추론해볼수있다. 보고에의하면동맥경화증의경우, 동맥경화병변에침윤된면역세포에서 MMP-1, MMP-2, MMP-9 등의발현이증가된다고알려져있으며 (Magnus et al., 2010) 이러한 MMPs 발현의증가는 collagen과같은세포외기질을분해하며동맥경화증의발달과관련된면역세포의침윤을증가시키는것으로알려져있다. 따라서본연구의결과로유추하면동맥경화발달시단핵구가대식세포로분화하고분화된대식세포는 MMPs 발현이증가하고이렇게증가된 MMPs에의해동맥경화병변으로의이동이촉진될가능성을제시할수있다. 본연구에서는 PKC 활성물질로알려진 PMA를이용하여단핵구를대식세포로분화유도하여 MMPs의발현양상을확인하였다. 따라서 PKC 활성억제제처리시 PMA 처리에의한 MMPs의발현에영향이있을것이라예상을하였다. 하지만 PKC의활성을억제하였음에도불구하고 MMPs 발현에는큰변화가없었다 (Fig. 3). 이결과로단핵구에서대식세포로분화유도시나타나는 MMPs의발현증가에 PKC는관여하지않으며따라서 다른세포신호전달체계의관여여부를확인할필요성이제시되었다. 따라서다양한신호전달물질에특이적으로작용하는억제제를사용하여 PMA 처리에의한 MMPs 발현에관여하는신호전달체계를조사하였으며그결과 PMA 처리에의한 MMPs의발현과관련된신호전달체계가 MMP 별로다소차이가있으나공통적으로 MEK1을매개로신호전달이이루어짐을확인할수있었다 (Fig. 4). 이것은대식세포로분화시 MEK1을경유하는신호전달체계에의해 MMPs 발현이증가한다는것을보여주는결과이다. 따라서대식세포의 MEK1을선택적으로억제하는물질을발굴, 환자에처리하면동맥경화병변으로의대식세포이동을감소시켜동맥경화발달이억제될가능성도추론해볼수있다. MMPs가염증성질환, 종양발달등각종질환발병에관여한다는사실이최근지속적으로보고되고있고따라서대식세포를포함한각종세포에서 MMPs 발현, 활성화, 기능에대한많은연구가진행되고있다. 본연구결과가염증성질환등에중요한역할을하는단핵구및대식세포에서의 MMPs 발현조절및기능연구에대한기초자료로활용될것으로기대한다. Acknowledgements 이논문은 2011 년도정부 ( 교육과학기술부 ) 의재원으로한국연구재단의지원을받아수행된해외우수연구기관유치사업연구임 ( ). REFERENCES Amorino GP, Hoover RL. Interactions of monocytic cells with human endothelial cells stimulate monocytic metalloproteinase production. Am J Pathol : Andrea S, Ulrike K, Michaela K, Johannes D, Sonja F, Jelena A. Expression pattern of matrix metalloproteinases in human gynecological cancer cell lines. BMC Cancer : Andrew CN. Metalloproteinases expression in monocytes and macrophages and its relationship to atherosclerotic plaque instability. Arterioscler Thromb Vasc Biol : Ardans JA, Economou AP, Martinson JM, Zhou M, Wahl LM. Oxidized low-density and high-density lipoproteins regulate the production of matrix metalloproteinase-1 and -9 by activated monocytes. J Leukoc Biol :

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