DOI:10.5125/jkaoms.2010.36.6.481 All-trans retinoic acid 가면역세포의 Toll-like receptor 5 발현에미치는영향 김기형 1 박상준 2 1 CK 치과병원, 2 인제대학교의과대학부산백병원구강악안면외과 Abstract (J Korean Assoc Oral Maxillofac Surg 2010;36:481-9) Effects of all-trans retinoic acid on expression of Toll-like receptor 5 on immune cells Ki-Hyung Kim 1, Sang-Jun Park 2 1 CK Dental Hospital, 2 Department of Oral and Maxillofacial Surgery, Pusan Paik Hospital, College of Medicine, Inje University, Busan, Korea Introduction: TLR-5, a member of the toll-like receptor (TLR) family, is a element of the type I transmembrane receptors, which are characterized by an intracellular signaling domain homolog to the interleukin-1 receptor. These receptors recognize microbial components, particularly bacterial flagellin. All-trans retinoic acid (atra, tretinoin), a natural metabolite of vitamin A, acts as a growth and differentiation factor in many tissues, and is also needed for immune functions. In this study, THP-1 human macrophage-monocytes were used to examine the mechanisms by which atra regulated the expression of TLR-5. Because the molecular mechanism underlying this regulation at the transcriptional level is also unclear, this study examined which putative transcription factors are responsible for TLR-5 expression by atra in immune cells. Materials and Methods: This study examined whether atra induces the expression of TLR-5 in THP-1 cells using reverse transcription-polymerase chain reaction (RT-PCR), and which transcription factors are involved in regulating the TLR-5 promoter in RAW264.7 cells using a reporter assay system. Western blot analysis was used to determine which signal pathway is involved in the expression of TLR-5 in atra-treated THP-1 cells. Results: atra at a concentration of 10 nm greatly induced the expression of TLR-5 in THP-1 cells. Human TLR-5 promoter contains three Sp-1/GC binding sites around -50 bp and two NF-kB binding sites at -380 bp and -160 bp from the transcriptional start site of the TLR-5 gene. Sp-1/GC is primarily responsible for the constitutive TLR-5 expression, and may also contribute to NF-kB at -160 bp to induce TLR-5 after atra stimulation in THP-1 cells. The role of NF-kB in TLR-5 expression was further confirmed by inhibitor pyrrolidine dithiocarbamate (PDTC) experiments, which greatly reduced the TLR-5 transcription by 70-80%. Conclusion: atra induces the expression of the human TLR-5 gene and NF-kB is a critical transcription factor for the atra-induced expression of TLR-5. Accordingly, it is conceivable that retinoids are required for adequate innate and adaptive immune responses to agents of infectious diseases. atra and various synthetic retinoids have been used therapeutically in human diseases, such as leukemia and other cancers due to the antiproliferative and apoptosis inducing effects of retinoids. Therefore, understanding the molecular regulatory mechanism of TLR-5 may assist in the design of alternative strategies for the treatment of infectious diseases, leukemia and cancers. Key words: Tretinoin, Toll-like receptor 5, Cellular immunity, NF-kappa B [paper submitted 2010. 7. 27 / revised 2010. 11. 20 / accepted 2010. 12. 13] Ⅰ. 서론 Toll-like receptor (TLR) 는원래초파리의발생과정에관여하는유전자로처음클로닝이되었으며, 이후많은연구를통하여초파리의 innate immune response 에도관여한다는사실이밝혀졌다 1. 포유류에서도 Toll 과유사성을가진 박상준 614-735 부산광역시부산진구개금동 633-165 인제대학교의과대학부산백병원구강악안면외과 Sang-Jun Park Department of Oral and Maxillofacial Surgery, Pusan Paik Hospital, College of Medicine, Inje University 633-165 Gaegum-dong, Busanjin-gu, Busan, 614-735, Korea TEL: +82-51-890-6363 FAX: +82-51-895-9650 E-mail: ds5nki@hanmail.net 유전자를찾게되었으며, 사람에서 TLR 가 1997 년에처음클로닝되어현재까지총 13 종류의 TLR 유전자들을발견하여 TLR family 를이루고있다 2,3. TLR 은 type I transmembrane signaling molecule 로 innate immune system 을담당하는세포에서주로발현된다. 이들세포에서 TLR 은미생물의특이적인구조를인식해서면역반응을일으키는신호를전달한다. TLR family 에속하는수용체는일반적으로 extracellular region 에 leucine-rich repeat (LRR) motif 가있으며, interleukin (IL)-1 수용체와구조가비슷한 cytoplasmic domain 이존재한다. TLR 은 extracellular domain 을통해서 pathogen-associated molecular patterns (PAMP) 나 microorganism-associated molecular patterns (MAMP) 를인식하며, 그결과 innate immune 세포에서염증성세포활성화를유 481
J Korean Assoc Oral Maxillofac Surg 2010;36:481-9 도한다 4. Vitamin A 는배발생 (embryogenesis) 의조절및면역계의활성등을포함하는수많은생물학적효과들을나타낸다 5,6. Vitamin A 유도체인 all-trans retinoic acid (atra, RA or tretnoin) 는정상적인세포성장, 분화, 발달, 재생산, 형태형성그리고항상성유지와같은다양한생물학적과정에작용하며 7, 암세포의무분별한증식을억제하고분화를유도하거나세포자연사를유발하여암의성장을억제하는것으로알려져있다 8. 또한면역계에작용하여면역활성을증가시킨다고알려져있다. Israel 등 9 에의하면 atra 를제대혈에서분리한 mononuclear cells 과 polyclonal B-cell activator 와반응하여 IgM 의생산을증가시킨다고보고하였다. 이와같은면역글로불린분비의증가는 T 및 B 세포에대한 atra 의효과에의해나타난다고알려져있다 10,11. 또한 vitamin A 결핍생쥐에서분리한 lymphocytes 를 in vitro 에서자극하였을때 interferon (IFN)-r 의생산을증가시키는데반하여 IL-4 및 IL-5 의생산은감소시켰다 12. 이와는반대로 vitamin A 나 atra 는 IFN-r 의생산을감소시키고 IL-5, IL-4 및 IL-10 의생산은증가시킨다 13. 이와같이 vitamin A 결핍은 Th1 면역반응을증가시키고, vitamin A 또는 retinoid 의처리는 Th2 반응을증가시킨다고알려졌다 14. 이러한 atra 의작용개념은면역체계전반에특이적으로작용하는것으로생각된다. atra 의세포내작용은핵내에존재하는수용체인 retinoic acid receptor (RAR) 와 retinoid X receptor (RXR) 에의해서매개된다 15. 이들 RAR 와 RXR 는 steroid hormone receptor superfamily 에속하며각각 3 개의 subtype 으로 RAR α, β, γ 와 RXRα, β, γ 가있다 16. 이들수용체와 atra 의결합은선택적으로이루어지는데, 9-cis retinoic acid 와 atra 는 RAR 와결합하며, 13-cis retinoic acid 는 RXR 와결합하여작용한다 17. 핵내에서 atra 와결합한 RA 수용체복합체는전사조절자로작용하여특정유전자부위인, retinoic acid response elements 와결합하여여러유전자의발현을증가또는감소시켜세포성장을조절한다. 아직까지 atra 의작용기전에대한명확하게밝혀지지않았으며, 몇몇연구자들에의해서 activator protein (AP)-1 의활성에의해세포성장억제가유도된다고보고된바가있다 18. 그러나 vitamin A 유도체가이들생물학적작용의조절에관여하는기작은많은부분에있어서불분명하다. 최근연구에서 Liu 등 19 은 atra 가 primary human monocytes 에서 TLR2 의발현을감소시킨다고보고하였다. 그러나다른 TLR 의발현에관여하는지, 그작용기전에관해서는여전히불분명하다. 따라서본연구에서는 human monocytic leukemia cell line (THP-1) 및 mouse leukemia monocyte macrophage cell line (RAW264.7) 면역세포주에 atra 를처리하여 24 시간배양한후 RNA 를분리하고, TLR 유전자들의발현을 reverse transcription-polymerase chain reaction (RT-PCR) 을이용하 여측정하고, atra 에의해그발현이유도되는 TLR 을대상으로 atra 가이들유전자의발현에작용하는신호전달경로및전사조절에관여하는조절인자를밝히고자하였다. 1. atra Ⅱ. 연구재료및방법 atra (Sigma chemical Co., St Louis, MO, USA) 를 dimethyl sulfoxide (DMSO) (Sigma chemical Co., St Louis, MO, USA) 에녹여 100 μm stock solution 으로만들어차광하여 -80 에보관하였다. Stock solution 은사용하기직전에 0.5% fetal bovine serum (FBS) 배지에원하는농도로희석하여사용하였다. 2. 세포주배양과처리 사람과생쥐의면역세포주인 THP-1 과 RAW264.7 은한국세포주은행에서구입하였다. 이들세포주는 Rosewell Park Memorial Institute (RPMI) 1640 배지에 10% FBS 와 antibiotics (Gibco BRL, Grand island, NY, USA) 를첨가하여, 37, 5% CO2 의조건하에배양기에서배양하였다. 배지는 48 시간마다갈아주었다. 실험군세포를배양접시에분주하고, 다음날실험에따라적절한농도의 atra 를배지에첨가하여처리하였다. 재조군세포에는 atra 대신에 0.001% DMSO 를처리하였다. 3. RT-PCR 1) Primer 제작 Gene bank data 를기본으로 PCR Primer 를 Table 1 과같이주문제작하였다. 2) Total RNA 분리각각의조건에서수확한세포에 TRIZol reagent (Invitrogen, Carlsbad, CA, USA) 를 1 ml 씩넣고 5 분동안흔든후전체양의 0.2 volume 에해당하는 chloroform 을첨가한후 15 초동안 tube 를흔든후 3 분간둔다. 이혼합물을 4 에서 15 분간 12,000 rpm 으로원심분리한후상층액을취하여, 전체양의 0.5 volume 의 2-propanol 을첨가하고이를다시 4 에서 15 분간두었다가 12,000 rpm 으로원심분리하였다. 상층액을제거하고얻은침전물에 70% ethanol 500 μl 을넣어 12,000 rpm 에서 5 분간원심분리시키고진공건조하였다. 3) cdna 합성얻어진 RNA 를 30 μl 의 diethyl pyrocarbonate-distilled water (DEPC-DW) 에용해시키고, 2 μg 의 RNA 를 42 에서 482
All-trans retinoic acid 가면역세포의 Toll-like receptor 5 발현에미치는영향 Table 1. Oligonucleotide primer sequence used in this study and products size of the amplified RT-PCR Oligonucleotides Sequence (5' 3') Product size htlr-1 F : CGT AAA ACT GGA AGC TTT GCA AGA 889 bp R : CCT TGG GCC ATT CCA AAT AAG TCC htlr-2 F : GGC CAG CAA ATT ACC TGT GTG 614 bp R : CCA GGT AGG TCT TGG TGT TCA htlr-3 F : ATT GGG TCT GGG AAC ATT TCT CTT C 319 bp R : GTG AGA TTT AAA CAT TCC TCT TCG C htlr-4 F : CTG CAA TGG ATC AAG GAC CA 622 bp R : TCC CAC TCC AGG TAA GTG TT htlr-5 F : CAT TGT ATG CAC TGT CAC TC 445 bp R : CCA CCA CCA TGA TGA GAG CA htlr-6 F : TAG GTC TCA TGA CGA AGG AT 1108 bp R : GGC CAC TGC AAA TAA CTC CG htlr-7 F : AGT GTC TAA AGA ACC TGG 527 bp R : CTT GGC CTT ACA GAA ATG htlr-8 F : CAG AAT AGC AGG CGT AAC ACA TCA 637 bp R : AAT GTC ACA GGT GCA TTC AAA GGG htlr-9 F : TTA TGG ACT TCC TGC TGG AGG TGC 331 bp R : CTG CGT TTT GTC GAA GAC CA htlr-10 F : CAA TCT AGA GAA GGA AGA TGG TTC 659 bp R : GCC CTT ATA AAC TTG TGA AGG TGT hgapdh F : TGA TGA CAT CAA GAA GGT GG 244 bp R : TTA CTC CTT GGA GGC CAT GT mtlr-5 F : GCT CAA ACA CCT GGA TGC TCA 248 bp R : TCC CAC CAC CAC CAC GAT GA mgapdh F : TTC ACC ACC ATG GAG AAG GC 210 bp R : GGC ATG GAC TGT GG TCAT GA (RT-PCR: reverse transcription polymerase chain reaction, TLR-5: Toll-like receptor-5, GAPDH: glyceraldehyde 3-phosphate dehydrogenase) 1 μl reverse transcriptase (TaKaRa, Shiga, Japan), 10 buffer 2 μl, 10 mm dntp 2 μl (dntp mix), oligo dt primer 1 μl, RNase inhibitor 0.5 μl, 25 mm MgCl 2 4 μl 를넣고최종반응량이 20 ul 되게조절하여합성하였다. Carlsbad, CA, USA) 을이용하여합성하였으며, Real-time RT-PCR 은 SYBR Supermix kit 와 icycler system (both from Bio-Rad icycler, Bio-Rad, Hercules, CA, USA) 를이용하여 manufacturer's instructions 에따라수행하였다. 4) PCR RT-PCR 에서얻어진 1 μl 의 cdna 를조건설정후증폭시켰다. PCR 의조건은다음의기본조건을기준으로 i) 94 에서 5 분 (initial denaturation), ii) 94 에서 30 초 (denaturation), 58 에서 30 초 annealing), 72 에서 30 초 (extension), iii) 72 에서 7 분 (final extension) 으로수행하였으며, 2 단계를 30 회반복증폭하였다. 또한예상 PCR 산물의크기에따라 extension 시간을조절하였다. 5) Real-time PCR 얻어진 total RNA 에 RNase-free DNase (RQ1 DNase, Promega, Madison, WI, USA) 를처리하여 genomic DNA 를제거한후 RNA 를정량하였다. First-strand cdna 합성은 SuperScript First-Strand Synthesis System (Invitrogen, 6) 전기영동 (electrophoresis) Ethidium bromide 1 μg/ml 을함유하는 1.5% agarose gel 에 10 μl 의 reaction mixture 를전기영동하여자외선조사로확인하였다. 4. Western blot Six well plates 에 90% confluence 를이룬 THP-1 세포를떼어내원심분리하여세포를회수하였다. 이세포침전물에 100 μl 의 lysis buffer (1% Triton X-100, 20 mm Tris-HCl (ph 7.5), 150 mm NaCl, 10% glycerol, 0.1 mm ethylenediaminetetraacetic acid (EDTA), 10 mm sodium fluoride, 1 mm sodium orthovanadate, 1 mm phenylmethanesulfonyl fluoride (PMSF), 3 μg/ml aprotinin, 2 μg/ml pepstatin, 1 μg/ml leupeptin) 를 483
J Korean Assoc Oral Maxillofac Surg 2010;36:481-9 첨가한후 4 에서 20 분간 lysis 시켰다. 13,000 rpm, 4 에서 10 분동안원심분리하여얻은상등액을 Bradford dyebinding assay (Bio-Rad, Hercules, CA, USA) 로단백질농도를결정하였다. 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel 에 50 μg 의단백질를전기영동하고, semidry blotter 를이용하여 nitrocellulose membrane (Schleicher & Schuell, Keene, NH, USA) 에이동시켰다. 그리고비특이적인결합을줄이기위하여, 1 시간동안차단용액으로반응시키고, 각각의특이적인항체인 ERK, JNK, p38 MAPK 및 phospho-erk, JNK, p38 MAPK 를사용하여반응시켰다. 각각의단백질들은 HRP-conjugated antirabbit IgG (1:4,000) 으로탐지하였다. Membranes 은반응사이에 0.05% Tween 20 이함유된 Tris-buffered saline (10 mm Tris, 150 mm NaCl) 으로세척하였다. Chemiluminescence 의양은 LAS-3000 SYSTEM (Fuji Photo Film, Fuji, Japan) 으로측정한다. 5. Promoter constructs 제조 THP-1 세포에서 DNeasy Tissue Kit (Qiagen, GmhH, Germantown, MD, USA) 를이용하여 human genomic DNA 를분리하였다. Genomic DNA 로부터표적유전자를 PCR 로증폭하였고증폭된 fragment 는 TA-vector 로 cloning 하였다. TLR-5 의 Promoter region 에서크기가다른 promoter region 을적당한제한효소를이용하여 luciferase reporter vector 인 pgl3 basic vector 에 cloning 하였다. Construct 의제작에사용된 primer sequence 는다음과같다. htlr-5pf1: 5'-CCGGTACCGTCTGGAAGGCTCCTCTCT-3'; htlr-5pf2: 5'-CCGGTACCACTGTGGCTCCCAGCTCTT-3'; htlr-5pf3 : 5'-CCGGTACCCCAGCTCCCAAACATAT-3'; htlr-5pf4: 5'-CCGGTACCACTGTGGAAGTCTGCAGAG-3'; htlr-5pr: 5'-CCAAGCTTGGCTCCTCCCGGACGCAAAA-3'. TLR-5 유전자의 5'-비전사 promoter 영역내전사인자결합영역은 GenomeNet (http://motif.genome.jp) 의 sequence motif search program을이용하여분석하였다. 6. Transient transfection 과 luciferase assay Plasmid DNA 는 QIAprep Spin Miniprep Kit (Qiagen, GmhH, Germantown, MD, Germany) 를이용하여준비하였다. 24 well plate 에 RAW264.7 세포를 60-70% confluence 가되게배양한다. Plasmid DNA 0.8 μg 과 Lipofectamine 2000 (Invitrogen, CA, USA) 2 μl 를각각 Opti-MEM (GIBCO, NY, USA) 50 μl 에희석한다. 그리고희석된 DNA 와 Lipofectamine 2000 을혼합한뒤, 상온에서 30 분반응시킨다. 세포가배양된 plates 는 serum-free medium 으로씻어주고 Opti-MEM 400 μl 를넣어준후, 희석된혼합액을세포에넣어준다. 37, 6 시간배양후, 배지를제거하고 10% FBS 가들어있는신선한배지를넣어주고, atra 를처리하여 24 시간동안배양하였다. Promega assay system (Promega, WI, USA) 의 passive lysis buffer 를이용하여세포를 lysis 하고 dual luciferase reporter assay (Promega, WI, USA) 방법으로 luciferase 활성을측정하였다. Luciferase assays 는 Wallac 1420 multilabeling counter (Perkin Elmer, CT, USA) 를사용하여결과를얻었다. Ⅲ. 연구결과 1. 면역세포에서 atra 에의한 TLR 유전자의발현분석 atra 가 TLR 의발현을유도하는기전을조사하기위하여사람 monocyte-macrophage 계통세포인 THP-1 세포를이용하였다. THP-1 세포는기본적으로 10 종의 TLR 을다양한농도로발현하고있었다.(Fig. 1. A) 이세포에 10-8 M (10 nm) 농도의 atra 를 24 시간처리한후 TLR 의발현변화를 RT-PCR 로확인하였다. atra 는 TLR-5 와 -10 의발현을크게유도하는반면에 TLR-2 의발현은억제하였으며, 나머지 TLR 의발현에는크게영향을주지않았다.(Fig. 1. A) atra 에의한 TLR-2 발현의감소는 Liu 등 19 이보여주었던결과와유사하였다. TLR-5 의발현을 Real-time PCR 을이용하여정량분석한결과 atra 가처리되었을때그발현이 7 배이상증가되었음을확인할수있었다.(Fig. 1. B) 2. 사람면역및암세포들에서 atra 에의한 TLT-5 의발현분석 THP-1 세포를이용한선행연구에서 atra 가 TLR-5 의발현을크게유도하였기때문에이결과가모든세포에서공통적으로일어나는가를확인하기위하여다양한사람세포에 atra 를처리하여보았다. TLR-5 는사람면역세포 (Molt4, EBV-B, Raji, U937) 와암세포 (Huh7, HepG2, SKBR3, HBL- 100, Hela, 293T, A549, A431, Colo201) 들은기본적으로 TLR-5 를다양한농도로발현하고있었다.(Fig. 2) 이들세포를 10-8 M 농도의 atra 로 24 시간동안처리한후 RT-PCR 분석한결과 TLR-5 의발현은면역세포인 EBV-B 및 U937 에서소량증가하였다. 그러나나머지암세포주에서는 TLR-5 의발현에영향을주지않았다.(Fig. 2) 이결과에서 atra 는주로면역세포에작용하여 TLR-5 의발현을유도한다는것을확인할수있었다. 3. 생쥐면역세포에서 TLR-5 의발현분석 atra 에의한 TLR-5 의발현이이종간에도동일한현상을나타내는지확인하기위하여생쥐 monocyte-macrophage 계통면역세포인 RAW264.7 세포에동일한조건으로처리하였다. atra 로처리한 RAW264.7 세포는 THP-1 세포와 484
All-trans retinoic acid 가면역세포의 Toll-like receptor 5 발현에미치는영향 유사하게생쥐 TLR-5 의발현을유도하였다.(Fig. 3. A) 이결과는사람 THP-1 세포와생쥐 RAW264.7 세포에 atra 처리시동일한전사조절이이루어지고있음을의미한다. atra 처리시활성화되는전사조절인자를예측하기위하여 NF-kB, CRE, AP-1, SRE, c-jun 및 c-myc 전사조절인자결합부위를포함하는 luciferase 벡터를 RAW264.7 세포에 트랜스펙션 ( 형질전환 ) 시킨후 atra 를처리하여발현정도를측정하여보았다. atra 는 NF-kB 결합부위를포함하는 plasmid 의발현을 3 배이상증가시켰다. 또한, SRE 와 c-myc 을포함하는 plasmid 의발현을 2 배이상증가시켰으나, CRE, AP-1, c-jun 을포함하는 plasmid 의발현에는크게영향이없었다.(Fig. 3. B) 이결과는 atra 에의한 TLR-5 유전 A B Fig. 1. Effect of all-trans retinoic acid on the expression of Toll-like receptors in THP-1 cells. A. THP-1 cells were stimulated with a 10-8 M concentration of RA for 24 hours. Total RNAs were isolated from the cells and analyzed for human TLR mrna expression by RT-PCR. B. Gene expression of human TLR-5 was analyzed at the same condition by real-time PCR. (DMSO: dimethyl sulfoxide only, RA: retinoic acid, TLR: Toll-like receptor, GAPDH: glyceraldehyde 3 phosphate dehydrogenase, THP: human monocytic leukemia cell line, RT-PCR: reverse transcription polymerase chain reaction) Fig. 2. Effects of all-trans retinoic acid on the expression of human TLR-5 gene on the various human cell lines. The various human immune and cancer cell lines including Molt4, EBV-B, Raji, U937, Huh7, HepG2, SKBR3, HBL-100, Hela, 293T, A549, A431, Colo201 cell were stimulated with a 10-8 M concentration of atra for 24 hours. Total RNAs were isolated from the cells and analyzed for human TLR-5 mrna expression by RT-PCR. (DMSO: dimethyl sulfoxide only, RA: retinoic acid, GAPDH: glyceraldehyde 3 phosphate dehydrogenase, TLR: Toll-like receptor, RT-PCR: reverse transcription polymerase chain reaction) 485
J Korean Assoc Oral Maxillofac Surg 2010;36:481-9 A A B B Fig. 3. All-trans retinoic acid (atra) induces the expression of mouse TLR-5 gene on RAW264.7 cells. A. The mouse RAW264.7 cells were stimulated with a 10-8 M concentration of atra for 24 hours. Total RNAs were isolated from the cells and analyzed for mouse TLR-5 mrna expression by RT-PCR. B. Raw264.7 cells were transiently co-transfected with various promoter constructs luciferase vector such as NF-kB, CRE, AP-1, SRE, c-jun, and c-myc with prl CMV. After 6 hours of transfection, the cells were left untreated or treated with 10-8 M atra for the last 24 hours. Relative luciferase activity was determined as described in materials and methods. Results are represented as means ±SD of a representative experiment performed in triplicate. (DMSO: dimethyl sulfoxide only, RA: retinoic acid, GAPDH: glyceraldehyde 3 phosphate dehydrogenase, TLR: Toll-like receptor, RAW: mouse leukemia monocyte macrophage cell line, RT-PCR: reverse transcription polymerase chain reaction, CMV: cytomegalovirus, SD: standard deviation) Fig. 4. Effect of various signaling inhibitors on all-trans retinoic acid (atra)-induced TLR-5 expression. A. THP-1 cells were pretreated for 0.5 hour with the indicated inhibitors and then stimulated with 10-8 M atra in the presence or absence of the same inhibitor for an additional 24 hours. Total RNAs were prepared and analyzed by RT-PCR for TLR-5 or β-actin. (NT: no treatment, DMSO: dimethyl sulfoxide only, PDTC: pyrrolidine dithiocarbamate (NF-kB inhibitor), SP: SP600125 (JNK inhibitor), PD: PD98059 (extracellular signal-regulated kinase inhibitor), SB: SB203580 (p38 inhibitor), LY: LY294002 (PI3K inhibitor), AG: AG490 (JAKII inhibitor), GAPDH: glyceraldehyde 3 phosphate dehydrogenase, TLR: Toll-like receptor, THP: human monocytic leukemia cell line, RT-PCR: reverse transcription polymerase chain reaction) 자의발현에 NF-kB, SRE, c-myc 전사조절인자가단독또는공동으로작용할가능성을보여준다. 4. TLR-5 발현에관여하는신호전달인자분석 atra 에의한 TLR-5 유전자의발현에 NF-kB, SRE, c-myc 전사조절인자중어떤인자가관여하는지알아보기위하여다양한종류의신호전달저해제 (signal inhibiter: PDTC (NF-kB inhibitor), SP: SP600125 (JNK inhibitor), PD: PD98059 (ERK inhibitor), SB: SB203580 (p38 inhibitor), LY: LY294002 (PI3K inhibitor), AG: AG490 (JAKII inhibitor)) 를전처리한후 atra 에의한 TLR-5 의발현을조사하였는데, atra 에의해유도된 TLR-5 의발현은 NF-kB 저해제인 pyrrolidine dithiocarbamate (PDTC) 의처리에의해서만감소되었다.(Fig. 4) 이결과는 TLR-5 의유전자발현에전사조절인자 NF-kB 가중요하게작용하고있음을보여주는것이다. 세포를수확하여 ERK, JNK 및 p38 MAPK 의활성을분석하여보았다. atra 는 ERK 를 120 분에서 phosphorylation 시켰다. 또한 JNK 도 15 분에활성화되어 120 분까지내신호전달경로를분석하기위하여 atra 처리후주어진시간단위별로지속적으로 phosphorylation 을증가시켰으나 p38 MAPK 의활성에는영향을미치지않았다.(Fig. 5) 이결과에서 tumor necrosis factor (TNF)-α 나 Fas ligand (FasL) 와같은사이토카인이초기 ( 약 15 분 ) 에 ERK 와 JNK 를최대로활성화시키는데반해 atra 는 120 분에최대활성을보여후반부에작용함을알수있었다. 486
All-trans retinoic acid 가면역세포의 Toll-like receptor 5 발현에미치는영향 A B Fig. 5. Effects of all-trans retinoic acid (atra) on the activation of ERK, JNK, and p38 MAPK in THP-1 cells, THP-1 cells were serum-starved for 12 hours, stimulated with atra (10-8 M) for the indicated times, and then lysed. Cell lysates were resolved by SDS-PAGE and then subjected to Western blotting withan antibody against phosphorylated ERK, JNK, and p38 MAPK. The same membrane was stripped and reprobed with anti-erk, anti-jnk, anti-p38 MAPK, and anti-actin antibody. (THP: human monocytic leukemia cell line, NT: no treatment, RA: retinoic acid, ERK: extracellular signal-regulated kinase, JNK: MAPK: SDS-PAGE: ) 5. 사람 TLR-5 포로모터의재조합및특성분석 atra 에의해발현이유도되는사람 TLR-5 유전자발현의중요한전사조절인자를밝히기위하여 TLR-5 유전자의 5 상위프로모터지역을분석하여보았다. TLR-5 promoter 1.3-kb 단편을사람 genomic DNA 로부터 PCR 을통해증폭하여얻었고, luciferase reporter plasmid 인 pgl3 basic vector 로재조합하였다. RAW264.7 세포에 TLR-5 promoter construct 와 prl CMV plasmid 를함께형질전환하여 Luciferase assay 로 promoter 활성을측정하였다. Promoter 활성은 atra (10 nm) 를 24 시간동안처리한후측정하였고, 처리하지않은형질전환체와비교하였다. TLR promoter 염기서열을 -1,300 및 -1,200 을포함하는 construct 들은 atra 처리시 luciferase gene 활성이대략 2 배높게유도되었다. -360 을포함하는 construct 는 atra 처리시 luciferase gene 활성이대략 2.5 배높게유도되었으나 -150 을포함하는 construct 는 1.5 배로감소시켰다.(Fig. 6. A) 그러므로 TLR-5 promoter 부위에는 -380 과 -160 에 atra 에의해유도된전사조절인자의결합부위가존재할것으로사료되며, THP-1 에서 atra 에의한 TLR-5 의활성에중요한전사조절인자일것으로 Fig. 6. Promoter constructs and delineation of human TLR-5 promoter region. A. THP-1 cells were transiently cotransfected with various promoter constructs or empty luciferase vector with prl CMV to compare transfection efficiencies. After transfection for 6 hours the cells were left untreated or treated with 10-8 M atra for the last 24 hours. Relative luciferase activity was determined as described in materials and methods. Results are represented as means ±SD of a representative experiment performed in triplicate. B.Nucleotide sequence of the promoter region of human TLR-5 gene. The 1,300 bp sequence of the 5 -flanking region of TLR-5 is shown. The putative transcription start site is indicated by +1 as shown mrna sequences from GenBank accession number NM_003268. The arrow indicates the transcription start site. Underlined sequences are possible transcription factor binding sites, as predicted by GenomeNet. (THP: human monocytic leukemia cell line, NT: no treatment, RA: retinoic acid, TLR: Toll-like receptor, CMV: cytomegalovirus, SD: standard deviation) 판단되었다. Promoter 분석프로그램인 GenomeNet 의 sequence motif search 를이용하여 TLR-5 유전자의 5 상위프로모터지역을분석한결과 -380 과 -160 사이에는 NFkB/Rel 결합부위가존재하는것으로밝혀졌으며 (Fig. 6. B), 이결과는 NF-kB construct 및 inhibitor PDTC 처리결과와잘일치함을알수있었다. Ⅳ. 고찰 Vitamin A 와그대사체인 atra 나 9-cis-retinoic acid 는광범위한면역강화효과 (immunopotentiating effects) 를가지고있기때문에매우유용한물질로간주되어왔다 20-22. 많은연구들은 atra 가 T 및 B 세포의활성에특히효과가있는것으로보고하고있다 11,23,24. B-cell Ig 의생산에 atra 의면역조 487
J Korean Assoc Oral Maxillofac Surg 2010;36:481-9 절효과들은 T 세포 11,25 나다른세포들 23,26,27 에의해생산된사이토카인과같은 soluble factors 에의해나타난다는보고도있다. 그러나 atra 가면역세포에직접작용하여 TLR-2 의활성을억제한다는보고도있다 19. 이연구에서는 atra 가면역세포에작용하여유전자발현을유도하는기전을이해하기위하여사람 monocytes-macrophages 계통의 THP-1 세포에서 TLR 의발현에미치는영향을확인하고그작용기작을밝히고자하였다. atra 를면역세포인 THP-1 에처리하였을때 TLR-2 의발현은현저히감소하였다. 이는 Lui 등 19 이수행해서얻은결과와일치하였다. 또한 atra 는 TLR-5 및 -10 의발현을크게유도하였으며, 나머지 TLR 의발현에는영향이없었다. 동일한유도기작이다양한세포에서공통적으로일어나는기작인지확인하기위하여사람면역세포및암세포에동일한방법으로처리한결과면역세포계통에서만유사한발현유도가발생됨을알수있었다. atra 가 THP-1 세포에서 TLR-5 유전자의발현을 7 배이상유도하였기때문에 TLR-5 유전자를대상으로 atra 의작용기작을분석하고자하였다. THP-1 세포에 TLR-5 promoter constructs 를다양한 transfection reagent 로형질전환하였으나그효율이 5% 에도미치지않아동일한 monocytes-macrophages 계통의생쥐 RAW264.7 세포를이용하였다. RAW264.7 세포에 atra 를처리한결과 THP-1 세포와동일하게 TLR-5 유전자발현이유도됨을확인할수있었으며, 형질전환효율도 30% 에달하는것으로확인되었다. TLR-5 의 promoter 부위를검색한결과 -1,100 bp 부위에 RAR-related orphan receptor α2 결합부위가존재함을확인하고이부위가 atra 의작용에중요하게작용할것으로예상하였다. 그러나 RAW264.7 세포에 -1,300 bp 의 construct 를이용한전사조절실험에서이부위는전사조절에크게작용하지않는것으로확인되었다. 이를해결하기위하여기존에알려진다양한 promoter construct (NF-kB, CRE, AP- 1, SRE, c-jun, c-myc) 를형질전환시킨후 atra 를처리하였을때 NF-kB 의활성을 3 배이상유도함을확인할수있었다. 또한 NF-kB 의 inhibitor 인 PDTC 를처리하였을때 TLR- 5 유전자의발현을현저히감소시켰다. 이자료를근거로 TLR-5 의 promoter 부위를검색한결과 NF-kB 결합부위가 2 곳이존재함을확인할수있었다. 이자료를근거로각각의 promoter construct 를제작하고전사조절실험을한결과전사개시부위에서 -380 bp 에존재하는 NF-kB 부위는 atra 에영향이없었으며, -160 bp 지역에위치한 NF-kB 결합부위가중요하다는것을확인할수있었다. 또한 -50 bp 이내에존재하는 Sp-1/GC 부위및 NF-kB 부위가 TLR-5 유전자의 basal level 발현에중요할것으로생각된다. Chen 등 28 에의하면 atra 는 THP-1 세포에서수용체인 RXR-α 및 RAR-α 의발현을저해한다고하였으며, 이연구의예비실험에서도유사한결과를보여주었다. 이자료를기초로 THP-1 세포에존재하는 atra 유도신호전달경로를분석하기위하여 ERK, JNK 및 p38 MAPK 의활성을분석하여보 았다. atra 는 ERK 및 JNK 의활성을 120 분에최대활성을보여 TNF-α 나 FasL 와같은사이토카인이초기에 ERK 와 JNK 를최대로활성화시키는것과는다른결과를보이고있어후기에작용함을알수있었다. 이상의결과를확증하기위하여추가적으로 TLR-5 protein 의세포표면발현및 electrophoretic mobility shift assay (EMSA) 를통한 NF-kB 의실질적인결합을증명하여야할것이다. Ⅴ. 결론 이연구에서 atra 는면역세포인 THP-1 에작용하여 TLR- 5 및 TLR-10 의발현을크게유도하였으며, TLR-2 의발현은억제하였으나나머지 TRL 에는영향이없었다. TLR-5 유전자발현을유도하는전사조절인자의분석에서 atra 는 NF-kB 의활성을유도하여 TLR-5 유전자의발현을유도함을확인할수있었다. 또한신호전달분석에서 atra 는 ERK 와 JNK 을활성화하였으며, 활성시간은사이토카인에의한유도시간보다는늦게나타났다. TLR-5 의 promoter 염기서열분석에서 -160 bp 부위에위치한 NF-kB 가 atra 에의한유전자의활성에중요하며, -50 bp 전후에위치한 Sp- 1/GC 부위가 TLR-5 유전자의기본활성에중요할것으로사료된다. 이결과는면역활성을유도하는 vitamin A 유도체의임상활용및 TLR-5 의 ligand 인 flagellin 을이용한면역활성연구에유용한정보를줄것으로예상된다. References 1. Hashimoto C, Hudson KL, Anderson KV. The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein. Cell 1988;52:269-79. 2. Medzhitov R, Preston-Hurlburt P, Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997;388:394-7. 3. Kaisho T, Akira S. Toll-like receptor function and signaling. J Allergy Clin Immunol 2006;117:979-87; quiz 988. 4. Lee SJ, Lee S. Toll-like receptors and inflammation in the CNS. Curr Drug Targets Inflamm Allergy 2002;1:181-91. 5. Goodman DS. Vitamin A and retinoids in health and disease. N Engl J Med 1984;310:1023-31. 6. Lotan R. Immunomodulatory effects of retinoids. J Nutr Growth Cancer 1986;3:57-65. 7. Lotan R. Retinoids in cancer chemoprevention. FASEB J 1996; 10:1031-9. 8. Hofmann C, Eichele G. Retinoids in development. In: Sporn MB, Roberts AB, Goodman DS, eds. The retinoids: biology, chemistry and medicine. 2nd ed. New York, NY: Raven Press; 1994: 387-442. 9. Israel H, Odziemiec C, Ballow M. The effects of retinoic acid on immunoglobulin synthesis by human cord blood mononuclear cells. Clin Immunol Immunopathol 1991;59:417-25. 10. Wang W, Ballow M. The effects of retinoic acid on in vitro immunoglobulin synthesis by cord blood and adult peripheral blood mononuclear cells. Cell Immunol 1993;148:291-300. 11. Ballow M, Wang W, Xiang S. Modulation of B-cell immunoglobulin synthesis by retinoic acid. Clin Immunol Immunopathol 488
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