대한피부연구학회지제 14 권, 제 1 호원저 29 Korean Journal of Investigative Dermatology 2007 ; Vol. 14, No. 1, pp. 29-35 Keratinocyte 에서 Rtinoic acid 가 Human β Dfensin-2 과 LL-37 의발현에미치는영향 김연진 안지영 서성준 홍창권 중앙대학교의과대학피부과학교실 The Effect of Retinoic Acid on Expression of Human Beta Defensin-2 and LL-37 in Keratinocyte Yeon Jin Kim, Ji Young Ahn, Seong Jun Seo, and Chang Kwan Hong Department of Dermatology, Chung-Ang Univerisity College of Medicine, Seoul, Korea Human skin is able to mount a fast response against harmful injury through the rapid production of inducible antimicrobial peptide (AMP) such as the human β-defensins (hbd), cathelicidin-family termed LL-37. Keratinocyte secrete AMPs upon stimulation with exogenous factor such as lipopolysaccharides (LPS), UV radiation and proinflammatory cytokines-tumor necrosis factor (TNF)-α and interleukin (IL)-1. In psoriasis patients, antimicrobial peptides tend to be expressed abundantly in the epidermal layer of the skin. Retinoic acid (RA) modulates immunological and inflammatory response and it also affects epidermal cell growth and differentiation by activation the retinoid acid receptor. RA is effective and safe in the treatment of psoriasis. This study aimed to investigated the hbd-2 and LL-37 expressions of human keratinocytes after exposure to the RA. Keratinocytes were cultured and to assess the expression of defensin-2 and LL 37 in the control group that was not treated with any stimulants, the experiment group treated with LPS, TNF-α, or UVB each, the experiment group treated again with RA, reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical stainings were performed. Based on our experiment results, it was found that hbd2 and LL-37 expression had been regulated by stimulants-uv, LPS, TNF-α and RA in keratinocyte. We found retinoic acid can down-regulate both hbd and LL-37. Key words : Human beta defensin-2, LL-37, Retinoic acid, Keratinocyte 서론피부는외부에대한일차적인보호막역할이외에도다양한형태의면역반응을나타내는대표적인장기이다. 피부의면역반응에는피부에존재하는여러세포들과국소림프절이관여하고이외에도내인성항균펩타이드를생산하여여러유해한미생물들을중화시키고사멸시키는역할을수행 한다 1,2. 항균펩타이드는호흡기, 비뇨기와피부상피등의여러조직에서분리된다 3-5. 인간의항균펩타이드중 defensin과 cathelicidin이중요하며 defensin에관해서가장널리알려져있다 3,4,6,7. 항균펩타이드가아토피피부염에서는발현이감소하고심상성건선환자의피부에서는발현이증가하는것은이미밝혀진바있다 7,8. 아토피피부염과건선이모두피부장벽에결 저자연락처 : 서성준, (156-755) 서울동작구흑석동 224-1, 중앙대학교의과대학피부과학교실, Tel: 02) 6299-1525 / Fax: 02) 838-2359 / E-mail: drseo@hanafos.com
30 대한피부연구학회지 : 제 14 권제 1 호 2007 손이오는질환임에도불구하고아토피피부염에서건선과비교하여더감염성질환이증가하는것은항균펩타이드의발현의저하에서그원인을찾고있다 9,10. 항균펩타이드의저하는아토피피부염에서증가되어있는 Th-2 싸이토카인에의한억제때문이며, 각질형성세포에 IL-4, IL-13을투여할때 hbd의증가가억제됨이보고되었다. 건선은 Th1 관련질환으로 IL-6, IL-8이증가되어있으며, 항균펩타이드의하나인 LL-37은 IL-6에결합부위를가지고있어건선환자에서발현이증가되어있다. 이로인한각질형성세포분화의증가로상피는두꺼워져있다 11,12. Retinoic acid는각질형성세포의분화에영향을미치며, 건선환자에서분화, 과증식을억제시키므로상피증식상태를정상화시킨다. 따라서 retinoic acid는건선치료제로널리사용되고있다. 본연구는건선에서항균펩타이드의발현및생산이증가하는기전을건선치료제인 retinoic acid를투여함으로써규명하려하였고, 더나아가항균펩타이드의분비조절인자가무엇인지그리고피부에서의다른기능의존재유무를알아보려하였다. 재료및방법 1. 각질형성세포배양사람의각질형성세포를 KBM (Keratinocyte Basal Medium) (Gibco, Carlsbad, CA, USA), 100 cm 3 petridish에서 37, 5% CO 2 의조건으로배양시켰다. 배양된각질형성세포는 2 10 5 개 /ml 으로나누어편평한바닥의 10 cm 2 polystyrene plate에넣고, 세포를 starvation 시킨뒤실험조건에맞게 UVB 20 mj/cm 2, Lipopolysaccharide (LPS; Sigma, St. Louis, MO, USA) 5 μg/ml, TNF-α (Sigma, St. Louis, MO) 100U/ ml, Retinoic Acid (Sigma, St. Louis, MO) 10-7 μg으로처리한후 6시간, 12시간, 24시간동안배양한후세포를수확하였다. 2. Ultraviolet B 조사 (UVB) 기존의연구자료를바탕으로 20 mj/cm 2 를조사하였다. 280-320 nm wave로 313 nm wave를 peak wave로하는 Philips TL 20W/12 (Eindhoven, Netherlands) 를사용하였다. 광량은 Waldmann UV-meter (Waldmann, Villigen-Schwenningen, Germany) 를통해모니터하였다. 3. 역전사중합효소연쇄반응 (reverse transcription polymerase chain reaction, RT-PCR) 1) Primer 제작 Gene bank data를기본으로 PCR Primer는 DNA synthesizer (Pharmacia, Bj?rkgatan, Uppsala, Sweden) 를사용하여합성하였다. hbd-2 (128bp) : 5'-ATC TCC TCT TCT CGT TCC TC-3' (sense), 5'-ACC TTCTAG GGC AAA AGA CT-3 (anti-sense) LL-37 (203bp) : 5'-CTG ATG CCT CTT CCA GGT GT-3' (sense), 5'-GAG GGA GCC CTT TCT GAA TC-3' (anti-sense) GAPDH (593bp) : 5'-CCA CCC ATG GCA AAT TCC ATG GCA-3' (sense), 5'-GGT GCT GCT TGT TAG GAG GTC AAG TAA AGG GC-3' (anti-sense) 2) Total RNA 분리각각의조건에서수확된세포에 TRIZol reagent (Invitrogen, Carlsbad, CA, USA) 를 1mL 씩넣고 5분동안흔든후전체양의 0.2 volume에해당하는 chloroform을첨가한후 15초동안 tube를흔든후 3분간둔다. 이혼합물을 4 에서 15분간 12,000 rpm으로원심분리시킨뒤상층액을취하여, 전체양의 0.5 volume의 2-propanol을첨가하고이를다시 4 에서 15분간두었다가 12,000 rpm 으로원심분리하였다. 상층액을제거하고얻은침전물에 70% ethanol 500 μl을넣어 12,000 rpm에서 5분간원심분리시키고진공건조시켰다. 3) cdna 합성얻어진 RNA를 DEPC-DW 30 μl에용해시키고, 2 μg의 RNA를 42 에서 1 μl reverse transcriptase (TaKaRa, Shiga, Japan), 10Xbuffer 2 μl, 10mM dntp 2 μl (dntp mix), oligo dt primer 1 μl, RNase inhibitor 0.5 μl, 25 mm MgCl 2 4 μl를넣고역전사시켰다. 4) 중합효소연쇄반응 RT-PCR에서얻어진 2 μl의 cdna를조건설정후증폭시켰다. PCR의조건은다음과같다. i) 94 에서 5분 (initial denaturation), ii) 94 에서 1분 (denaturation), iii) 59 에서 1분 (annealing), iv) 72 에서 1분 (extension), v) 72 에서 10분 (final extension) 으로하여 2단계에서 4단계까지 35회반복하였다. 5) 전기영동 (Electrophoresis) Ethidium bromide 1 μg을함유하는 1.5% agarose gel에 20 μl의 reaction mixture를섞어전기영동하여자외선조사
김연진등 :Keratinocyte 에서 Rtinoic Acid 가 Human β Dfensin-2 과 LL-37 의발현에미치는영향 31 로확인하였다. 6) 정량분석 (Quantitative analysis) 발현된양을정량적으로분석하기위해밀도계 (densitometer) 를이용하여 GAPDH 발현량에대한 hbd-2 유전자의양 (hbd-2/gapdh), LL-37 유전자의양 (LL-37/ GAPDH) 을절대적인값으로표시하였다. 7) 통계학적검정각각의실험은모두 3회실시하여나온결과의평균값및표준편차를이용하였으며, 통계학적유의성검정은 ANOVA test로하여 p값이 0.05 이하일때통계학적의의가있다고판정하였다. 4. Western blotting 세포를50 mm Tris-Cl (ph 8.0), 150 mm NaCl, 0.02% sodium azide,100 μg/ml phenylmethanesulfonyl fluoride (PMSF), 1 μg/ml aprotinin, 1% TritonX100가포함된 buffer 에서분해시킨후 4 에서 30분간 12,000 rpm으로원심분리시켰다. 상층액을취하여 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 에서 loading 시켰다. 분리된 protein을 nitrocellulose membrane (Osmonics, Minnesota, MN, USA) 으로옮기고 Tris-buffered saline tween 20 (TBST) 으로 3 번씻어내고 5% skim milk로 block 시켰다. Membrane에 goat anti HBD-2 polyclonal antibody (1:1000 in BSA, SantaCruz, Delaware, CA, USA), goat anti human LL-37 polyclonal antibody (1:1000 in 5% bovine serum albumin, SantaCruz, Delaware, CA) 를붙여 4 에 overnight 동안두고 TBST로 3번씻어내었다. 실온에서 1시간동안 secondary mouse anti-goat peroxidase conjugated antibody (1:2000 in blocking solution, SantaCruz, Delaware, CA, USA) 를붙여두었다. TBST로씻어낸뒤 ECL solution (SantaCruz, Delaware, CA, USA) 에 3분간노출시킨후 X-ray film으로확인하였다 (Roche, Indianapolis, IN, USA). 5. 면역조직화학염색세포를 coverslip (Nunc, Rochester, NY, USA) 에서배양후 10분간 4% paraformaldehyde로고정하고 30분동안 PBS 로 3번 washing 하였다. 3% hydrogen peroxide로 endogenous peroxidase를불활성화시키기위해 10분정도실온에둔후 PBS로 3번씻어준다. 0.2% TritonX100을 cell에침투시키고 PBS로씻어낸후 20분간 3% BSA로 block시키고, 세포에 primary antibody인 goat anti-hbd 2 polyclonal antibody (1 : 100 in 3% BSA), goat anti human LL-37 polyclonal antibody (1:1000 in 5% bovine serum albumin, SantaCruz, Delaware, CA) 를붙인후 4 에서 overnight 시킨후 PBS로세번씻어내고 mouse anti-goat peroxidase-conjugated antibody (1 : 200 in PBS) 를붙여실온에서 1시간두었다. 3 번의 washing 후세포를 3, 3 -diaminobenzidine (DAKO, Glostrup. Denmark) 을이용하여염색하였다. 1. β-defensin 결과 결 Fig. 1. RT-PCR. The expressions of hbd-2 mrna (A), LL-37 mrna (B) in keratinocytes were upregulated when stimulated with LPS, TNF-α, UVB irradiation at 6, 12, and 24 hours. HBD-2 mrna, LL-37 mrna expressions were decreased after treatment with retinoic acid. *: Statistically significant between UV and RA+UV (p<0.01). #: Statistically significant between LPS and RA+LPS (p<0.01). +: Statistically significant between TNF-α and RA+TNF-α (p<0.01). 과 1) 역전사중합효소연쇄반응 (RT-PCR) LPS, UVB 조사, TNF-α로처리한실험군은정상대조군보다 β-defensin mrna 의발현이증가되었고위실험군에 retinoic acid를재처리한실험군에서발현이감소됨을역전사중합효소연쇄반응 (RT-PCR) 에서관찰할수있었다 (Fig. 1A). 통계학적유의성검정은 ANOVA test로하여 p값이 0.01 이하로의의가있었다. A B
32 대한피부연구학회지 : 제 14 권제 1 호 2007 A B Fig. 2. Western blotting. Expression of hbd-2 protein (A), LL-37 protein (B) in keratinocytes were evaluated by Western blotting study using polyclonal antibody to hbd-2,ll-37 at 24 hours post stimulation. The expression of hbd-2,ll-37 were decreased after additional treatment of retinoic acid as compared with those stimulated with LPS, TNF-α, UVB irradiation respectively in keratinocytes. A B Fig. 3. Immunostainings for hbd-2(a), LL-37 (B) were more intense on LPS, TNF-α treated, UV irradiated groups than in normal control. In additional groups treated with retinoic acid expression of hbd-2 was less intense than those of simply exposed to stimulants groups at 24 hours. (A) hbd-2(24hr). Control UV LPS TNF-α RA RA+UV RA+LPS RA+TNF-α. (B) LL-37(24hr). Control UV LPS TNF-α RA RA+UV RA+LPS RA+TNF-α. 2) Western blotting LPS, UVB 조사, TNF-α로처리한실험군은정상대조군보다 β-defensin 단백질의발현이증가되었고위실험군에 retinoic acid를재처리한실험군에서발현이감소됨을 Western blotting에서관찰할수있었다 (Fig. 2A). 3) 면역조직화학염색 LPS, UVB 조사, TNF-α로처리한실험군은정상대조군보다 β-defensin의발현이증가되었고위실험군에 retinoic acid를재처리한실험군에서발현이감소됨을면역조직화학염색에서관찰할수있었다 (Fig. 3A). 2. LL-37 결과 1) 역전사중합효소연쇄반응 (RT-PCR) LPS, UVB 조사, TNF-α로처리한실험군은정상대조군보다 LL-37 mrna의발현이증가되었고위실험군에 retinoic acid를재처리한실험군에서발현이감소됨을역전사중합효소연쇄반응 (RT-PCR) 에서관찰할수있었다 (Fig. 1B). 통계학적유의성검정은 ANOVA test로하여 p값이 0.01 이하로의의가있었다. 2) Western blotting LPS, UVB 조사, TNF-α로처리한실험군은정상대조군보다 LL-37 단백질의발현이증가되었고위실험군에 retinoic acid를재처리한실험군에서발현이감소됨을 Western blotting에서관찰할수있었다 (Fig. 2B). 3) 면역조직화학염색 LPS, UVB 조사, TNF-α로처리한실험군은정상대조군보다 LL-37의발현이증가되었고위실험군에 retinoic acid 를재처리한실험군에서발현이감소됨을면역조직화학염색에서관찰할수있었다 (Fig. 3B). 고찰항균펩타이드는세균, 바이러스, 곰팡이등에대한넓은항균작용을가지는작은분자량의단백질이다 1,6. 항균펩타이드는첫번째면역단계에작용하며, host repair와 adaptive
김연진등 :Keratinocyte 에서 Rtinoic Acid 가 Human β Dfensin-2 과 LL-37 의발현에미치는영향 33 immune response의관계에중요역할을한다. 인간에서항균효과를나타낸다고알려진항균펩타이드로는 salivary histatins, lactoferrin, defensin, cathelicidin, secretory leukocyte protease inhibitor(slpi), neutrophil gelatinase associated lipocalin (NGAL) 등이있으나이중에서 defensin과 cathelicidin이중요하며 defensin에관해서가장널리알려져있다 3,4,6,7. Defensin은저분자량의시스테인 (cysteine) 이풍부한양이온펩타이드로세균, 바이러스및진균에대해서광범위한항균작용을나타내며, 이외에도단핵구에대한화학주성효과를통해감염에대한세포매개성면역반응을증강시키기도하며, 표피세포와섬유모세포에대한증식및분화과정에영향을미쳐창상치유를촉진하는기능도있다. Defensin 은이중화학결합 (disulfide bond) 위치, 유전자의위치, 조직의분포에따라 α와 β 두가지로분류한다 13-15. β defensin (hbd) 은현재까지 4가지가알려져있는데 hbd-1은혈액투석의과정에서처음발견되었으며소변과자궁경부점액에서분리되어비뇨생식기계의항균효과에일익을담당하는것으로알려져있다. hbd-2는건선환자의각질에서처음분리되었고미생물과같은유해인자. 사이토카인등의전염증성자극, 각질형성세포의분화에의해유도발현되어피부나상피세포의방어인자로작용한다. 최근건선환자의각질에서처음분리된 hbd-3는그람음성세균과그람양성세균에대해넓은범위의항균효과를나타내며, 이는상피조직뿐만아니라심장, 편도선, 골격근등에도존재하는것으로알려져있다. hbd-4는피부에서의발현은아직밝혀지지않았으며위의전정부에상대적으로높게발현되어있다고알려져있다 5,16. 이러한 β-defensin의발현의조절은대부분이 NF-κB로설명하고있다 12,17. IL-1β, TNF-α와 LPS, UV 조사, 바이러스등의자극은 NF-κB의활성화를통하여염증성병변을일으킨다 11.18. 자극에의한염증성병변의경우 NF-κ B의부착부위를가진 hbd-2는유도발현되어지고, 부착부위가없는 hbd-1은발현되지않는것으로설명된다. Cathelicidin은 cathelin 전구체기를가지고있기때문에명명되어졌다. 분자구조는강한전구체부분인 NH3 말단기와구조적으로다양성을나타내는양성 COOH 말단기로구성되어있다. 인간에서 LL-37은 cathelicidin에속하는항균펩타이드로처음시작하는아미노산이 2개의 leucine이며 37개의아미노산으로구성되어있다 19-21. Cathelicidin의항균작용은 elastase나 proteinase-3 등의단백분해작용에의한 cathelin 기의분해에의해활성화된다 22. 염증성자극이나미생물침범에의해발현이증강되며피부나점막의상피세포에존재한다. 그람양성, 음성세균과바이러스에대한항균 작용을보이며창상치유에도역할을담당하며호중구나단핵구 T세포, 비만세포에화학주성인자로작용한다. 초파리에서 Toll의자극은 NF-κB의유도로연결되어 defensin, cecropins, drosomycin 등을포함한항균펩타이드의활성화에이른다. 초파리에서의결과는포유류에서항균펩타이드의유도기전의실마리를제공한다. 최근기관지상피에서 LPS가 hbd-2를유도되는것이보고되었고, 이것은초파리에서와동일한 Toll 기전의항균펩타이드활성화의가능성을보여준다 3,23. Mookherjee 등 24 은 LL-37의발현이 TLR에의해조정된다고하였고, LL-37이 TLR에서 NF-κB 의유전자발현변화에영향을미친다고하였다. LPS, UVB 조사, TNF-α는 NF-κB 활성을자극한다. 또한상피에서 inhibitor of NF-ΚB(IκBα) 의결핍은상피의두꺼워짐과건선유사피부병변을나타낸다는보고가있다 25. 이는 NF-ΚB의활성화로인한항균펩타이드의유도기전을뒷받침한다. 기존의보고와같이 LPS, UVB 조사, TNF-α로처리한실험군은정상대조군보다 β-defensin과 LL-37의발현이증가되었고위실험군에 retinoic acid를재처리한실험군에서발현이감소됨을관찰할수있었다. Retinoic acid는비타민 A의자연대사물로, 좌창치료와과각화증이동반되는여러피부질환치료에국소도포제로사용되어왔다 26. Kaidbey 등 27 은정상피부에 RA를국소도포하여각질층이정상두께의반으로감소되어각질층의투과성이증가하는것을관찰하였다. Retinoic acid의건선환자에서의임상적치료효과에도불구하고아직까지치료작용기전은확립되어있지않다 28. Retinoic acid 는세포막의 retinoic acid 수용체의발현이증가되어이들수용체가 AP-1(activated protein-1) 을구성하는 Fos나 Jun 단백질과상호작용하여화합물을이루어 DNA와의결합능력을잃게되므로 AP-1의활성도가상실된다고보고된바있다 29-31. AP-1은여러발암물질, 증식촉진인자및 cytokine 등의자극에반응하여활성도가증가하는것으로알려져있다. AP-1은 Fos와 Jun 양자에의해 encode된 protein dimer로유전자발현과정중전사과정의개시에관여하는인자로 metallothioneins, plasminogen activator, collagenase 등과같은 DNA-damage-inducible(DDI) 유전자들의전사를조절하는데관여한다 32,33. 발암물질이나증식촉진인자등이여러 signal transduction 과정을통해자극되더라도 AP-1 활성도증가로통합되는결과로된다는여러보고가있다 34-36. Retinoic acid 가 AP-1의활성도를억제하는지여부는밝혀져있지않지만, 여러보고에서처럼본실험에서 retinoic acid에의한 AMP의발현감소는 AP-1의활성도감소로인한증식촉진의감소로인한것으로추측된다. 관련사이토
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