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1 J. Exp. Biomed. Sci. 12 (2006) Molecular Characterization of Ischemia-Responsive Protein 94 (irp94) Response to Unfolded Protein Responses in the Neuron Seung-Whan Kim 1, Kisang Kwon 3, Kee-Sun Shin 4, Seung-Ho Kim 2 and O-Yu Kwon 3 1 Department of Emergency Medicine Chungnam National University Hospital, aejon , Korea. 2 Department of Emergency Medicine, Yonsei University of Medicine, Seoul , Korea. 3 Department of Anatomy College of Medicine Chungnam National University, aejon , Korea. 4 Korea Research Institute of Bioscience and Biotechnology, aejon , Korea he ischemia-responsive 94 gene (irp94) encoding a 94 kda endoplasmic reticulum resident protein was investigated its molecular properties associated with unfoled protein responses. First, the expression of irp94 mrna was tested after the reperfusion of the transient forebrain ischemia induction at the central nervous system in three Mongolian gerbils. Second, irp94 expression in PC12 cells, which are derived from transplantable rat pheochromocytoma cultured in the DMEM media, was tested at transcriptional and translational levels. he half life of irp94 mrna was also determined in PC12 cells. Last, the changes of irp94 mrna expression were investigated by the addition of various ER stress inducible chemicals (A23187, BFA, tunicamycin, D and H 2 O 2 ) and proteasome inhibitors, and heat shock. High level expression of irp94 mrna was detected after 3 hours reperfusion in the both sites of the cerebral cortex and hippocampus of the gerbil brain. he main regulation of irp94 mrna expression in PC12 cells was determined at the transcriptional level. he half life of irp94 mrna in PC12 cells was approximately 5 hours after the initial translation. he remarkable expression of irp94 mrna was detected by the treatment of tunicamycin, which blocks glycosylation of newly synthesized polypeptides, and H 2 O 2, which induces apoptosis. When PC12 cells were treated with the cytosol proteasome inhibitors such as ALLN (N-acetyl-leucyl-norleucinal) and MG 132 (methylguanidine), irp94 mrna expression was increased. hese results indicate that expression of irp94 was induced by ER stress including oxidation condition and glycosylation blocking in proteins. Expression of irp94 was increased when the cells were chased after heat shock, suggesting that irp94 may be involved in recovery rather than protection against ER stresses. In addition, irp94 expression was remarkably increased when cytosol proteasomes were inhibited by ALLN and MG 132, suggesting that irp94 plays an important role for maintaining the ERAD (endoplasmic reticulum associated degradation) function. Key Words: irp94, Chaperone, Endoplasmic reticulum, Ischemia, Neuron 서 허혈 (ischemia) 이란조직에혈류공급이중단되어서세포손상이일어나는것을말하며뇌허혈 (cerebral ischemia) 은뇌졸중또는중풍으로잘알려져있다. 그리고일단허혈이한번일어난후에혈액이재관류 (reperfusion) 하여도신경세포의손상은치명적이다. 뇌허혈은혈류결핍의양상및부위에따라전뇌허혈증 (global cerebral ischemia), 전뇌허혈 론 * 논문접수 : 2006년 5월 1일수정재접수 : 2006년 5월 17일 교신저자 : 권오유, ( 우 ) 대전광역시중구문화동 6, 충남대학교의과대학해부학교실 el: , Fax: oykwon@cnu.ac.kr 증 (forebrain ischemia), 국소성뇌허혈증 (focal cerebral ischemia) 등으로나눌수있다. 실험동물모델에서일과성전뇌허혈 (transient forebrain ischemia) 은허혈에특히취약한부위로알려진해마 (hippocampus) 의 CA1 부위에선택적신경세포사 (selective neuronal death) 를유도하는것으로보고되고있다 (Pulsinelli et al., 1982; Wolf et al., 1992). 이와같은신경세포사는일과성전뇌허혈후에수일간전개되기때문에, 급성에너지소실에의한단순신경세포괴사 (simple neuronal necrosis) 와는달리, 성숙현상 (maturation phenomenon) 혹은지연성신경세포사 (delayed neuronal death) 라고부른다. 최근분자생물학이발달하면서실험적전뇌허혈유도후의유전자나단백질발현과같은신경세포의스트레스반응이많이연구, 보고되고있다 (Morimoto et al., 1994). 즉, 일과성전뇌허혈유도후 immediate early genes,

2 apoptosis-related gene, epidermal growth factor (EGF), faciogenital dysplasia gene product (FGD), apoptosis promoter gene (APG), sulfated glycoprotein-2, interleukin-1 beta, heat shock protein 70 (hsp70), hsp72, fibroblast growth factor-receptor, endoplasmic reticulum protein 72 (Erp72), brain-derived neurotrophic factor, cyclooxygenase-2 등많은유전자들의활성화및단백질유도가보고되었다 (Nowak, 1991; Blumenfeld et al., 1992; akami et al., 1992; akami et al., 1993; Wiessner et al., 1993; Yabuuchi et al., 1994; Higashi et al., 1995; Sommer et al., 1995; Chen et al., 1996; Ohtsuki et al., 1996; Mochizuki et al., 1998; sukahara et al., 1998; Xue et al., 1998; Kawahara et al., 1999). 그러나이같은각종인자들이허혈유도과정에서어떤역할을하는지, 신경세포의방어수단으로서어떻게작용하는지, 허혈의진행과정에서단백질분해에어떻게작용하는지, 이웃한신경세포에게어떤종류의신호를어떻게전달하는지등과같은문제는아직까지분명하게밝혀져있지않고있다 (Higashi et al., 1995). 이들중일부유전자는신경세포의괴사를촉진시키지만, 반면에어떤유전자들은일과성전뇌허혈에서신경세포의생존을돕기도한다 (Mochizuki et al., 1998). 예를들어일과성허혈후에는 apoptosis-effector gene 인 bax의발현이증가하여신경세포의괴사를유도하는반면, c-jun 같은암유전자나 retinoic acid-responsive gene 등의유전자는허혈에대한내성에관여한다는사실이보고되었다 (Sommer et al., 1995; Chen et al., 1996). 그러나많은연구보고에도불구하고허혈로유도되는신경세포의변성과방어에대해서는아직도분자생물학적인기전이명확히밝혀지지않고있다. 1999년 Yagita 에의해처음보고된 ischemia-responsive protein 94 kda (irp94) 는일과성전뇌허혈에빠진백서의뇌에서 differential display-polymerase chain reaction (DD-PCR) 방법으로분리된유전자로서, hsp110 family에속하지만지금까지보고된것과는전혀다른분자특성을보인다 (Yagita et al., 1999, 2001). 백서의정상조직중 irp94의발현이확인되는곳은폐, 위, 간, 비장, 신장, 고환, 뇌로알려져있다. irp94 는백서의총경동맥을임시로결찰하여허혈을유도했을경우 15분째에가장강하게발현하며, 10분허혈후재관류를시켰을때에는 12시간째까지발현이증가한다 (Koh et al., 2000). 배양세포를대상으로 irp94의샤페론 (chaperone) 성질을알아본 AP-depletion 실험에서는, AP 결핍 2시간째부터강한발현을보였으며, 소포체 (endoplasmic reticulum; ER) 샤페론으로잘알려진 Erp72보다민감한반응을보여허혈상태가또하나의소포체스트레스로작용함을알수있었다. 최근의 Fischer rat thyroid epithelial cell line (FRL-5 cells) 갑상선배양세포를사용한연구에서는 irp94 발현이소포체스트레스에의해서유도되며갑상샘자극호르몬 (thyroid sti- mulating hormone; SH) 에의해더욱강하게유도되는것으로밝혀졌다 (Kim et al., 2001). 지금까지의 irp94에관해서는유전자의발현위치, 호르몬및소포체스트레스에의한발현등이연구되었지만, 실제로 irp94가허혈성손상으로유도된세포성외부환경의변화에반응하여어떤시기와위치에발현하는지는아직명확히밝혀지지않았다. 뇌허혈에대한가장대표적인분자생물학적연구는세포내칼슘의변화에관한것이다. 칼슘대사항상성의장애는소포체내의스트레스대항반응과신호전달기능이상에관련된일련의반응으로나타나, 정상적이지못한단백질접힘 (folding) 과당쇄부가 (glycosylation) 의결함이동시에발생하여이들이스트레스유발인자 (stress-inducible factor) 가되어 unfolded protein response (UPR) 가유도되어각각의스트레스단백질유전자가작동하게된다. 최근의분자생물학적기법에기반을둔신경조직의허혈성손상연구들에서는허혈로인해소포체내의환경변화가일어나소포체내에서합성되는분비단백질의접힘및조합 (assembly) 이비정상적으로되는것이중요한원인으로밝혀지기시작하였다. 이러한관점에서신경세포내의 UPR에의한 irp94 유전자의발현변화가허혈유도와진행과정에어떤영향을미치는지를분명하게하는것이뇌허혈손상의기전을이해하는데큰도움을줄것이다. 본연구에서는백서의 irp94 cdna의 501 bp DNA 단편을분자탐색자 (molecular probe) 로사용하여 Northern blotting 방법을통해 irp94 mrna의열충격및각종소포체스트레스유발약물들에대한발현정도, mrna의세포내반감기, 발현조절특이성, 허혈후재관류때의뇌조직에서의발현변화, 프로테오좀억제제에대한반응등을관찰하여 irp94 유전자가신경세포의소포체스트레스에서의역할을규명하고자하였다. 실험의결과는허혈손상과관련한 irp94의역할에대한정보제공과함께허혈손상의진단과치료에서새로운가능성을여는데기초자료로이용될수있을것이다. 재료및방법 1. Gerbil 의뇌허혈유도대동맥에서분지되는척추동맥이없어서뇌허혈실험에많이사용되는수컷 Mongolian gerbil을 1.5% 할로탄과 N 2 O/ O 2 (7:3) 의혼합가스로흡인마취시킨후전경부를정중절개하여수술현미경하에서양측총경동맥을노출하였다. 총경동맥을협자 (clip) 를사용하여 30분동안결찰하여일과성전뇌허혈을유도하였다. 협자로는약 60 g의압력을가진신경외과에서혈관수술에사용하는 Sugita mini-temporary clip 을사용하였다. 마취상태에서 30분간의허혈과 3시간동안의재관류과정을거친 gerbil의뇌를신속하게적출하여대

3 뇌피질, 소뇌, 해마, 척수부위에서 total RNA를추출하였다. 대조군과실험군으로는각각세마리씩의 gerbil을사용하였고대조군은허혈및재관류과정을제외한모든마취, 실험조작을실험군과동일하게시행하였다. 2. PC12 세포배양및시약류백서의갈색세포종에서유래한 PC12 세포를 37 의온도와 90% 이상의습도가유지되는환경에서 10% horse serum, 5% fetal bovine serum, 50 units penicillin 을포함한 Dulbecco's Modified Eagle's Media (DMEM) 에서 1주일에 3회배지를교환하면서배양하였다. 실험에사용하는세포는약 70% 의세포충실도 (confluence) 를유지하도록 0.5% horse serum과 0.25% fetal bovine serum이포함된 DMEM에서하루동안배양시켜사용하였다. 본실험에사용된시약중에서특별하게언급하지않은시약류는모두 Sigma Chemical Co. (MO, USA) 의것을사용하였다. 3. R-PCR 및 Northern blotting Gerbil의뇌조직과 PC12 세포로부터의 total RNA 분리에는 RNAzol M B Kit (el-est, Inc. X, USA) 를사용하였다. 뇌조직은약 5~10 mg의조직을 RNAzol M B 800 µl와함께 1.5 ml tube에넣고얼음위에서덩어리가없어질때까지 homogenization 시킨다음 RNA 추출용액 200 µl를첨가하여약 30초간교반후상온에서 5분간방치하였다. PC12 세포는차가운 phosphate-buffered saline (PBS) 로 3회세척후 PBS 800 µl와함께상온에서 5분간방치하여용해시켰다. 이후 12,000 x g (4 ) 에서 15분간원심분리하여얻은 500 µl의상층액에 500 µl의 isopropanol를첨가하여 4 에서 15분간방치후다시 12,000 x g (4 ) 에서 15분간원심분리하여튜브의바닥에서백황색의침전물을얻었다. 이침전물에 75% ethanol 500 µl를첨가하고잘교반하여 7,500 x g (4 ) 에서 8분간원심분리하여 RNA를얻었으며 total RNA의질량은 spectrophotometer로측정하였다. RNA를다룰때는항상장갑을착용하였고 diethylpyrocarbonate (DEPC) 처리된물을사용하였으며, -20 에서 1 mm의 ethylene diamine tetraacetic acid (EDA) (ph 7) 를함유한멸균증류수에보관하였다. Northern blotting을위해 irp94 유전자 (GenBank accession No. AF077354) 의단편을 R-PCR 방법으로얻은 501 bp를 random primer kit로사용하여 32 P를표식하여 molecular probe 로사용하였다. PCR primer 로는 irp94 유전자의 coding region 중 PCR 산물이 501 bp가만들어지도록 forward primer (20- mer); 5'-CAGGAGCCCACCAGA-3', reverse primer (20- mer); 5'-GCACCGCCCCCA-3' 를제작하였다. Gerbil 뇌조직의 total RNA를주형으로사용하여전술한두길잡이를이용하여 31회의중합효소연쇄반응을시행하여 501 bp DNA 단편을얻었다. 이것을 A 플라스미드 (Promega, WI, USA) 에 ligation하여 JM109 competent bacteria 100 µl와 10 ng의재조합플라스미드를섞은후얼음위에 10분방치하고, 42 에서 50초동안열충격을준다음다시 2분동안얼음에방치하였다. 이후상온의 SOC 배지 900 µl을첨가하여 60분간 37 에서 225 rpm으로원심분리하여 JM109를 X-gal과 IPG가포함된 ampicillin agar plate에서하룻밤동안배양하였다. 배양균주에서 white colony를분리배양하여플라스미드의유무를확인하고, 제한효소로소화시켜우무겔 (agarose gel) 에전기영동하여 501 bp의 irp94 DNA 단편을가진플라스미드임을확인하고, 탐색자로사용하기위하여플라스미드를 Wizard Miniprep Kit (Promega, WI, USA) 로대량분리하였다. 삽입된 irp94 DNA의재확인을위해서는 DNA 염기순서분석을시행하여 irp94임을재확인하였다. otal RNA를변성시켜 formaldehyde gel에서분획하였다. 즉, 50% formamide / 2.2 M formaldehyde가포함된 1 MOPS buffer (20 mm MOPS, 5 mm Sodium acetate, 1 mm EDA, ph 7.0) 에 RNA 20 µg을용해시킨다음 65 에서 5분간가열하여상온에서방치한후 1% formaldehyde gel에서분석하였다. 전기영동은 gel을물로여러번닦아서 formaldehyde를제거하고 PVDF Western Blotting Membrane (Schleicher & Schuell BioScience, NH, USA) 에모세관현상을이용하여하룻밤동안시행하였다. PVDF membrane은 5 SSC로희석시킨 20 SSC [174 g NaCl (3 M), 88.2 g sodium citrate (0.3 M), ph 7] 로가볍게세척하고, 68 에서 2시간동안열을가하여 RNA 를고정시켰다. 이후 Quick hybridization solution (Stratagen, WA, USA) 으로 62 에서 30분간 prehybridization 시키고, 32 P 로표식한변성탐색자를사용하여 62 에서 12시간동안중합반응을마친다음 20 SSPE (1L) [175.3 g NaCl (3 M), 27.6 g NaH 2 PO 4 (0.2 M), 40 ml 0.5 M EDA] 를 6 SSPE로희석하여 0.5% SDS를섞어 15분동안 2회, 1 SSPE / 0.5% SDS (37 ) 로 2회세척하고 0.1 SSPE / 0.1% SDS로 60분간 60 에서세척하였다. 필름감광은 Kodak-OMA AR을사용하여 -80 에서하룻밤동안실시하였다. 4. irp94 mrna 의발현특이성및세포내반감기배양한 PC12 세포에전사의특이적억제제인 actinomycin D (AD) 0.2 µg/ml, 유전암호해석의특이적억제제인 cycloheximide (CH) 2 µg/ml을각각 2시간, 10시간씩처리한후 total RNA를얻어 Northern blotting을시행하였다. 배양한 PC12 세포에전사효소인 RNA polymerase II의특이적억제제인 5,6-dichlo-1-β-D-ribofuranosylbenzimidaxole (DRB) 25 µg/ml 을처리하여 mrna의합성을저지시킨후 8시간까지 2시간간격으로세포내에남아있는 total RNA의양을 Northern blotting으로측정하여시간에따른감소정도를통해반감기

4 C C : cerebral cortex 1: AD-0.2 µg/ml/2 hrs 2: cerebellum 2: AD-0.2 µg/ml/10 hrs U 3: hippocampus 4: spinal cord 3: CH-2 µg/ml/2 hrs 4: CH-2 µg/ml/10 hrs Fig. 1. Northern blot analysis of irp94 mrna in Mongolian gerbil brain. he upper panel in each Northern blotting shows irp94 mrna resulting ischemia induction, middle panel is without ischemia induction. he lower panel shows rat rrna as a loading control. C, control; line 1~4 ordinary, 1, cerebral cortex; cerebellum, hippocampus, spinal cord. Fig. 2. Northern blot analysis showing the effects of actinomycin D and cycloheximide on irp94 mrna. Confluent PC12 cells were treated with actinomycin D or cycloheximide for indicated time, respectively. he lower panel shows rat rrna as a loading control. C, control; line 1-4 ordinary, AD-0.2 µg/ml/2 h; AD-0.2 µg/ml/10 h; CH-2 µg/ml/2 h; CH-2 µg/ml/10 h. N-acetyl-leucyl-norleucinal (ALLN) 25 µm과 methylguanidine (MG 132) 25 µm를각각 37 에서 30분씩처리하여 total RNA를얻어 Northern blotting을시행하였다. 를산출하였다. 결 과 5. irp94 mrna 의소포체스트레스유도약물및열충격에의한발현 PC12 세포에소포체내의칼슘을교란시켜서단백질의정상적인접힘을방해하는 Ca ++ 이온운반체 (ionophor) 인 A µm, 소포체에서 Golgi complex로신생분비단백질의이동을차단하는 brefeldin A (BFA) 10 µg/ml, 신생분비단백질의당쇄부가 (N-glycosylation) 를방해하여단백질접힘을방해하는 tunicamycin 2 µg/ml, 강력한환원제로서신생단백질의각 thiol기 (-SH) 가이황화결합 (-S-S-) 으로형성되는것을방해하는 dithiothreitol (D) 3 mm, 소포체내의산화스트레스를유도하여정상적인단백질의접힘및조립을방해하는 H 2 O 2 23 µg/ml의 5가지소포체스트레스유도약물을처리하여 37 에서 3시간동안배양하였다. 이후차가운 PBS로 3회세척한후 RNAzol M B Kit을사용하여 total RNA를얻어 Northern blotting 을시행하였다. 그리고발현이증가하는약물에대해서는다양한약물처리시간에따른발현양상을알아보는추가실험을하였다. PC12 세포에 45 /15분의열충격을가한뒤에 37 에서 0시간, 1시간, 5시간경과후각각의 total RNA를얻어 Northern blotting을시행하였다. 6. 소포체와세포질간의의사소통에서 irp94 mrna 의역할 PC12 세포의세포질프로테오좀 (proteasome) 억제제인 일과성전뇌허혈 / 재관류후 gerbil 뇌조직에서의 irp94 mrna 발현부위를조사하였다. 총경동맥의일시결찰에의한 30분간의일과성전뇌허혈및 3시간재관류의결과, 대조군에서는특이적인발현양상을관찰할수없었으나실험군에서는대뇌피질과해마부위에서 irp94 mrna의강한발현을관찰할수있었다 (Fig. 1). irp94 mrna의발현특이성을알아보았다. 전사억제제인 AD, 유전암호해독억제제인 CH 처리모두가 PC12 세포의 irp94 mrna 발현을약화시켰다. 또한이러한발현약화는 AD 처리군에서더욱현저하여 irp94 mrna의발현은유전암호해독수준보다는전사수준에서조절됨을알수있었다. CH 처리군에서보인대조군보다약한 irp94 mrna의발현은 PC12 세포의전체적인유전암호해석저하로인한부작용으로사료된다 (Fig. 2). irp94 mrna의세포내반감기를측정하였다. irp94 mrna는시간이경과하면서점점약한발현을보였다. DRB 처리전의발현정도를 100% 로하여시간에따른발현정도를도표로표시할때, 세포내반감기는약 5시간임을알수있었다 (Fig. 3). 각종소포체스트레스유도약물에대한 irp94 mrna의발현을조사하였다. PC12 세포는 5가지약물중 tunicamycin 처리에가장강한 irp94 mrna의발현을보였다. H 2 O 2 에도발현이증가하였으나나머지약물에서는대조군에비해괄목할만한발현을보이지않았다 (Fig. 4). 신생분비단백질의당쇄부가방해에의한 irp94 mrna의발현을조사하였다. 신생단백질의당쇄부가 (N-glycosylation) 를특이적으로방해

5 C C (DRB 25 µg/ml, 0 h after) 1: 2 hrs after 1: 1 hr 2: 3 hrs 2: 4 hrs after 3: 5 hrs 3: 6 hrs after 4: 8 hrs after 4: 12 hrs % of remained mrna % of remained mrna Fig. 5. he expression of irp94 against glycosylation inhibition in PC12 cells. Confluent PC12 cells were treated with N-glycosylation inhibitor of tunicamycin during indicated times, and purificated each total RNA was used in Northern blotting. : tunicamycin 4 µl/ml treatment, : rrna (control). C ime after induction with DRB (h) (h) Fig. 3. Estimation of irp94 mrna half life in a cell. Confluent PC12 cells were treated with DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole), which is a specific inhibitor of RNA polymerase II. otal RNA was purificated after indicated times. he DRB treatment prevents new mrna synthesis and permits the monitoring of intracellular residue mrnas. 1: 1 hr 2: 3 hrs 3: 5 hrs C : A23187 Fig. 6. he expression of irp94 against oxidative stress in PC12 cells. Confluent PC12 cells were treated with oxidative stress inducible H 2 O 2 during indicated times, and purificated each total RNA was used in Northern blotting. : H 2 O 2 23 µl/ml treatment, : rrna (control). 2: BFA 3: unicamycin 4: D 5: H 2 O 2 Fig. 4. he expression of irp94 against ER stress inducible drugs in PC12 cells. Confluent PC12 cells were treated with each ER stress inducible drug, and the purificated total RNA was used in Northern blotting. : reatment of each ER stress inducible drug, : rrna (control; lower panel). 하는 tunicamycin 4 µl/ml를 1시간, 3시간, 5시간, 12시간동안처리하였을때에 irp94 mrna는점점강하게발현되었다 (Fig. 5). 산화스트레스에대한 irp94 mrna의발현을조사하였다. 대조군에비해 H 2 O 2 처리 3시간경과시 irp94 mrna 가강하게발현하는것을확인할수있었다 (Fig. 6). 열충격 에대한 irp94 mrna의발현을조사하였다. 열충격 (45 에서 15분 ) 처리직후에비하여일정한시간 (1시간, 5시간 ) 이경과할수록 irp94의발현은더욱강해졌다 (Fig. 7). 세포질프로테오좀억제제에대한 irp94 mrna의발현을조사하였다. 대조군에비하여 ALLN, MG 132 투여군각각에서강한발현이관찰되었다 (Fig. 8). 고찰최근까지허혈에따르는장기상해는, 에너지대사의붕괴에의한일련의세포죽음으로생각되었다. 허혈에서뇌조직뿐만아니라심장과신장에서열충격단백질 (heat shock protein; hsp) 의발현이보고된이래, 허혈에의한세포죽음을허혈스트레스에대한세포의응답현상으로이해할필요성이제기되었다. 허혈은여러종류의화학스트레스로구성된복합

6 C C 1 2 Heat shock 1: 45 for 15 min 1: ALLN 2: 45 for 15 min/1 h 2: MG 132 3: 45 for 15 min/5 h Fig. 7. he expression of irp94 against heat shock in PC12 cells. Confluent PC12 cells were treated with heat shock during indicated times, and chase 0 h, 1 h and 5 h at the control conditions. he purificated each total RNA was used in Northern blotting. : Heat shock teatment, : rrna (control). Fig. 8. he expression of irp94 against proteasome inhibitors in PC12 cells. Confluent PC12 cells were treated with proteasome inhibitors (Alln or MG 132) during indicated times, and purificated each total RNA was used in Northern blotting. : ALLN or MG 132 treatment, : rrna (control). 스트레스로서각각다른세포내소기관들을표적으로하고있다. 예를들어, 생체반응으로발생하는활성산소와자유기체 (free radical compound) 는 DNA 장애를유발하며세포내의 AP 감소는세포질의단백질변성을유발하여세포질의기능을상실하게만든다. 이러한허혈스트레스에대응하기위하여세포는각각의세포내소기관에서다양한스트레스단백질의발현을유도한다. 신경세포의허혈손상에대한방어메커니즘은아직명확하게밝혀지지못했다. 지금까지의연구를살펴보면세포내칼슘의항상성 (homeostasis) 유지가중요하여세포내칼슘의교란은허혈손상과깊은관계를가진다는것을알수있었다. 신경세포의허혈손상시항상성의장애가발생하는곳은세포질과 mitochondria의칼슘이온의농도변화에기인한세포의항상성의장애가중요한시발점으로여겨졌지만, 최근의연구에의하면정상적인기능을위하여세포질보다몇배의활동도 (activity) 가필요한소포체내의칼슘농도의저하로인한소포체단백질의정상적인접힘과공정화 (processing) 의장애로신경세포의사멸이유도된다는것이대체적인통설이다 (Paschen and Doutheil, 1999; Paschen, 2000; Paschen, 2001; Paschen and Frandsen, 2001). 또한현재까지의분자세포생물학적연구결과에따르면허혈손상시소포체내의환경변화 ( 단백질의당쇄합성저해, 단백질의이황화결합형성저해, 칼슘고갈, 분비단백질의소포체에서 Golgi 복합체로의이동저해, AP 고갈유도 ) 에의한소포체내분비당단백질의접힘및조합과정의불량이결정적인원인으로밝혀지기시작하였다. 이상의과정에서허혈자극이소포체의기능이상을초래하게되는기전을종합적으로살펴보면평상시에는에너지가소모되는과정으로세포질에서소포체내강 (ER lumen) 으로칼슘이온을역으로이동시키는능동적인 ER Ca 2+ -APase (SERCA) 에의해서소포체와세포질사이에 급격한칼슘이온농도의차이가존재하게되는데허혈손상이발생하면소포체내의칼슘항상성의이상에직접적인영향을끼치게되고소포체내에저장되어있던칼슘이유리되면서 oxygen/glucose-deprivation (OGD) 작용에의해서세포질내에존재하는칼슘이온의활성도의증가를유발하여허혈손상에대한일련의다양한유전자의발현및억제작용을촉발한다는것이다 (Paschen, 2000). 일반적으로세포속에는복잡한구조를가진수많은단백질이존재하며, 이들중대부분은세포골격단백질과같이 AP 분자가있어야만정상적인구조를가질수있다. 허혈이라는복합스트레스는세포내에너지대사를저해하여 AP의고갈, 혐기성해당작용에의한산성도 (ph) 저하, 활성산소의발생을유도하고이러한자극이더욱세포내단백질변성을촉진시키게된다. 이러한환경변화에대한방어인자로서소포체샤페론이유도되어세포내분비단백질과막단백질의비가역적인변성을억제하게된다. 신경세포에일과성뇌허혈손상이가해진상황하에서, 허혈로인한유전암호해독과정의억제 (shut-down) 가초기재관류시에는 unfolded protein의합성을봉쇄하고 aggregation을억제하여신경세포를보호하는방향으로나타나고, 재관류의기간이길어질수록신경세포보호단백질 (neuroprotective protein) 인 XBP1 proc 와 GRP78과같은유전자의새로운합성을방해하여신경세포에해로운작용을보이는데이와같은점에서유전암호해독을억제하는작용은궁극적으로신경세포의괴사를유발하는과정에서신경세포의보호작용과독성작용을다함께나타내는양면적인역할을담당하게된다 (Paschen, 2003). 또한최근의연구에의하면소포체스트레스가유도되면소포체내에축적되는비정상적인단백질들이일정비율로분해된다는개념인 endoplasmic reticulum associated degradation (ERAD) 이확립되었다. 소포체내에서유전암호해

7 독되는단백질의약 30% 가비정상적으로접힌 (unfolded, misfolded 혹은 malfolded) 단백질이지만내, 외부의자극에의해서과도하게축적되지않으면질병으로이어지지는않는다. 그이유는소포체내에서정상적인단백질로접혀지지못한것들은세포질의프로테오좀과긴밀한관계를유지하여분해되어버림으로써일정수준이하로유지된다. 이러한 ERAD 메커니즘이정상적으로작동되지못하면과도한비정상적인단백질이소포체내에축적됨으로써유발되는 endoplasmic reticulum storage disease (ERSD) 가된다. 본연구의대상인 irp94가허혈스트레스의방어인자로서의가능성을보여준다할수있다. 즉 irp94는허혈이라는세포의손상원인에대해신생단백질의생산을동반하는역동적인항스트레스반응의일부를담당하고있다. 본연구결과에서얻은 irp94의허혈손상에서의역할은다음과같이정리할수있다. irp94가허혈및재관류손상에가장취약한부위인대뇌피질과해마부위에서강한발현을보인점은허혈스트레스의방어인자로서의가능성을보여주는결과라할수있다. 그리고 irp94 유전자의발현조절은전사단계에서이루어지며세포내의반감기는약 5시간이라는것도알수가있었다. 소포체스트레스유도약물중에서신생단백질의당쇄부가형성을특이적으로억제하는 tunicamycin에대해가장강한발현을보인것은당단백질이소포체내에서정확한구조를가지기위해서는단백질에부가되는당쇄가필수적이라는것을의미한다. 소포체내에서정상적인단백질접힘이일어나지않으면이들이계속적으로축적됨으로인하여더욱강한샤페론의발현이유도되는것으로보인다. 허혈손상시에는많은자유기 (free radical) 가생산되고소포체가각종스트레스를받을때도자유기들이생체고분자들과반응하여괴사혹은세포고사에이르게되나실질적으로산화스트레스가세포내에서어떤기능을조절하는지는정확히알수없는상태이다. 특히 PC12 세포에서는아무것도확인된것이없었는데이번연구에서강한발현을보인것은 irp94의세포고사와의연관성도유추해볼수있게하였다. 열충격후에일정한시간이지날수록강한발현을보인것은 irp94가소포체스트레스로부터세포를보호하는샤페론기능보다는일단변성이된혹은일부변성된단백질의수복 (repair) 과정에관여하기때문이지않을까하는추정을가능케한다. 정상적이지못한단백질접힘을보이는분비당단백질들이세포내에계속축적되는것은세포자신에게는심각한세포독성으로작용한다. 일반적인세포의경우약 30% 정도는정상적이지못한단백질접힘현상이소포체내에서일어난다고한다. 그렇다면이들세포독성으로부터어떻게세포가생존을유지할수있을까하는것이의문이다. 이에대한새로운개념이 ERAD이다. 즉, 소포체내에축적되는비정상적인단백질의축적이세포질에존재 하는프로테오좀과밀접한관계를가짐으로써소포체내를항상일정하게유지한다는것이다. 본실험에서확인된것은세포질에존재하는프로테오좀을불활성화시켰을때에소포체내에존재하는 irp94의발현이증가한다는사실이었다. 이는소포체샤페론인 irp94의분자생리적특성을고려할때에세포질의프로테오좀의불황성화로인하여소포체내에비정상적인단백질접힘을보이는단백질들이분해되지못하고계속축적되기에이를처리하기위하여 irp94의발현이증가하는것으로생각할수있다. 이상의결과는 irp94가뇌허혈의유도및방어기전에어떤기능을가지는지를알기위한기본적인분자생물학적정보를제공한다. 이는뇌허혈의예방및치료제의개발에새로운가능성을줄일것이다. 특히, 응급의학적으로흔한외상에의한뇌손상을포함한다양한형태의뇌병변시에관찰할수있는반응인 spreading depression (SD) 은초점성허혈손상시조직손상의정도를심화시키고손상조직에서포도당이풍부한세포외액의축적과알칼리성의부종액이형성되면서신경조직의비가역적인손상을유도하게된다 (Hermann et al., 1998; Paschen, 2003). 이와같은허혈성손상조직에서대사장애가다르게나타나는영역별로손상기전이차이가있으므로단계별손상에초점을맞추어야효과적으로신경조직손상을최소화할수있는동시에손상기전을억제할수있는방법도개발될수있을것이다. 감사의글이논문은 2004년도충남대학교병원공동연구비에의하여수행되었음. REFERENCES Blumenfeld KS, Welsh FA, Harris VA, Pesenson MA. Regional expression of c-fos and hsp70 mrna following hypoxiaischemia in immature injury in the rat brain. J Cereb Blood Flow Metab : Chen J, Zhu RL, Nakayama M, Kawaguchi K, Jin K, Stetler RA et al. Expression of the apoptosis-effector gene, bax, is upregulated in vulnerable hippocampal CA1 neurons following global ischemia. J Neurochem : Hermann DM, Mies G, Hossmann KA. Effects of a traumatic neocortical lesion on cerebral metabolism and gene expression of rats. Neuroreport : Higashi, Nakai A, Uemura Y, Kikuchi H, Nagata K. Activation of heat shock factor-1 in rat brain during cerebral ischemia or after heat shock. Mol Brain Res : Kawahara N, Mishima K, Higashiyama S, aniguchi N, amura

8 A, Kirino. he gene heparin-binding epidermal growth factor-like growth factor is stress-inducible: its role in cerebral ischemia. J Cereb Blood Flow Metab : Kim SW, Yoo IS, Koh HS, Kwon OY. Ischemia-responsive protein (irp94) is up-regulated by endoplasmic reticulum stress. Z naturforsch [C] : Kirino, sujita Y, amura A. Induced tolerance to ischemia in gerbil hippocampus neurons. J Cereb Blood Flow Metab : Koh HS, Moon IS, Lee YH, Shong M, Kwon OY. Expression of an HSP110 family, ischemia-responsive protein (irp94), in the rat brain after transient forebrain ischemia. Z Naturforsch [C] : Mochizuki R, akeda A, Sato N, Kimpara, Onodera H, Itoyama Y, et al. Induction of midkine expression in reactive astrocytes following rat transient forebrain ischemia. Exp Neurol : Morimoto RI, issieres A, Georgopoulos C. he biology of heat shock proteins and molecular chaperones. Postischemic stress response in brain. Cold Spring Harbor Laboratory Press; pp Nowak S Jr. Localization of 70 kda stress protein mrna induction in gerbil brain after ischemia. J Cereb Blood Flow Metab : Ohtsuki, Kitagawa K, Yamagata K, Mandai K, Mabuchi, Matsushita K, et al. Induction of cyclooxygenase-2 mrna in gerbil hippocampal neurons after transient forebrain ischemia. Brain Res : Paschen W. Disturbances in calcium homeostasis within the endoplasmic reticulum may contribute to the development of ischemic cell damage. Med Hypoth : Paschen W, Doutheil J. Disturbances of the functioning of endoplasmic reticulum: a key mechanism underlying neuronal cell injury? J Cereb Blood Flow Metabol : Paschen W. Role of calcium in neuronal cell injury: which subcellular compartment is involved? Brain Res Bull : Paschen W. Dependence of vital cell function on endoplasmic reticulum calcium level: implications for the mechanisms underlying neuronal cell injury in different pathological states. Cell Calcium : Paschen W, Frandsen A. Endoplasmic reticulum dysfunction: a common denominator for cell injury in acute and degenerative diseases of the brain? J Neurochem : Paschen W. Endoplasmic reticulum: a primary target in various acute disorders and degenerative diseases of the brain. Cell Calcium : Paschen W. Mechanism of neuronal cell death: diverse roles of calcium in the various subcellular compartments. Cell Calcium : Pulsinelli WA, Brierley JB, Plum F. emporal profile of neuronal damage in a model of transient forebrain ischemia. Ann Neurol : Sommer C, Gass P, Kiessling M. Selective c-jun expression in CA1 neurons of gerbil hippocampus during and after acquisition of an ischemia-tolerant state. Brain Pathol : akami K, Iwane M, Kiyota Y, Miyamoto M, sukuda R, Shiosaka S. Increase of basic fibroblast growth factor immunoreactivity and its mrna level in rat brain following transient forebrain ischemia. Exp Brain Res : akami K, Kiyota Y, Iwane M, Miyamoto M, sukuda R, Ogarashi K, et al. Upregulation of fibroblast growth factor-receptor messenger RNA expression in rat brain following transient forebrain ischemia. Exp Brain Res : sukahara, Iihara K, Hashimoto N, Nishijima, aniguchi. Increase in levels of brain-derived neurotrophic factor mrna and its promoters after transient forebrain ischemia in the rat brain. Neurochem Int : Yabuuchi K, Minami M, Katsumata S, Yamazaki A, Satoh M. An in situ hybridization study on interleukin-1 beta mrna induced by transient forebrain ischemia in the rat brain. Brain Res Mol Brain Res : Yagita Y, Kitagawa K, aguchi A, Ohtsuki, Kuwabara K, Mabuchi, et al. Molecular cloning of novel member of the HSP110 family of genes, ischemia-responsive protein 94 kda (irp94), expressed in rat brain after transient forebrain ischemia. J Neurochem : Yagita Y, Kitagawa K, Ohtsuki, anaka S, Hori M, Matsumoto M. Induction of the HSP110/105 family in the rat hippocampus in cerebral ischemia and ischemic tolerance. J Cereb Blood Flow Metab : Wiessner C, Back, Bonnekoh P, Kohno K, Gehrmann J, Hossmann KA. Sulfated glycoprotein-2 mrna in the rat brain following transient forebrain ischemia. Brain Res Mol Brain Res : Wolf PA, Cobb JL, D'Agostino RB. Epidemiology of stroke. In: Stroke: pathophysiology, diagnosis, and management. 2nd ed. New York: Churchill Livingstone; pp Xue JH, Fukuyama H, Nonoguchi K, Kaneko Y, Kido, Fuku

9 moto M, et al. Induction of apg-1, a member of the heat shock protein 110 family, following transient forebrain ische- mia in the rat brain. Biochem Biophys Res Commun :