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1 대한안과학회지 년제 권제 8 호 J Korean Ophthalmol Soc ;(8):9- pissn: eissn: Original Article 망막신경절세포에서고장성및고오스몰스트레스에의한긴장성반응유도결합단백질의발현 Expression of TonEBP by Hypertonic and Hyperosmolar Stress in RGC- Cells 우종은 권민영 정수월 우제문 Jong Eun Woo, MD, Min Young Kwon, Su Wol Chung, PhD, Je Moon Woo, MD, PhD 울산대학교의과대학울산대학교병원안과학교실, 울산대학교자연과학대학생명과학부 Department of Ophthalmology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Korea School of Biological Sciences, College of Natural Science, University of Ulsan, Ulsan, Korea Purpose: In order to determine whether the Tonicity responsive enhancer binding protein (TonEBP) is expressed by hypertonic and hyperosmolar stress, TonEBP expression was investigated in the retinal ganglion cell (RGC) line, RGC- cells. Methods: After RGC- cells were cultured by Staurosporine, TonEBP expression was measured with Western immunoblotting analysis and real-time reverse transcription-polymerase chain reaction in mm NaCl, mm mannitol, mm glucose, or mm glucose at, 6,, and hours after exposure to each environment. Results: In this study, the protein expression of TonEBP was determined to be statistically significantly checked in mm NaCl after, and 6 hours, in mm mannitol after 6 hours, and in mm glucose after, and 6 hours. TonEBP messenger Ribonucleic acid (mrna) expression was determined to be statistically significantly checked in mm NaCl after hours, in mm mannitol after, and hours, and in mm glucose after, and hours. Conclusions: These results suggested that TonEBP was expressed by hypertonic and hyperosmolar stress at the protein and mrna levels. Further studies are nedded to determine the role of TonEBP and the mechanism of expression and regulation of TonEBP. J Korean Ophthalmol Soc ;(8):9- Key Words: Hyperosmolar stress, Hypertonic stress, Retinal ganglion cell death, TonEBP expression Received: Revised:... Accepted:. 6.. Address reprint requests to Je Moon Woo, MD, PhD Department of Ophthalmology, Ulsan University Hospital, #877 Bangeojinsunhwando-ro, Dong-gu, Ulsan 68-7, Korea Tel: , Fax: limbus68@naver.com * This study was presented as an e-poster at the th Annual Meeting of the Korean Ophthalmological Society. 신경퇴행 (neurodegeneration) 은많은중추신경계질환에있어주요한특징으로여겨지고있는데녹내장과색소망막염등의망막질환에서도망막신경절세포층 (retinal ganglion cell layer) 의결손이진행되는것으로알려졌다. 녹내장의경우안압의상승이녹내장의위험요인들중가장자세히알려진요인중하나로안압을낮추는것이시야결손의진행을늦출수있다는사실이알려졌기때문에지난수십년간녹내장의치료는주로안압의하강에초점이맞춰졌다., 최근에는녹내장의가장주요한병태생리학적특징인망막신경절세포층의점진적인손상을방지하기위한신경보호기전을규명하는데에관심이모아지고있다., 망막신경절세포가여러가지원인에의해스트레스를받게되면여러가지단백질활성효소들과신호전달체계에변화가일어나게된다. 허혈상태에빠지게되면 calcium/calmodulin-dependent protein kinase II, mitogen activated protein c The Korean Ophthalmological Society This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 9

2 - 대한안과학회지 년제 권제 8 호 - kinase (MAPK), extracellular signal-regulated kinase (ERK), protein kinase B (Akt), 그리고 protein kinase C (PKC) 등이활성화된다. - 또한 ATP의결핍, 산화스트레스, glutamate 의증가는미토콘드리아와관련된경로를통하여신경세포의사멸을유발하게된다. 최근에는당뇨망막병증또한망막신경절세포층의결손이유발되는신경퇴행성질환으로여겨지고있어그기전에관한연구가활발히진행중이다. 당뇨망막병증의신경퇴행에대한기전은다음의두가지가설에의해제기되었다. 먼저혈액-망막장벽 (blood-retinal barrier) 의파괴로인한혈관투과성의증가로인해망막내의세포외체액 (extracellular fluid) 의조절실패로인해황반부종등을유발하게된다. 또한당뇨는망막에서세포사멸을증가시키게되는데고혈당증, 산화스트레스, 그리고당화반응등의환경에서신경퇴행이일어나는것으로알려졌으나그기전에대해서는몇가지가설이제시되었을뿐아직까지명확하게밝혀진바는없다., 고장성과같은환경에서세포는위축되어스트레스를받게되며세포내염 (salt) 의증가로세포손상이초래된다. 세포가고장성환경에노출되면일차적으로세포내에존재하는물이세포외부로이동하여세포의위축 (shrinkage) 이일어나게되고세포내삼투농도는증가하게되는데 K+, Cl-와같은이온과물이세포내로재흡수되어세포의크기는원래대로회복되지만세포내삼투농도는증가된상태로유지된다. 6 최근의연구결과에의하면고장성은 double-stranded DNA break (DSDB) 를유발하여결과적으로세포자멸사를유발하게되는데신장수질에서고장성에의해활성화된 TonEBP (Tonicity responsive enhancer binding protein) 는여러유전자들의전사 (transcription) 를활성화시켜 inositol, betaine, taurine, sorbitol 등의 organic osmolytes의세포내축적에관련된유전자및 Hsp7의발현을증가시켜고장성에의한스트레스로부터세포를보호하는데중요한역할을담당하고있다. 따라서본연구에서는당뇨망막병증환자와같은고혈당으로인해발생할수있는고장성및고오스몰농도환경에노출되었을때 RGC- 세포에서의 TonEBP의발현정도에대해알아보고자하였다. 대상과방법 RGC- 세포배양과분화및고장성스트레스유발 RGC- 세포주를선택적으로분리, 분주 (ATCC, Manassas, VA) 하여 RIPA buffer (Tris/Cl (ph 7.6); mmole/l, EDTA; mmole/l, NaCl; mmole/l, b-glycerophosphate; mmole/l, NaF; mmole/l, Na VO ;. mmole/l, NP-;.%, sodiumdeoxycholate;.%) 에서 시간동안배양하였고 Staurosporine (Sigma, S9) 을 nm 농도로 시간처리하여분화시킨후고장성및고오스몰농도스트레스를유발하기위해 mm NaCl, mm mannitol, mm glucose, mm glucose를, 6,, 시간처리하여단백질을추출하였다. 대조군은 RGC- 세포주를 Staurosporine으로분화하기전단계와분화후고장성및고오스몰농도를가하지않은군으로선정하였고고장성및고오스몰농도스트레스를가한실험군과의단백질및 mrna의발현정도를비교하였다. Western Immunoblotting 이실험은 회에걸쳐시행되었으며첫번째실험에서는 mm NaCl, mm mannitol을, 6,, 시간처리하였고 TonEBP와 GAPDH (glyceraldehyde--phosphate dehydrogenase) 의발현정도를확인하였으며두번째실험에서는 mm glucose, mm glucose를, 6,, 시간처리하여단백질을추출하였으며 TonEBP와 β-actin의발현정도를확인하였다. GAPDH 는배양된 RGC- 세포에 % SDS-polyacrylamide gel을만든후 protease inhibitors (Roche Applied Science, Mannheim, Germany) 를처리한다음각 well에 cell lysates ug을 loading하였다. 차항체로는 NFAT (TonEBP, :,, donated from Kwon HM, Ph. D, UNIST) 와 GAPDH (:,, Santa Cruz, CA) 를사용하였으며 차항체로는 rabbit (:,) 을사용하여항원항체반응을유도하였으며기질로는 Pierce BCA protein assay kit (Thermo Scientific, Rockford, IL) 제품을사용하였다. 각각의항원항체반응사이에는반드시 회씩의세척과정을거쳤으며배양후 Konica SRX-A detection agent를사용하여감광시켰다. Real-time PCR TonEBP의발현정도를 mrna 수준에서확인하기위해 real-time PCR을시행하였는데첫번째실험에서는 mm NaCl 및 mm mannitol을, 6,, 시간처리한후 TonEBP의발현정도를확인하였으며두번째실험에서는 glucose mm의환경에서, 6,, 시간처리한후 TonEBP의발현정도의변화를확인하였다. Total RNA는 TRIzol reagent (Invitrogen, Life technologies, Carlsbad, CA) 를사용하여추출하였으며 reverse transcription은 SuperScript TM IIIFirst-Strand Synthesis System (Invitrogen, CA) 을사용하여수행하였다. Primer는 Table 에언급한바와같은 sequence를사용하였으며 cdna는 9 C에서 96

- 우종은외 : 스트레스환경에서 TonEBP 의발현 - A TonEBP GAPDH + STD B + STD NaCl ( mm) Mannitol ( mm) -STD V 6 hours -STD V 6 hours TonEBP GAPDH C D TonEBP/GAPDH Protein (Fold change) TonEBP/GAPDH Protein (Fold

3 - 우종은외 : 스트레스환경에서 TonEBP 의발현 - A TonEBP GAPDH + STD B + STD NaCl ( mm) Mannitol ( mm) -STD V 6 hours -STD V 6 hours TonEBP GAPDH C D TonEBP/GAPDH Protein (Fold change) TonEBP/GAPDH Protein (Fold change) Figure. Hypertonic inducers increase Tonicity responsive enhancer binding protein (TonEBP) protein expression in RGC- cells (A, C: mm NaCl; B, D: mm mannitol; -STD: before preconditioning; V: just after the preconditioning; : hours after preconditioning; 6: 6 hours after preconditioning; : hours after preconditioning; : hours after preconditioning; * p <. by Mann-Whitney U-test). RGC = retinal ganglion cell; STD = staurosporine; GAPDH = glyceraldehyde--phosphate dehydrogenase. Table. Primer sequences for real-time PCR Gene name Sequence NFAT (TonEBP) GAPDH F -TTC ATC TCA TTG CTC AGC G- R -GGG AGA AGA TCA TAG ACA GAT TC- F -GGC ACA GTC AAG GCT GAG AAT G- R -ATG GTG GTG AAG ACG CCA GTA- F = forward; R = reverse; PCR = polymerase chain reaction; NFAT = nuclear factor of activated T-cells ; TonEBP = tonicity responsive enhancer binding protein; GAPDH = glyceraldehyde- -phosphate dehydrogenase. 분간배양한후이후 9 C에서 초간및 6 C에서 분간배양하여총 cycles를수행하였다. TonEBP와 GAPDH의모든 real-time quantitative PCR은 iq SYBR Green Supermix (Bio-Rad, Hercules, CA) 를사용하여수행하였다. PCR 결과는측정된 Ct값을기반으로 β-actin값으로보정한후 - Ct 방법을사용하여계산하였다. 모든측정값은비모수검정인 Mann-Whitney U-test로검정하여발현정도의차이에대한통계적유의성에대하여검정하였으며통계분석에사용된프로그램은 Microsoft Excel 과 SPSS. (IBM SPSS Co.) 를이용하였다. RGC- 세포주를배양하여고장성스트레스에노출하였을때 TonEBP가단백질수준에서발현되는것을 Western blotting을통해확인할수있었다. mm NaCl 및 mm mannitol을, 6,, 시간처리하였을때 TonEBP가발현되어감광되는것을확인할수있었다 (Fig. ). 감광여부를히스토그램으로분석해보았을때 mm 및 mm NaCl을 6시간처리시감광도가가장높았으며시간이경과할수록감광도는감소하는추세를보였다. 통계적으로는 mm NaCl 시간, 6시간처리했을때와 mm mannitol 6시간처리했을때발현정도에있어유의한차이를보였다 (p<. by Mann-Whitney U-test). 다음으로 mm 및 mm glucose를처리한실험에서도 TonEBP는처리후부터 시간후까지비교적일정하게발현되는것을확인할수있었으나대조군과비교하여통계적유의성을보이지는않았다. mm glucose를처리했을때에는 TonEBP의발현정도가처리 시간후가가장높았고그이후에는시간이지날수록감소하는추세를보였다. 통계적으로는 mm glucose 처리후 시간및 6시간후에서유의성을보였다 (Fig. ). 결 Western Immunoblotting 과 Real-time PCR TonEBP의발현정도를 mrna 수준에서확인하기위해 real-time PCR을수행하였다. 먼저 mm NaCl 과 mm 97

다음으로 mm glucose를, 6,, 시간처리한실험에서는통계적으로 시간, 시간경과후의발현정도가유의성있게나타났으며 glucose 처리후 시간째증가했다가이후 6시간째감소후시간이지나면서다시증가하는양상을보였다 (Fig. ). B β-actin TonEBP/GAPDH Protein (Fold change) Figure.

4 - 대한안과학회지 년제 권제 8 호 - mannitol을, 6,, 시간처리후각시점에서의 TonEBP 발현정도를 real-time PCR을통해확인하였다. mm NaCl 처리 시간및 mm mannitol 처리 시간및 시간후에서통계적유의성을보였다. 두경우모두약물처리후 시간후에서발현정도가가장높았으며이후 6시간후에감소했다가시간이지나면서다시조금씩증가하는 A + STD Glucose ( mm) Glucose ( mm) -STD V 6 6 hours TonEBP 경향을보였다 (Fig. ). 다음으로 mm glucose를, 6,, 시간처리한실험에서는통계적으로 시간, 시간경과후의발현정도가유의성있게나타났으며 glucose 처리후 시간째증가했다가이후 6시간째감소후시간이지나면서다시증가하는양상을보였다 (Fig. ). B β-actin TonEBP/GAPDH Protein (Fold change) Figure. High glucose increases Tonicity responsive enhancer binding protein (TonEBP) protein expression in RGC- cells ( mm glucose, mm glucose; A: Western blotting; B: Histogram; -STD: before preconditioning; V: just after the preconditioning; : hours after preconditioning; 6: 6 hours after preconditioning; : hours after preconditioning; : hours after preconditioning; * p <. by Mann-Whitney U-test). RGC = retinal ganglion cell; STD = staurosporine; GAPDH = glyceraldehyde--phosphate dehydrogenase. Fold induction mrna (TonEBP/ actin) β- STD Glucose hours Figure. High glucose increases Tonicity responsive enhancer binding protein (TonEBP) messenger Ribonucleic acid (mrna) expression in RGC- cells (-STD: before preconditioning; +STD and -glucose: just after the preconditioning; : hours after preconditioning; 6: 6 hours after preconditioning; : hours after preconditioning; : hours after preconditioning; * p <. by Mann-Whitney U-test). RGC = retinal ganglion cell; STD = staurosporine. A B Fold induction mrna (TonEBP/ β-actin ) Fold induction mrna (TonEBP/ β-actin ) STD NaCl hours STD Mannitol hours Figure. Hypertonic inducers increase Tonicity responsive enhancer binding protein (TonEBP) messenger Ribonucleic acid (mrna) expression in RGC- cells (A: mm NaCl; B: mm mannitol; -STD: before preconditioning; +STD and -NaCl/-mannitol: just after the preconditioning; : hours after preconditioning; 6: 6 hours after preconditioning; : hours after preconditioning; : hours after preconditioning; * p <. by Mann-Whitney U-test). RGC = retinal ganglion cell; STD = staurosporine. 98

5 - 우종은외 : 스트레스환경에서 TonEBP 의발현 - 고찰 망막신경절세포 (retinal ganglion cell) 의손상또는죽음은결국비가역적인시야결손또는시력저하를유발하므로이를예방하기위해망막신경절세포가여러스트레스환경에노출될때이를보호하는기전에대한연구가활발히진행되고있으며그중당뇨병과같은만성대사성질환에있어서고혈당으로인한고장성환경에의한스트레스가유발될때망막신경절세포를보호하기위한세포보호기전에대한연구가최근조명받고있으며분자유전학적인관점에서그기전을밝혀내고자많은연구들이이루어지고있다. 본연구또한그기전중하나로알려진 TonEBP의발현에대해실험적으로고장성환경에서 mrna 및단백질수준에서 TonEBP가발현된다는것을확인할수있었다. TonEBP는 Rel family에속하는전사조절인자로서고장성환경에의해활성화된 TonEBP는 vasopressin-regulated urea transporter (UT-A) 및 Hsp7 단백질의발현을증가시켜스트레스로인한세포사멸을막아주는데중요한역할을담당하고있는것으로알려졌다. 7,8 아직까지 TonEBP 의활성기전에대해서는아직명확히밝혀지지않았으나보고된바에따르면 TonEBP의 transactivation domain이고장성에의해활성화된다고알려졌으며인산화가크게증가하는데주로 serine과 tyrosine이인산화되는것으로보고되었다. 또한 TonEBP의발현으로인해 sodium/myo-inositol cotransporter (SMIT) 및 aldose reductase (AR) 가활성화되는것으로알려졌는데뇌손상또는당뇨합병증이일어난조직에서증가되어있는것으로알려졌으나그기전에대해서는명확히알려진바가없다. 본연구에서는당뇨망막병증환자에서발생할수있는고혈당에의한고장성환경에서망막신경절세포가죽음에이르는과정을방지하기위해 TonEBP와같은전사조절인자를활성화시킨다는추측하에실험적으로고장성환경내에서 TonEBP의발현여부를확인한것이다. 아직까지는 TonEBP의발현과관련하여고장성스트레스외에다른조절기전에대해서는알려진바가없으나신장 (kidney) 을제외한뇌 (brain), 간 (liver), 고환 (testis), 흉선 (thymus) 과같은내분비조직, 생식기관, 면역조직에서관찰된바가있어고장성에대한세포의보호기능외에도다른기능이 TonEBP에존재할것으로예상되고있다. 9- 먼저본실험에서 TonEBP의단백질수준에서의발현여부를보기위해 Western blotting을시행한실험에서는 mm NaCl 처리, 6시간후와 mm mannitol 처리 6시간후, 그리고 mm glucose 처리, 6시간후에서통계적으로유의한발현정도를보였다. 이를종합해보면 TonEBP의단백질발현정도는고장성환경의농도에의존적으로증가할것이라고예상된다. 모든약물처리후초기인, 6시간후에유의하게발현된부분과 mm glucose 를처리했을때는통계적으로유의한발현정도를보이지않았고 mm glucose를처리했을때유의한발현정도를보인점이이를뒷받침해주고있다. 따라서단백질의발현정도는농도가높을수록즉고장성인환경일수록잘발현될것으로생각한다. 본실험에서는단백질발현정도를확인할수있는 Western immunoblotting에서 mm 및 mm glucose를처리했을때농도의존적으로단백질이발현되는것을확인하였고단백질발현전단계인 mrna 수준에서도 mm glucose에서 mrna가발현되는것을확인하였기때문에 mm glucose에서는 real-time PCR 을시행하지않았다. 다만 mm glucose의경우에는단백질발현정도에있어통계적으로유의한결과를보이지않은부분에대해서는 TonEBP의발현이유도되는 glucose의고장성농도가얼마인지에대한추가적인실험이필요할것으로생각한다. 따라서농도별로좀더세분화하여추가실험을해본다면 TonEBP의발현이어느농도에서부터발현되는지, 그리고발현정도가농도의존성인지에대해더많은결과들을알수있을것이다. 다음으로생각해볼수있는것은 real-time PCR에서는다른경향성을보이고있다는점이다. 물론통계적으로모두유의한것은아니지만 mm Nacl, mm mannitol, mm glucose 세경우모두에서초기 시간경과후에 mrna 발현도가가장높았고이후 6시간후에는발현도가가장낮았으며이후시간이지날수록다시증가하는경향을보인점이다. 이는 Western blotting에서는초기에만발현되고 시간에이르러서는발현이모두감소했지만 real-time PCR에서는초기에발현되고중간에감소했다가 시간후에다시 mrna의발현도가증가했다는점이다. 따라서 TonEBP의 mrna 전사가단백질발현까지모두이어지지않았다고추측해볼수있으며이와관련한활성기전에있어다른조절메커니즘에의해 TonEBP의발현이중간에조절되고있다는가능성을제기해볼수있다. 다만아직까지 TonEBP의활성기전및그역할에대해서정확히알려진바가없기에이에대한추가적인연구가필요할것으로생각한다. 현재까지보고된바에따르면 TonEBP는고장성 (hypertonic) 스트레스환경에서발현되어세포사멸을막아주는역할을하고있는것으로알려졌으나다른역할로는 nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) 와같은전사인자들을활성화시키는데영향을끼치는것으로도알려졌다. NF-κB는 vascular endothelial growth factor (VEGF), tumor necrosis factor-α 99

6 - 대한안과학회지 년제 권제 8 호 - (TNF- α), Endothelin- 등을활성화시켜망막내신생혈관 형성및황반부종을촉진시키는것이확인되었다. 따라서 당뇨망막병증과같은경우발생할수있는염증반응의유 발및조절메커니즘에서 TonEBP 가관여하는역할에대한 추가적인연구가필요할것으로생각한다. -8 본연구에서는 RGC- 세포가고장성스트레스환경에 노출되었을때 TonEBP 를발현시킨다는것을 mrna 및 단백질수준에서확인하였다. 다만본실험은고장성스트 레스환경에서 TonEBP 가발현되었다는사실만확인하였 을뿐, 그조절기전에대해서는본실험만을통해서는밝 힐수없었으며앞서언급한대로 TonEBP 가발현되기시 작하는 glucose 의고장성농도는얼마인지, 그리고고장성 농도별로 TonEBP 가발현되는정도에대한추가실험을 통해 TonEBP 의발현정도와고장성농도간의상관관계 에대해알아보아야할것이다. 그리고 mrna 수준에서의 발현정도와단백질수준에서의발현정도가시간대에따 라차이가나는점에대해서도추가적인실험및고찰을 통해 TonEBP 가발현되는데있어서고장성스트레스외 에다른요인및다른전사조절인자들이관여하는지에대 한연구가필요할것으로생각한다. 또한본실험은세포 수준에서수행하였기때문에실제동물모델에서도같은 결과를보일수있을지에대한추가실험도필요할것으 로생각한다. REFERENCES ) The Advanced glaucoma intervention study (AGIS): 7. The relationship between control of intraocular pressure and visual field deterioration. The AGIS Investigators. Am J Ophthalmol ; :9-. ) Comparison of glaucomatous progression between untreated patients with normal-tension glaucoma and patients with therapeutically reduced intraocular pressures. Collaborative Normal-Tension Glaucoma study group. Am J Ophthalmol 998;6: ) Chen JZ, Kadlubar FF. A new clue to glaucoma pathogenesis. Am J Med ;: ) Flammer J, Haefliger IO, Orgül S, Resink T. Vascular dysregulation: a principal risk factor for glaucoma damage? J Glaucoma 999;8:-9. ) Matsumoto S, Shamloo M, Matsumoto E, et al. Protein kinase C-gamma and calcium/calmodulin-dependent protein kinase II-alpha are persistently translocated to cell membranes of the rat brain during and after middle cerebral artery occlusion. J Cereb Blood Flow Metab ;:-6. 6) Speechly-Dick ME, Mocanu MM, Yellon DM. Protein kinase C. Its role in ischemic preconditioning in the rat. Circ Res 99;7: ) Piccoletti R, Bendinelli P, Arienti D, Bernelli-Zazzera A. State and activity of protein kinase C in postischemic reperfused liver. Exp Mol Pathol 99;6:9-8. 8) Padanilam BJ. Induction and subcellular localization of protein kinase C isozymes following renal ischemia. Kidney Int ;9: ) Tanaka C, Nichizuka Y. The protein kinase C family for neuronal signaling. Annu Rev Neurosci 99;7:-67. ) Chen L, Hahn H, Wu G, et al. Opposing cardioprotective actions and parallel hypertrophic effects of delta PKC and epsilon PKC. Proc Natl Acad Sci USA ;98:-9. ) Whitlock NA, Agarwal N, Ma JX, Crosson CE. Hsp7 upregulation by HIF- signaling offers protection against retinal ischemia in rats. Invest Ophthalmol Vis Sci ;6:9-8. ) McNaughts KS, Carrupt PA, Altomare C, et al. Isoquinoline derivatives as endogenous neurotoxins in the etiology of Parkinson's disease. Biochem Pharmacol 998;6:9-. ) Cunha-Vaz J, Faria de Abreu JR, Campos AJ. Early breakdown of the blood-retinal barrier in diabetes. Br J Ophthalmol 97;9: ) King GL, Brownlee M. The cellular and molecular mechanisms of diabetic complications. Endocrinol Metab Clin North Am 996; :-7. ) Barber AJ, Nakamura M, Wolpert EB, et al. Insulin rescues retinal neurons from apoptosis by a phophatidylinositol -kinase/aktmediated mechanism that reduces the activation of caspase-. J Biol Chem ;76:8-. 6) Hoffmann EK, Dunham PB. Membrane mechanisms and intracellular signaling in cell volume regulation. Int Rev Cytol 99; 6:7-6. 7) Nakayama Y, Peng T, Sands JM, Bagnasco SM. The TonE/TonEBP pathway mediates tonicity-responsive regulation of UT-A urea transporter expression. J Biol Chem ;7: ) Woo SK, Lee SD, Na KY, et al. TonEBP/NFAT stimulates transcription of HSP7 in response to hypertonicity. Mol Cell Biol ;:7-6. 9) Cha JH, Woo SK, Han KH, et al. Hydration status affects nuclear distribution of transcription factor tonicity responsive enhancer binding protein in rat kidney. J Am Soc Nephrol ;:-. ) Lopez-Rodriguez C, Aramburu J, Jin L, et al. Bridging the NFAT and NF-kappaB families: NFAT dimerization regulates cytokine gene transcription in response to osmotic stress. Immunity ; :7-8. ) Zhang Z, Ferraris JD, Brooks HL, et al. Expression of osmotic stress-related genesin tissues of normal and hypoosmotic rats. Am J Physiol Renal Physiol ;8:F ) Favale NO, Casali CI, Lepera LG, et al. Hypertonic induction of COX expression requires TonEBP/NFAT in renal epithelial cells. Biochem and Biophys Res Commun 9;8:-. ) Roth I, Leroy V, Kwon HM, et al. Osmoprotective transcription factor NFAT/TonEBP modulates nuclear factor-kappab activity. Mol Biol Cell ;:9-7. ) Woo SK, Lee SD, Na KY, et al. TonEBP/NFAT stimulates transcription of HSP7 in Response to Hypertonicity. Mol Biol Cell ;:7-6. ) Lee SD, Choi SY, Lim SW, et al. TonEBP stimulates multiple cellular pathways for adaptation to hypertonic stress: organic osmolyte-dependent and -independent pathways. Am J Physiol Renal Physiol ;:F77-. 6) Trama J, Lu Q, Hawley RG, Ho SN. The NFAT-related protein NFATL (TonEBP/NFAT) is induced upon T cell activation in a

7 - 우종은외 : 스트레스환경에서 TonEBP 의발현 - calcineurin-dependent manner. J Immunol ;6: ) Jauliac S, López-Rodriguez C, Shaw LM, et al. The role of NFAT transcription factors in integrin-mediated carcinoma invasion. Nat Cell Biol ;:-. 8) Trama J, Go WY, Ho SN. The osmoprotective function of the NFAT transcription factor in T cell development and activation. J Immunol ;69: = 국문초록 = 망막신경절세포에서고장성및고오스몰스트레스에의한긴장성반응유도결합단백질의발현 목적 : 고장성 (hypertonicity) 및고오스몰농도 (hyperosmolarity) 환경에서망막신경절세포 (RGC-) 의전사인자인 TonEBP (Tonicity responsive enhancer binding protein) 의발현을확인함으로써망막신경절세포의사멸을방지하는데있어 TonEBP 의발현이관여함을알아보고자하였다. 대상과방법 : RGC- 세포배양후고장성스트레스를유발하기위해 mm NaCl, mm mannitol, mm 및 mm glucose 를, 6,, 시간처리한후 Western immunoblotting 및 Real-time PCR 을시행하였다. 결과 : mm NaCl 처리, 6 시간및 mm mannitol 처리 6 시간후, 그리고 mm glucose 처리, 6 시간후에서 Western blotting 을통해 TonEBP 가발현되는것을확인할수있었으며 real-time PCR 을통한 TonEBP 의발현은 mm NaCl 처리 시간및 mm mannitol 처리, 시간후, 그리고 mm glucose 처리, 시간후에서통계적유의성을보였다. 결론 : Staurosporine 으로분화시킨 RGC- 세포를고장성스트레스환경에노출시켰을때 TonEBP 가발현되는것을단백질및 mrna 수준에서확인할수있었다. < 대한안과학회지 ;(8):9->

< D B9E8BEE7B5C820B8C1B8B7BDC5B0E6C0FDBCBCC6F7C1D6BFA1BCAD20C7E3C7F7C0FCC3B3C4A1BFCD F E206B696E C0C720BFAAC7D E687770>

< D B9E8BEE7B5C820B8C1B8B7BDC5B0E6C0FDBCBCC6F7C1D6BFA1BCAD20C7E3C7F7C0FCC3B3C4A1BFCD F E206B696E C0C720BFAAC7D E687770> 대한안과학회지제 49 권제 6 호 2008 J Korean Ophthalmol Soc 49(6):979-986, 2008 DOI : 10.3341/jkos.2008.49.6.979 배양된망막신경절세포주에서허혈전처치와 Protein kinase C 의역할 나경두 강성용 성공제 홍사민 전미진 김찬윤 연세대학교의과대학안과학교실, 시기능개발연구소 목적 : Staurosporin