Ataxia telangiectasia mutated(ATM)의 산화성 스트레스에 대한 세포보호작용 : heme oxygenase-1 유도

Dept. of Medical Science/박사Ataxia telangiectasia (AT) is caused by the mutational inactivation of ataxia telangiectasia mutated (ATM) gene which is involved in DNA repair process. A part of the clinical phenotype of AT is associated with oxidative stress. Compelling evidence shows the increased oxidative stress in human AT cells and neuronal tissues of ATM-deficient mice. However, it is unclear whether ATM itself is directly involved in sensing the increase of reactive oxygen species (ROS) or whether ATM regulates the expression of genes involved in oxidative stress responses. Heme oxygenase-1 (HO-1), whose main function is the degradation of heme, is known to be an important antioxidant enzyme. The role of HO-1 is cytoprotection against oxidant injury. Therefore, it is hypothesized that ATM may induce antioxidant enzyme such as HO-1 against oxidative stress. In the present study, A-T fibroblasts stably transfected with human full-length ATM cDNA (Atm+/+ cells) or empty vector (Atm-/- cells) were used. As a source of reactive oxygen species, hydrogen peroxide was treated to the cells. The cytoprotective effect of ATM against oxidative stress was investigated by determining the activation of nuclear factor-κB (NF-κB) and protein kinase C (PKC δ) as well as HO-1 induction. As a result,transfection of ATM inhibited ROS-induced cell injuries including apoptosis through the induction of HO-1 in Atm-/- cells. Transfection of ATM induces the expression of HO-1 which was mediated by PKC δ and NF-κB in H2O2–treated Atm-/- cells. ZnPP, an HO-1 inhibitor, and transfection of HO-1 siRNA increased ROS levels and apoptosis in H2O2-treated cells, whereas hemin, an HO-1 activator, decreased ROS levels and apoptosis. Transfection of a dominant-negative mutant I-κBa (MAD-3) decreased H2O2-induced HO-1 expression. Rottlerin, a PKC δ inhibitor, inhibited NF-κB activation as well as HO-1 expression. These results suggest that PKC δ is an upstream signaling for the activation of NF-κB, which induces HO-1 expression in H2O2-treated Atm+/+ cells. In conclusion, cytoprotective effect of ATM against oxidative stress is mediated by induction of antioxidant enzyme HO-1 via activation of PKC δ and NF-κB. The results may suggest the possible mechanisms how AT patients are vulnerable to oxidative stress.ope

Signal transduction for the expression of cyclooxygenase-2 and inducible nitric oxide synthase i

의과학과/석사[한글]Helicobacter pylori(H. pylori)는 다양한 위장관질환을 일으키는 원인균으로 알려져 있다. H. pylori 감염에 의해 증가하는 활성산소는 핵전사인자인 AP-1과 NF-κB의 활성을 증가시키고, 염증매개 유전자 발현에 기여한다고 알려져 있다. AP-1과 NF-κB는 염증 매개 효소인 inducible nitric oxide synthase(iNOS) 와 cyclooxygenase-2(COX-2)의 promoter 부위에 결합 부위를 갖고 있다. 본 연구에서는 한국인 십이지장 궤양 환자의 점막에서 분리한 H. pylori 균주인 HP99를 사람 위 상피세포주인 AGS에 감염시켜 H. pylori에 의한 AGS 세포의 핵전사조절인자의 활성 변동을 규명하고자 하였다. 아울러 핵전사조절인자의 활성을 염증매개효소의 발현 변동에 의해 규명하고자 하였다. 먼저 핵전사조절인자의 활성을 측정한 결과, NF-κB와 AP-1의 활성 증가를 확인하였다. 그리고 H. pylori 감염에 의해 COX-2 및 iNOS의 발현이 증가됨을 확인하였다. H. pylori에 의한 AGS 세포의 COX-2 및 iNOS 발현 증가가 어떤 핵전사인자에 의하여 매개되는지 여부를 알아보기 위하여, IκB 변이 유전자인 MAD3 또는 c-Jun 및 Ras의 dominant negative 유전자인 TAM67, Ras N-17 유전자를 세포내로 transfection 시킴으로서 NF-κB, AP-1 그리고 Ras의 활성화를 각각 억제시킨 결과 COX-2 및 iNOS 발현이 억제되었다. 이상의 결과로 보아 위 상피세포에서 H. pylori는 Ras, AP-1 그리고 NF-κB를 활성화시키고, 이는 COX-2 및 iNOS의 발현을 유도함을 알 수 있었다. 또한 이러한 결과로 iNOS의 생성물인 nitrite와 COX-2의 생성물인 PGE2 단백양이 증가함을 알 수 있었다. [영문]Helicobacter pylori infection leads to gastroduodenal inflammation, peptic ulcer, and gastric carcinoma. Oxygen radicals are important regulators in Helicobacter pylori (H. pylori)-induced gastric disease, COX-2 and iNOS might be regulated by oxidant-sensitive transcription factors, NF-κB and AP-1. Cyclooxygenase-2(COX-2) and inducible nitric oxide synthase(iNOS) are important enzymes that mediate inflammatory processes. In addition, the binding sites for NF-κB and AP-1 are found in the promoter region of COX-2 and iNOS gene. Present study was aimed to investigate whether H. pylori-induced expressions of COX-2 and iNOS are regulated by NF-κB and AP-1 in gastric epithelial AGS cells, and whether the transcriptional regulation of COX-2 and iNOS are inhibited by transfection with mutant genes for Ras(ras N-17), c-Jun(TAM67), and IκBα(MAD3). As a result, H. pylori induced activation NF-κB and AP-1 and thus COX-2 and iNOS expressions were induced in gastric epithelial cells. Transfection with mutant genes for Ras(ras N-17), c-Jun(TAM67), and IκBα(MAD3) inhibited H. pylori-induced COX-2 and iNOS expression in AGS cells. In conclusion, H. pylori induced the expression of mRNA and protein of COX-2 and iNOS via activation of ras, NF-κB, and AP-1.ope