단핵구에서 TNF-a Gene의 전사 활성을 증가시키는 HIF-1과 NF-kB의 기능적 상호작용

박민정,정재훈,이선민,옥순정,김혜림,김형회 대한수혈학회 2013 大韓輸血學會誌 Vol.24 No.1

배경: 종양 괴사 인자 알파(TNF-α)는 광범위하고 다양한 면역 조절 기능을 수행하는 다면발현(pleiotropic) 사이토카인이다. 단핵구와 대식세포는 염증 및 면역 조절에 중추적인 역할을 한다. NF-κB와 HIF-1은 다른 방식으로 TNF-α 유전자발현을 증가하는 것으로 알려져 있다. 방법: NF-κB와 HIF-1의 세포 내 발현을 위해인간 단핵구 세포주인 U937 세포를 표준전기천공방법으로 형질 전환시켰고, 두 단백질의 상호작용을 확인하기 위해 mammalian two-hybrid assay와Co-immunoprecipitation assay로 분석하였다. 또한, NF-κB와 HIF-1이 binding 하는 TNF-α 유전자의 프로모터를 확인하기 위해 chromatin immunoprecipitation 분석을 시행하였다. 결과: 두 전사인자 NF-κB와 HIF-1의 직접 단백질 상호 작용에 의해 프로모터 활성도에 따른TNF-α 유전자 발현이 협동적으로 증가하는 것으로 나타났다. 저산소 상태에서 HIF-1과 NF-κB에의한 TNF-α 프로모터의 활성도가 상당히 증가되었고, tandem NF-κB/HIF-1 binding site가 강력한인핸서로 작용하는 것이 TNF-α 프로모터 내에서확인되었다. NF-κB의 Rel도메인과 HIF-1의 N-TD 도메인은 물리적으로 서로 연결되어야 했다. Hypoxia 치료에서도 생체 내 NF-κB와 HIF-1 단백질상호 작용이 상당히 증가되었고, 두 전사 인자는hypoxia 자극에 의해 염색질 TNF-α 프로모토에결합되었다. 결론: 이러한 결과는 TNF-α gene활성화를 위한 다양한 세포외 신호 전달이 NF-κB/HIF-1 신호경로를 통해 전사 조절이 될 수 있음을 나타낸다. Background:Tumor necrosis factor alpha (TNF-a) is a pleiotropic cytokine fulfilling a broad variety of immunoregulatory functions. Monocytes and macrophages play a pivotal role in inflammation and immune regulation. NF-kB and HIF-1 are known to increase expression of the TNF-a gene in a separate way. Methods:Human monocytic leukemia, U937 cells, were transfected using the standard electroporation method for intracellular expression of NF-kB and HIF-1. We performed analysis using the mammalian two-hybrid assay and co-immunoprecipitation assay for detection of protein interaction of both proteins. In addition, chromatin immunoprecipitation analysis was performed for examination of NF-kB and HIF-1 binding on the TNF-a gene promoter. Results:Here we show that NF-kB and HIF-1 cooperatively induced an increase in expression of the TNF-a gene dependent on promoter activity by the direct protein interaction of these two transcription factors. Hypoxia signaling induced marked enhancement of the transactivation of TNF-a promoter by HIF-1 and NF-kB. A tandem NF-kB/HIF-1 binding site was identified within the TNF-a promoter, which acted as a strong enhancer element. Physical association of the Rel domain of NF-kB and the N-TD domain of HIF-1 was required. Hypoxia treatment also resulted in a significant increase in the protein interaction of NF-kB and HIF-1 in vivo. Both transcription factors were recruited on the chromatin TNF-a promoter dependent on hypoxia stimuli. Conclusion:The results of this study indicate that a variety of extracellular signals for activation of TNF-a gene expression might converge on the transcriptional regulation through the NF-kB/HIF-1 signaling pathway.

위상피세포주에서 타우린 포합 담즙산에 의한 Nuclear Factor-Kappa B 매개 Interleukin-8의 발현

백순구,서정인,김현수,이동기,권상옥,김혜영,김원호 대한소화기학회 2002 대한소화기학회지 Vol.39 No.3

목적: 담즙은 세정 작용에 의하여 상피 세포막의 구성성분인 지질을 용해시키고 수소 이온을 세포내로 역류시켜 점막 방어벽을 손상시키는 것으로 알려져 있으나 담즙산매개 위상피세포 손상에 관여하는 세포 신호전달 경로에 대해서는 밝혀진 바가 없다. 본 연구는 간에서 합성되어 장으로 분비되는 담즙산중에서 비교적 고농도로 존재하는 타우로콜릭산(TC)과 타우로케노데옥시콜릭산(TCDC)을 이용하여 위상피 세포주를 자극한 후 대표적 친염증성 싸이토카인으로 알려진 interleukin-8 (IL-8)의 발현여부와 IL-8 유전자의 핵심적인 전사조절인자로 알려진 nuclear factor kappa B (NF-B)의 억제제를 이용하여 IL-8의 발현에 대한 NF-의 역할을 규명하고자 하였다. 대상 및 방법: AGS Kato Ⅲ 위상피세포주에 TC와 TCDC를 각각 고농도와 저농도로 투여한 뒤 시간별로 NF-IL-8 및 IL-8의 발현을 정량적으로 분석하였고 NF-kB의 억제제인 레바미피드와 PDTC를 전처리하거나 NF-kB의 anti-sense (AS) oligonucleotide (ODN)를 위상피세포주에 트랜스펙션한 후 NF-kB의 활성도와 IL-8의 발현의 변화를 분석하였다. 결과: AGS 와Kato Ⅲ 위상피세포주에 TC 나TCDC 자극 후IL-8의 농도는 시간별, 농도별로 증가하였고 TC와 TCDC 자극 24시간 후 각각 3.3배, 4.9배의 증가를 나타내었다. AGS 와Kato Ⅲ 세포주에서 TC나 TCDC 자극 15 분 후부터 NF-kB의 활성도가 증가하기 시작하여 30∼60분 사이에 최대로 활성화 되었다가 120분 후부터는 감소하였다. 담즙산의 농도가 증가할수록B .NF-kB의 활성도가 높았다. AGS 와Kato Ⅲ 세포주에서 NF-kB의 억제제인 레바미피드와 PDTC를 전처리하였을 때와 AGS 세포주에서 NF-kB의 p-50 subunit에 대한 AS-ODN 처리를 통하여 NF-kB 활성을 억제하였을 때 NF-kB의 활성화 감소와 더불어 IL-8의 발현이 유의하게 감소하였다. 결론: 타우린 포합담즙산에 의한 위상피세포 손상의 세포 신호전달경로로 NF-kB 매개에 의한 IL-8의 발현의 활성화가 중요한 역할을 하며 담즙 역류성 위염의 가능성 있는 분자 생물학적 기전으로 생각하였다. Background/Aims: The molecular mechanism of gastric epithelial injury induced by bile acid remains poorly understood. The aims of this study were to examine whether IL-8 was expressed by the stimulation of human gastric epithelial cells (AGS and Kato III) with taurine-conjugated bile acids, taurocholic acid (TC) or taurochenodeoxycholic acid (TCDC), and to evaluate the role of Nuclear Factor-Kappa B (NF-B) on the expression of IL-8. Methods: After the gastric epithelial cells were treated with TC or TCDC, time courses of were determined by electrophoretic mobility shift assay (EMSA) and NF-B binding activity and IL-8 secretion ELISA. To evaluate the role of NF-B on the expression of IL-8, IL-8 levels were assessed after pretreatment with rebamipide BNF-anti-sense (AS) oligonucleotides (ODN) for p50 subunit of NF-B. Results: TCor pyrrolidine dithiocarbamate (PDTC), known as inhibitors, or phosphorothioate-modified or TCDC stimulation increased IL-8 secretion in a time and dose-dependent manner. Moreover, AGS and Kato Ⅲ cells treated with TC or TCDC dose-dependently induced a prominent NF-B binding complex within 60 min. Pre-incubation of the cells with PDTC (100 μM), rebamipide (0.1 and 0.5 mM) or AS-ODN caused significant decreases in IL-8 secretion induced by TC or BTCDC. Conclusions: NF-mediated IL-8 expression may play an important role in the taurine-conjugated bile acid-induced gastric epithelial injury and may present a plausible molecular mechanism for the bile reflux gastritis.

Molecular evidence for the existence of lipopolysaccharide-induced TNF-α factor (LITAF) and Rel/NF-kB pathways in disk abalone (Haliotis discus discus)

De Zoysa, M.,Nikapitiya, C.,Oh, C.,Whang, I.,Lee, J.S.,Jung, S.J.,Choi, C.Y.,Lee, J. Academic Press 2010 FISH AND SHELLFISH IMMUNOLOGY Vol.28 No.5-6

The lipopolysaccharide-induced TNF-α factor (LITAF) and Rel family nuclear factor kappaB (Rel/NF-kB) are two important transcription factors which play major roles in the regulating inflammatory cytokine, apoptosis and immune related genes. Here, we report the discovery of disk abalone LITAF (AbLITAF) and Rel/NF-kB (AbRel/NF-kB) homologues and their immune responses. Full-length cDNA of AbLITAF consists of 441 bp open reading frame (ORF) that translates into putative peptide of 147 aa. Analysis of AbLITAF sequence showed it has characteristic LITAF (Zn<SUP>+2</SUP> binding domain with two CXXC motifs. Phylogenetic analysis results further revealed that AbLITAF is a member of LITAF family. AbRel/NF-kB is 584 aa protein that contains several characteristic motifs including Rel homology domain (RHD), Rel protein signature, DNA binding motif, nuclear localization signal (NLS) and transcription factor immunoglobulin - like fold (TIG) similar to their invertebrate and vertebrate counterparts. Tissue specific analysis results showed that both AbLITAF and AbRel/NF-kB mRNA was expressed ubiquitously in all selected tissues in constitutive manner. However, constitutive expression of AbLITAF was higher than AbRel/NF-kB in all tissues except mantle. Upon immune challenge by bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes) and viral hemoragic septicemia virus (VHSV), AbLITAF showed the significant up-regulation in gills while AbRel/NF-kB transcription was not change significantly. Based on transcriptional response against immune challenge, we could suggest that regulation of TNF-α expression may have occurred mainly by LITAF activation rather than NF-kB in disk abalone. The cumulative data from other molluscs and our data with reference to TNF-α, LITAF and Rel/NF-kB from disk abalone provide strong evidence that LITAF and NF-kB are independent pathways likely to occur throughout the Phylum mollusca.

Review : Regulation of nuclear factor-kB in autoimmunity

( Shao Cong Sun ),( Jae Hoon Chang ),( Jin Jin ) 영남대학교 약품개발연구소 2013 영남대학교 약품개발연구소 연구업적집 Vol.23 No.0

Nuclear factor (NF)-kB transcripition factors are pivotal regulators of innate and adaptive immune responses, and perturbations of NF- kB signaling contribute to the pathogenesis of immunological disorders. NF-kB is a well-known proinflammatory mediator, and its deregu-lated activation is associated with the chronic inflamma-tion of autoimmune diseases. Paradoxically, NF-kB plays a crucial role in the establishment of immune tolerance including both central tolerance and the peripheral func-ton of regulatory T (Treg) cells. Thus, defective or deregulated activation of NF-kB may contribute to au-toimmunity and inflammation, highlighting the impor-tance of tightly controlled NF-kB signaling. This review focuses on recent progress regarding NF-kB regulation and its association with autoimmunity.

Short-term feeding of baicalin inhibits age-associated NF-kB activation

Kim, Dae Hyun,Kim, Hyung Keun,Park, Secongjoon,Kim, Ji Young,Zou, Yani,Cho, Ki Ho,Kim, Young Suk,Kim, Dong Hyun,Yu, Byung Pal,Choi, Jae Sue,Chung, Hae Young 경희대학교 동서의학연구소 2006 東西醫學硏究所 論文集 Vol.2006 No.-

Baicalin is a flavonoid isolated from Scutellaria baicalensis and is known to affect multiple biological functions, including the inhibition of aldose reductase, HIV infection, and nitric oxide producing activity. Oxidative stress is considered a major cause of aging and various age-related diseases, and among the key cellular components exquisitely sensitive to oxidative stress is the transcription factor, nuclear factor-KB (NF-KB). In the present study, we attempted to elucidate the mechanisms underlying the suppression of age-related NF-KB activation by baicalin in kidney tissue from old rats. Results showed NF-KB activation and the upregulation of NF-KB targeting genes, hemoxygenase-1, inducible nitric oxide synthase (iNOS), and COX-2 with age. In contrast, the increased expression of these NF-KB targeting genes was effectively inhibited by baicalin. Baicalin was shown to inhibit the NF-KB cascade via three signal transduction pathways, NIK/IKK, extracellular signal-regulated kinase (ERK), and p38 mitogenactivated protein kinase (MAPK). Our results clearly indicated the anti-oxidative effects of baicalin on age-related redox imbalance. Thus, the significance of the current study is the new information revealing the anti-oxidative properties of baicalin and the role it plays in the regulation of age-related alterations.

pp60v-src에 의한 NF-kB 활성화에 대한 헤스페레틴과 나린제닌의 저해 효과

권오송,김보연,김경아,김민수,오현철,김범석,김영호,안종석 한국생약학회 2004 생약학회지 Vol.35 No.3

- The effects of hesperetin and naringenin on NF-kB activation were investigated in normal rat kidney cells transformed by temperature sensitive Rous Sarcoma Virus (tsNRK). The flavonoids, naringenin and hesperetin, significantly reduced v-Src-induced NF-kB activation as well as phosphorylation of Akt and GSK-3 in tsNRK cells, whereas these compounds did not effect on platelet-derived growth factor (PDGF)-induced NF-kB activation in NIH3T3g1 cells. In addition, the DNA binding activity of SP-1 was also reduced but that of AP-1 was not affected by the compounds. Our study suggests that Src-induced NF-kB activation could occur via Akt-GSK-3 pathway without IkBa degradation and that naringenin and hesperetin could be used in the treatment of cancer through the inhibition of NF-kB activation.

봉약침액(蜂藥鍼液)이 RAW 264.7 세포의 iNOS, TNF-α 및 NF-kB에 미치는 영향(影響 )

김군중,심성용,이성노,김기현,Kim, Goon-Joong,Sim, Sung-Yong,Lee, Seong-No,Kim, Kee-Hyun 대한약침학회 2003 Journal of pharmacopuncture Vol.6 No.2

Objective : The purpose of this study was to investigate the effects of Bee Venom on the lipopolysaccharide(LPS), sodium nitroprusside(SNP), hydrogen peroxide$(H_2O_2)$-induced expression inducilble nitric oxide synthetase(iNOS), tumor necrosis factor-${\alpha}$(TNF-${\alpha}$) and nuclear factor kappa B(NF-kB) in RAW 264.7 cells, a murine macrophage cell line. Method : The expressions of expression iNOS and TNF-${\alpha}$ were determined by western blotting with corresponding antibodies. The expressions of expression NF-kB was assayed by EMSA method. Results : 1. The 0.5, 1 and $5{\mu}g/mg$ of bee venom on LPS-induced expression of iNOS, the $5{\mu}g/mg$ of bee venom on SNP-induced expression of iNOS and the $1{\mu}g/mg$ of bee venom on $H_2O_2$-induced expression of iNOS compared with control were inhibited significantly. 2. The 0.5, 1 and $5{\mu}g/mg$ of bee venom inhibited significantly LPS and $H_2O_2$-induced expression of TNF-${\alpha}$ compared with control, respectively. The $0.5{\mu}g/mg$ of bee venom increased significantly SNP-induced expression of TNF-${\alpha}$ compared with control. 3. The $5{\mu}g/mg$ of bee venom on LPS-induced expression of NF-kB, the $0.5{\mu}g/mg$ of bee venom on SNP-induced expression of NF-kB and the 0.5, $5{\mu}g/mg$ of bee venom on $H_2O_2$-induced expression of NF-kB were inhibited significantly compared with control, respectively.

장시간의 일회성 지구성 운동 수행 시 노화에 의한 쥐 골격근의 NF-ĸB 신호전달 변화

김형준(Kim Hyung-Jun),권형태(Kwon Hyeong-Tae),손희정(Son Hee-jung),채정훈(Chai Jung-Hoon),어수주(Eo Su-Ju),김창근(Kim Chang-Keun),김효정(Kim Hyo-Jung) 한국체육과학회 2011 한국체육과학회지 Vol.20 No.3

Nuclear factor kappa B (NF- kB) is an important transcription factor of skeletal muscle atrophy. Several studies have been explored the possibile pathways which may be modulated with exercise and age. The purpose of this study was to examine the effect of one single bout of endurance exercise on nuclear factor kappa B (NF-kB) activation and MuRF1 expression in old rat. 32 rats were used and were divided into two groups by age; 20 weeks (n=16) vs 72 weeks (n=16). Each experimental group was further divided into two subgroups; Before exercise (n=8) and after exercise (n=8). All animals in exercise groups performed a 3 hr swimming exercise (30min x 6 bouts with 5min rest). Following the swimming exercise, the protein level of NF-kB (p65) subunit was increased in both old and young group(p<.05). The protein level of NF-kB (p65) was higher in old than young group before exercise (p<.05) and the expression of MuRF1 in only old group was increased with the swimming exercise(p<.05). On the other hand, In this study phospho-IkBa was unchnaged (p>.05). These results suggest that the expression of nuclear factor kappa B (NF- kB) with endurance exercise may a key role in muscle atrophy in aged group.

Review : Regulation of the Immune System by NF-kB and IkB

( Hsiou Chi Liou ) 생화학분자생물학회 2002 BMB Reports Vol.35 No.6

NF-kB/Rel transcription factor family participates in diverse biological processes including embryo development, hematopoiesis, immune regulation, as well as neuronal functions. In this review, the NF-kB/Rel signal transduction pathways and their important roles in the regulation of immune system will be discussed. NF-kB/Rel members execute distinct functions in multiple immune cell types via the regulation of target genes essential for cell proliferation, survival, effector functions, cell trafficking and communication, as well as the formation of lymphoid architecture. Consequently, proper activation of NF-kB/Rel during immune responses to allergens, auto-antigens, alto-antigens, and pathogenic infection is crucial for the integrity of host innate and adaptive immunity.

Molecular Target Therapy of AKT and NF-kB Signaling Pathways and Multidrug Resistance by Specific Cell Penetrating Inhibitor Peptides in HL-60 Cells

Davoudi, Zahra,Akbarzadeh, Abolfazl,Rahmatiyamchi, Mohammad,Movassaghpour, Ali Akbar,Alipour, Mohsen,Nejati-Koshki, Kazem,Sadeghi, Zohre,Dariushnejad, Hassan,Zarghami, Nosratollah Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.10

Background: PI3/AKT and NF-kB signaling pathways are constitutively active in acute myeloid leukemia and cross-talk between the two has been shown in various cancers. However, their role in acute myeloid leukemia has not been completely explored. We therefore used cell penetrating inhibitor peptides to define the contributions of AKT and NF-kB to survival and multi drug resistance (MDR) in HL-60 cells. Materials and Methods: Inhibition of AKT and NF-kB activity by AKT inhibitor peptide and NBD inhibitor peptide, respectively, resulted in decreased expression of mRNA for the MDR1 gene as assessed by real time PCR. In addition, treatment of HL-60 cells with AKT and NBD inhibitor peptides led to inhibition of cell viability and induction of apoptosis in a dose dependent manner as detected by flow cytometer. Results: Finally, co-treatment of HL-60 cells with sub-optimal doses of AKT and NBD inhibitor peptides led to synergistic apoptotic responses in AML cells. Conclusions: These data support a strong biological link between NF-kB and PI3-kinase/AKT pathways in the modulation of antiapoptotic and multi drug resistant effects in AML cells. Synergistic targeting of these pathways using NF-kB and PI3-kinase/AK inhibitor peptides may have a therapeutic potential for AML and possibly other malignancies with constitutive activation of these pathways.