Effect of Transcription Factor Decoy for NF- κB on the TNF-α Induced Cytokine and ICAM-1 Expression in Cultured HaCaT cells

( Kyu Suk Lee ),( Jee Ook Kim ),( Byung Chun Kim ),( Young Wook Ryoo ) 대한피부과학회 2003 Annals of Dermatology Vol.15 No.3

N/A Background: Psoriasis is the most prevalent T-cell-mediated inflammatory skin disease in humans. Numerous cytokines and adhesion molecules are expressed in the skin lesion of psoriasis such as TNF-α, IL-1, IL-6, VCAM-1 and ICAM-1. All of them contain at least one binding site for the transcription factor NF-kB. TNF-α activates NF-kB and many other transcription factors. Thus, transcription and expression of many genes involved in the inflammatory process may be influenced by TNF-α. Objective: The purpose of this study was to study the effect of synthetic double-stranded DNA with high affinity for the NF-kB binding site on the TNF-α induced proinflammatory cytokines and ICAM-1 gene expression in the HaCaT cells. Material and Methods: We examined whether inhibition of NF-kB activity by oligodeo-xynucleotides (ODN) decoy for NF-kB blocks TNF-α induced cytokines such as IL-lα, IL-1β, IL-6 and ICAM-1 expression with electrophoretic mobility shift assay (EMSA) and reverse transcription-polymerase chain reaction (RT-PCR). Results: In EMSA, TNF-α treatment (10 ng/ml) induced the activation of NF-kB. The NF-kB binding activity in the TNF-α treated HaCaT cells increased 5.0-fold compared to non-treated group. Next, we examined the effect of liposome mediated NF-kB decoy oligonucleotides(ODN) transfection. After transfection of the NF-kB decoy ODN, TNF-α increased NF-kB binding activity to 1 .9-foid of non-treated group. Accordingly the transfection of NF-kB decoy ODN inhibited the TNF-α induced NF-kB binding activity up to 63%. RT-PCR analysis revealed that the transfection of NF-kB decoy ODN inhibited TNF-α induced cytokines and ICAM-1 mRNA expression. Conclusion: Taken together, our results suggest the potential utility of NF-kB decoy technique for biologic therapy of psoriasis.

복강내 NF-κB decoy oligodeoxynucleotide를 이용한 수술 후 복강내 유착 발생 감소

안별님 ( Byul Nim Ahn,),송미현 ( Mi Hyun Song,),김주현 ( Ju Hyun Kim,),김경현 ( Kyung Hyun Kim ),박관규 ( Kwan Kyu Park ),최윤석 ( Youn Seok Choi ) 대한산부인과학회 2012 Obstetrics & Gynecology Science Vol.55 No.4

목적 쥐모델에서 nuclear factor κB decoy oligodeoxynucleotide (NF-κB decoy ODN) 복강내 주입을 이용한 수술 후 복강내 유착 발생 억제 효과를 알아보고자 하였다. 연구방법 40마리의 BALB/c 암컷 생쥐를 ketamine과 xylazine 복강내 주사를 이용하여 마취 후 개복하고 cytobrush를 이용하여 복막이 벗겨지는 상처를 입히고, 실험군은(n = 20) NF-κB decoy ODN (10 μg, 500 μL)을 복강내에 채우고, 대조군은(n=20) 생리 식염수 500 μL를 복강내에 채운 후 봉합하고 2주 뒤에 유착 정도를 판정하였다. 유착 정도는 유착이 없는 0등급에서 심한 정도에 따라 경증, 중등도, 중증 등급(1-3등급)으로 분류하였다. 결과 수술 후 첫날 실험군에서 2마리, 대조군에서 3마리의 생쥐가 사망하였다. 대조군은 경증과 중등도의 유착이 각각 2마리에서 관찰되었고 13마리에서 중증의 유착이 관찰되었으나, 실험군에서 4마리는 유착이 관찰되지 않았고, 6마리는 경증의 유착, 8마리는 중등도의 유착이 관찰되어 통계적으로 유의한 차이를 나타내었다(P<0.001). 결론 NF-κB decoy ODN를 복강내에 주입하여 NF-κB 작용을 차단하는 방법은 수술 후 복강내 유착 정도를 줄일 수 있다. Objective The objective of this study was to evaluate the effect of intra-peritoneal nuclear factor κB (NF-κB) decoy oligodeoxynucleotide on the prevention of postoperative intra-abdominal adhesion. Methods Forty female BALB/c mice were randomly assigned into 2 groups. The mice were anesthetized and then midline abdominal incision was made. Peritoneal injury was given using cytobrush until deperitonealization. The experimental group (n = 20) received NF-κB decoy oligodeoxynucleotide (NF-κB decoy ODN) in the peritoneal cavity before abdominal closure, while the control group (n = 20) received saline. At 2 weeks postoperation, after the mice were euthanized, the status of intra-abdominal adhesion was evaluated. The degrees of adhesion were graded into no (grade 0), mild (grade 1), moderate (grade 2), and severe adhesion (grade 3). Results Two mice in the experimental group and 3 mice in the control group were dead within 1 day postoperation. In the control group, 2, 2, and 13 mice showed mild, moderate, and severe adhesion, respectively. In the experimental group, 4 mice showed no adhesion, whereas, 6 and 8 mice showed mild and moderate adhesion, respectively. The difference of degrees of adhesion between the groups was statistically signifi cant ( P<0.001). Conclusion Intra-peritoneal NF-κB decoy ODN reduced the severity of postoperative intra-abdominal adhesion.

플라보노이드 합성 유도체인 DA-6034가 인간 대장 점막 상피 세포주에서 Lipopolysaccharide 및 Tumor Necrosis Factor-α 자극에 의한 NF-κB의 활성화에 미치는 영향

김지원 ( Ji Won Kim ),정용진 ( Yong Jin Jung ),정지봉 ( Ji Bong Jeong ),김병관 ( Byeong Gwan Kim ),이국래 ( Kook Lae Lee ),김주성 ( Joo Sung Kim ),정현채 ( Hyun Chae Jung ),송인성 ( In Sung Song ) 대한장연구학회 2005 Intestinal Research Vol.3 No.1

목적: Nuclear factor κB (NF-κB)는 전염증성 사이토카인의 유전자 발현을 조절하는 전사인자로서, NF-κB를 억제함으로써 점막의 염증반응을 줄이려는 치료법들이 시도되고 있다. 쑥에서 추출한 flavonoid 성분인 eupatilin에 화학적으로 측쇄를 연결하여 만든 화합물인 DA-6034는 염증성 장질환의 동물모델에서 기존의 치료제인 sulfasalazine 및 스테로이드와 유사한 치료효과를 보인다고 보고된 바 있다. 이에 본 연구에서는 인간 대장 점막 세포주를 이용하여 DA-6034의 항염증 작용기전을 규명하고자 하였다. 대상 및 방법: HT-29 세포를 10μg/mL LPS 및 10 ng/mL TNF-α로 자극하였으며, DA-6034는 자극 전 1시간 동안 전처치를 시행하였다. 자극 후 NF-κB의 DNA 결합능은 핵 추출물을 이용한 electrophoretic mobility shift assay (EMSA)를 통하여 확인하였으며, 자극 후 inhibitory protein κB (IκB) 분해는 세포질 추출물을 이용하여 Western blot을 시행하여 확인하였다. 전염증성 사이토카인인 TNF-α와 키모카인인 IL-8의 mRNA 발현은 역전사 PCR 법으로 분석하였고, TNF-α 및 IL-8 단백질 생성은 ELISA법으로 확인 하였다. 결과: HT-29 세포를 LPS 및 TNF-α로 자극하였을 때, 두 자극 모두에 대하여 IκB가 분해되면서 NF-κB의 DNA 결합능이 증가하는 양상을 보였으나, DA-6034로 전처치를 한 경우 IκB의 분해가 억제되었으며 NF-κB의 DNA 결합능도 감소하였다. DA-6034로 전처치를 시행한 경우 LPS 자극에 의한 TNF-α의 mRNA 발현과 단백질 생성 및 TNF-α 자극에 의한 IL-8의 mRNA 발현과 단백질 생성이 억제되었다. 결론: DA-6034는 인간 대장 점막 상피 세포주에서 IκB 분해 억제를 통하여 NF-κB의 활성화를 억제함으로써 항염증 작용을 나타낼 것으로 생한된다. Background/Aims: The nuclear factor-κB (NF-κB) is associated with expression of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8). DA-6034, a derivative of flavonoid, has shown a potent anti-inflammatory effect in IBD animal model. The aim of this study was to characterize the anti-inflammatory activity of DA-6034 in terms of regulation of NF-κB. Methods: HT-29 cells were stimulated with lipopolysaccharide (LPS) or TNF-α alone or pretreated with DA-6034. The influence of DA-6034 on NF-κB DNA binding activity and inhibitory protein κB (IκB) activity was determined using an electrophoretic mobility shift assay (EMSA) and Western blot analysis. The RT-PCR method was used to determine the degree of gene expression of TNF-α and IL-8. Production of TNF-α and IL-8 protein was measured by ELISA. Results: DA-6034 prevented NF-κB activation stimulated with LPS and TNF-α and inhibited LPS- and TNF-α-induced IκB degradation. LPS-induced TNF-α mRNA expression and TNF-α-induced IL-8 mRNA expression were significantly decreased by DA-6034 pretreatment as compared to the absence of DA-6034 pretreatment. Conclusions: These results suggest that the inhibitory effect of DA-6034 on the production of TNF-α in LPS-stimulated and IL-8 in TNF-α-stimulated HT-29 cells may involve transcription regulation by suppression of NF-κB activation by interfering with IκB degradation. (Intest Res 2005; 3:38-47)

Heme Oxygenase-1 is Involved in the Down-regulation of Nuclear Transcription Factor κB Activation in the Colonic Epithelium During Inflammation

윤기중(Ki Jung Yun),김유림(Yu Rim Kim),이홍재(Heung Jae Lee),김경숙(Kyoung Suk Kim),권영미(Young-Mi Kwon),최민규(Min Kyu Choi),오재민(JaeMin Oh),정연태(Yeun Tai Chung) 대한해부학회 2004 Anatomy & Cell Biology Vol.37 No.6

HO-1은 스트레스에 의해 유도되는 효소로 그 항염증작용에 대한 기전은 거의 밝혀지지 않고 있다. NF-κB 활성은 만성 염증성 대장염을 일으키는데 관여하는 매우 중요한 인자이다. 우리는 대장염에서 NF-κB 활성과 HO-1 사이의 관계에 대하여 조사하였다. 정상인에 비하여 염증성 대장염이나 acute pesudomembranous colitis를 가진 환자에서 대장의 상피조직의 HO-1의 발현이 현저히 감소하였다. 또한 사람 대장상피세포주인 HT-29세포에서 TNF-α와 IL-1β 등의 사이토카인들은 HO-1의 발현을 억제하였다. HO-1의 유도제인 CoPPIX는 TNF-α에 의한 NF-κB 활성을 억제하였다. 또한 HO-1의 대사물질인 일산화탄소와 빌리루빈 역시 TNF-α에 의한 NF-κB 활성을 억제하였다. 그러나 다른 대사물질인 철은 TNF-α에 의한 NF-κB 활성을 억제하는데 아무런 효과가 없었다. 재미있게도 CoPPIX는 TNBS에 의해 유도된 만성 대장염을 현저히 개선하였고 궤양성 대장염의 동물모델에서도 NF-κB 활성을 억제하였다. 이상의 결과로 CoPPIX는 NF-κB 활성의 억제를 통해서 TNBS에 의해 유도된 만성 대장염의 증상들을 개선시킨다고 생각되었고 HO-1은 만성 염증성 대장염을 치료하는데 중요한 타겟물질이라고 생각하였다. Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with anti-inflammatory activity, but the mechanisms underlying this activity are incompletely understood. Nuclear transcription factor κB (NF-κB) activation is an important factor in the pathogenesis of inflammatory bowel disease (IBD). We investigated the suppressive effects of HO-1 on the activation of NF-κB by pro-inflammatory cytokines in cultured colonic epithelial cells and by trinitrobenzene sulfonic acid (TNBS) in the colon of mice. The expression level of HO-1 in the colonic epithelium of a patient with inflammatory bowel disease and pseudomembranous colitis was lower than that in a healthy control subject. In cultured human colonic epithelial HT-29 cells, pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and IL-1β down-regulate HO-1 expression. The HO-1 inducer, cobalt protoporphyrin IX (CoPPIX), dramatically down-regulated NF-κB activation in HT-29 cells by TNF-α. In addition, bilirubin-a product of heme catabolism by HO-1-and the carbon monoxide donor tricarbonyldichlororuthenium (II) dimer also suppressed NF-κB activation by TNF-α. However, iron, another heme metabolite, did not suppress NF-κB activation by TNF-α. Furthermore, CoPPIX diminished the macroscopic and histopathological symptoms of TNBS-induced colitis and down-regulated NF-κB activation in mice. In conclusion, this study suggests that HO-1 plays an important role in the down-regulation of NF-κB activation, which is a key factor in the pathogenesis of IBD and is thus an excellent therapeutic target for the treatment of IBD.

결장직장암에서 Nuclear Factor-κB p65와 Nuclear Factor-κB p50 단백 발현과 임상병리학 인자와의 연관성

강기창(Gi Chang Kang),김범규(Beom Gyu Kim),박준석(Jun Suk Park),최유신(Yu Sin Choi),차성재(Sung Jae Cha),박성준(Sung Jun Park),장인택(In Taik Chang),박성일(Sung il Park),이태진(Tae Jin Lee),최영철(Young Cheol Choi) 대한외과학회 2008 Annals of Surgical Treatment and Research(ASRT) Vol.75 No.2

Purpose: Nuclear Factor-κB p65 (NF-κB p65) and nuclear Factor-κB1 p50 (NF-κB p65) have been shown to play roles in cell proliferation, apoptosis, cytokine production and oncogenesis. This study was designed to investigate the expressions of NF-κB p65 and NF-κB p50 proteins in premalignant lesions and colorectal adenocarcinoma. Methods: Paraffin sections of 20 normal mucosa specimens, 20 low grade tubular adenoma specimens, 20 high grade tubular adenoma specimens and 64 adenocarcinoma specimens were analyzed immunohistochemically for the expressions of NF-κB p65 and NF-κB p50 proteins. Results: The expressions of NF-κB p65 and NF-κB p50 proteins were significantly higher in the adenocarcinoma tissue compared with that in the normal mucosa, the low grade tubular adenoma and the high grade tubular adenoma tissues. The frequency of a NF-κB p50 expression was higher in the poorly differentiated histologic grade specimens, in the presence of nodal metastasis and in the high stage specimens. There were significant correlations between the NF-κB p65 and NF-κB p50 proteins. Conclusion: The expressions of NF-κB p65 and NF-κB p50 proteins may play a role in the pathogenesis of colorectal carcinoma.

Hsp90에 의한 NF-κB의 활성화를 촉진하는 IKKγ의 역할

이정아,김동완 한국생명과학회 2022 생명과학회지 Vol.32 No.3

NF-κB acts as a critical transcription factor in inflammation and innate immunity, and it is also closely involved in cell survival and tumorigenesis via induction of anti-apoptotic genes. In these processes, NF-κB cooperates with multiple other signaling molecules and pathways, and although many studies have demonstrated that Hsp90 regulates NF-κB activity, the exact mechanism is unclear. In this study, we investigated the relationship between Hsp90 and IKKγ in the regulation of NF-κB using expression plasmids of IKK complex components. Wild-type and deletion mutants of IKKγ were expressed together with Hsp90, and the combined regulatory effect of Hsp90 and IKKγ on NF-κB activation was assayed. The results show that Hsp90 activates NF-κB by promoting the phosphorylation and degradation of IκBα and that activation of NF-κB by NIK and LPS was increased by Hsp90. IKKγ elevated the effect of Hsp90 on NF-κB activation by increasing phosphorylation and degradation of IκBα. The positive regulation on NF-κB by Hsp90 and IKKγ was also proved in analysis with IKKβ-EE, the constitutively active form of IKKβ. In experiments with the deletion mutants of IKKγ, the N-terminal IKKβ binding domain, C-terminal leucine zipper, and zinc finger domains of IKKγ were found not necessary for the positive regulation of NF-κB activity. Additionally, the expression of pro-inflammatory cytokines was synergistically elevated by Hsp90 and IKKγ. These results indicate that inhibiting the interaction between Hsp90 and IKKγ is a possible strategic method for controlling NF-κB and related diseases. NF-κB는 염증과 선천성 면역에 중요한 전사인자이며 anti-apoptotic gene을 유도하여 세포의 생존과 발암에도 깊이 연관되어 있으며 많은 신호전달분자 및 신호전달회로와 연결되어있다. 한편, Hsp90는 NF-κB의 활성을 조절한다는 보고가 이루어졌으나 그 구체적인 기전은 알려져 있지 않다. 본 연구에서는 IKK compelx를 구성하는 인자들의 발현 plasmid를 이용하여 NF-κB의 활성조절에서 Hsp90와 IKKγ의 연관성 및 역할을 연구하였다. 그 결과 Hsp90는 IκBα의 인산화와 분해를 촉진하여 NF-κB를 활성화시켰고, NIK과 LPS에 의한 NF-κB의 활성화는 Hsp90에 의해 더욱 활성이 증가하였다. IKKγ는 Hsp90에 의해 증가된 IκBα의 인산화와 분해를 더욱 촉진함으로써 Hsp90의 NF-κB 활성화 작용을 상승시켰다. 이러한 Hsp90와 IKKγ에 의한 NF-κB의 활성화 현상은 항상 활성화 된 상태를 유지하는 IKKβ-EE mutant를 이용한 검토에서도 입증되었다. 또한 IKKγ의 deletion mutant를 이용한 검토에서 IKKγ의 N-말단에 위치하는 IKKβ 결합부위와 C-말단에 위치하는 leucine zipper 및 zinc finger 부위는 IKKγ와 Hsp90의 NF-κB에 대한 상호협력적 촉진작용에 필요하지 않았다. 또한 Hsp90에 의해 촉진된 세포내 pro-inflammatory cytokine들의 발현은 IKKγ에 의해 더욱 상승하였다. 이러한 결과로부터 Hsp90와 IKKγ의 상호작용을 차단한다면 NF-κB의 과다활성으로 인한 질병의 예방과 치료에 도움을 줄 수 있을 것으로 사료된다.

NF-κB 신호경로에서 CLK3의 새로운 음성 조절자로서의 기능

전별은(Byeol-Eun Jeon),권찬성(Chan-Seong Kwon),이지은(Ji-Eun Lee),우예린(Ye-Lin Woo),김상우(Sang-Woo Kim) 한국생명과학회 2022 생명과학회지 Vol.32 No.11

만성 염증은 종양의 발생 및 진행과 밀접하게 연관되어 있다. 핵인자 kappa B (NF-κB)는 5개의 전사인자로 구성되며 염증 반응에 필수적인 역할을 한다. 다양한 암에서 NF-κB의 조절장애가 보고되고 있으며 NF-κB 조절이 암 치료에 있어 핵심 표적이 된다. 본 연구에서는 CDC Like Kinase 3 (CLK3)를 NF-κB 신호전달 경로를 조절하는 새로운 키네이스임을 확인하였다. 우리는 CLK3가 정규 및 비정규 NF-κB 신호전달경로를 억제하는 것을 밝혔다. CLK3 과발현 또는 knock-down 세포주를 이용한 루시퍼레이즈 분석 결과, 이 키네이스는 TNFα와 PMA가 유도하는 정규 NF-κB 신호전달경로 활성을 억제하였다. 또한 CLK3 과발현은 잘 알려진 비정규 NF-κB 신호경로 유도제인 NF-κB-inducing kinase (NIK) 또는 CD40에 의한 NF-κB 활성을 저해하였다. 추가적으로 CLK3의 NF-κB 신호전달 저해기전을 설명하고자 TNFα 처리 후 웨스턴 블롯 분석으로 이 키네이스 영향권 내에 있는 NF-κB 신호경로 분자들을 식별하였다. 그 결과 CLK3가 TAK1, IKKα/β, p65, IκBα 및 ERK1/2-MAPK의 인산화/활성화를 저해하여 TNFα 처리로 유도된 NF-κB 및 MAPK 신호경로를 모두 억제함을 밝혔다. 앞으로의 연구는 CLK3가 정규 및 비정규 NF-κB 경로를 억제하는 기작을 밝히는데 초점을 맞출 것이다. 위 연구 결과들을 토대로 CLK3가 NF-κB 신호전달경로의 새로운 음성 조절자로써 기능함을 제시하였다. Chronic inflammation has been shown to be closely associated with tumor development and progression. Nuclear factor kappa B (NF-κB) is composed of a family of five transcription factors. NF-κB signaling plays a crucial role in the inflammatory response and is often found to be dysregulated in various types of cancer, making it an attractive target in cancer therapeutics. In this study, CDC-like kinase 3 (CLK3) was identified as a novel kinase that regulates the NF-κB signaling pathway. Our data demonstrate that CLK3 inhibits the canonical and non-canonical NF-κB pathways. Luciferase assays following the transient or stable expression of CLK3 indicated that this kinase inhibited NF-κB activation mediated by Tumor necrosis factor-alpha (TNFα) and Phorbol 12-myristate 13-acetate (PMA), which are known to activate NF-κB signaling via the canonical pathway. Consistent with data on the ectopic expression of CLK3, CLK3 knockdown using shRNA constructs increased NF-κB activity 1.5-fold upon stimulation with TNFα in HEK293 cells compared with the control cells. Additionally, overexpression of CLK3 suppressed the activation of this signaling pathway induced by NF-κB-inducing kinase (NIK) or CD40, which are well-established activators of the non-canonical pathway. To further examine the negative impact of CLK3 on NF-κB signaling, we performed Western blotting following the TNFα treatment to directly identify the molecular components of the NF-κB pathway that are affected by this kinase. Our results revealed that CLK3 mitigated the phosphorylation/activation of transforming growth factor-β-activated kinase 1 (TAK1), inhibitor of NF-κB kinase alpha/beta (IKKα/β), NF-κB p65 (RelA), NF-κB inhibitor alpha (IκBα), and Extracellular signal-regulated kinase 1/2-Mitogen-activated protein kinase (ERK1/2-MAPK), suggesting that CLK3 inhibits both the NF-κB and MAPK signaling activated by TNFα exposure. Further studies are required to elucidate the mechanism by which CLK3 inhibits the canonical and non-canonical NF-κB pathways. Collectively, these findings reveal CLK3 as a novel negative regulator of NF-κB signaling.

배양한 섬유아세포와 혈관내피세포에서 고혈당과 TNF-α에의한 NF-κB 의 활성화

김기훈 ( Ki Hun Kim ),김병천 ( Byung Chun Kim ),이규석 ( Kyu Suk Lee ) 대한피부과학회 2003 대한피부과학회지 Vol.41 No.4

N/A Background : A common endpoint of hyperglycemia dependent cellular changes is the generation of reactive oxygen intermediates (ROIs) and the presence of elevated oxidative stress. Therefore, oxidative stress is supposed to play an important role in the development of late diabetic complications. The transcriptional nuclear factor kB(NF-kB) can be activated by diverse stimuli such as cytokines, mitogens, oxidative stress, and lipids, leading to the transactivation of several genes that play important roles in the development of atherosclerosis, skin thickness, scleredema adultorum et at. Objective and Method : The dysfunction of endothelial cells and fibroblasts plays a role in late diabetic complication. This dysfunction may be caused or exacerbated by expression of many genes potently activated by the NF-kB. We have examined whether high glucose conditions to simulate the diabetic state can lead to the activation of NF-kB in cultured fibroblasts and endothelial cells Result : Within 3h incubation, high glucose (5, 10, 25 mmol/L) alone induced an increase in NF-kB activity in endothelial cells and fibroblasts. High glucose also enhanced NF-kB activity stimulated by TNF-a. Incubation with high glucose for 24h followed by stimulation with TNF-a led to a marked potentiation of NF-kB activation compared with normoglycemic (5 mmol/L) endothelial cells and fibroblasts exposed to TNF-a. An antioxidant thioctic acid and transcription factor decoy for NF-kB significantly suppressed the TNF-n induced NF-kB activity in both endothelial cells and fib-roblasts. Conclusion : These results suggest that high glucose or high glucose with TNF-n cause activation of NF-KB. And co-incubation with an antioxidant, thioctic acid, and transcription factor decoy produced the inhibition of glucose-induced NF-KB activation. Thus NF-KB activation is an early occurrence due to the elevations in glucose, which may elicit multiple pathways contributing to the origin of hyperglycemia- or diabetes-induced endothelial and fibroblast cell injury. In summary, our results for the first time suggest that therapeutic strategies involving inhibition of NF-kB activation induced by high glucose nay help avoid some of the complications of diabetes.(Korean J Dermatol 3003;41(4) : 423∼428)

Hsp70와 IKKγ에 의한 NF-κB 활성억제의 상승효과

김미정(Mi Jeong Kim),김가혜(Ka Hye Kim),김문정(Moon Jeong Kim),김진익(Jin Ik Kim),최혜정(Hye Jung Choi),문자영(Ja Young Moon),주우홍(Woo Hong Joo),김동완(Dong Wan Kim) 한국생명과학회 2016 생명과학회지 Vol.26 No.9

NF-κB는 anti-apoptotic gene을 유도하는 전사인자로서 대부분의 세포의 생존에 필요하다. 그러나 NF-κB가 많은 종류의 암세포에서 지속적으로 과다 활성화됨이 알려지면서 NF-κB의 활성억제가 암의 예방과 치료에 유효하다는 점이 알려지게 되었다. 한편, Hsp70가 NF-κB의 활성을 조절한다는 사실이 알려지면서 Hsp70를 이용한 암예방과 치료가 주목받게 되었으나 아직 Hsp70에 의한 NF-κB의 활성조절기전은 명확하지 않다. 본 연구에서는 Hsp70에 의한 NF-κB의 활성조절과정에서 IKK complex의 구성성분인 IKKγ의 역할을 검토하였다. IKKγ의 wild type과 deletion mutants를 이용하여 Hsp70와 관련된 NF-κB의 활성조절을 연구한 결과 Hsp70는 NF-κB의 활성화를 억제하였으며, 이러한 억제효과는 IKKγ가 과발현되었을 때 더욱 증가하였다. 또한 IKKγ의 N-말단의 IKKβ결합부위와 C-말단의 Leucine zipper 및 Zinc finger부위는 Hsp70와 연관된 NF-κB억제작용에 필요하지 않는 것으로 나타났으며, Hsp70와 IKKγ에 의한 NF-κB의 활성억제는 IκBα의 인산화와 분해를 저해함에 의해 일어나는 것으로 나타났다. 또한 RAW264.7 macrophage세포에서 LPS에 의한 COX-2의 발현유도는 Hsp70와 IKKγ가 동시에 발현 되었을 때 가장 효과적으로 억제되었다. 이상의 결과로부터 Hsp70에 의한 NF-κB의 활성억제작용은 IKKγ에 의해 상승됨을 알 수 있었으며, Hsp70와 IKKγ를 적절히 이용하면 NF-κB의 과다활성에 의해 발생하는 각종 질병의 예방과 치료에 도움을 줄 수 있을 것으로 기대된다. NF-κB acts as a critical transcription factor for the survival of cells via the induction of antiapoptotic genes. Constitutive activation of NF-κB in many types of solid tumors suggests that the inhibition of NF-κB might prevent or inhibit tumorigenesis. Although a number of studies demonstrated that Hsp70 regulated NF-κB activity, the exact mechanism is not clear. This study investigated the functional relationship of Hsp70 and IKKγ in the regulation of NF-κB activation using expression plasmids of components of the IKK complex. Wild-type and deletion mutants of IKKγ were expressed together with Hsp70, and the combined regulatory effect of Hsp70 and IKKγ on NF-κB activation was assayed. Hsp70 suppressed the activation of NF-κB in a reporter plasmid assay. Hsp70 also suppressed the phosphorylation and degradation of IκBα. The suppressive effect of Hsp70 on NF-κB activation was synergistically elevated by IKKγ. The N-terminal IKKβ binding site, C-terminal leucine zipper, and zinc finger domains of IKKγ were not necessary for the suppressive effect. Furthermore, Hsp70 and IKKγ synergistically suppressed the induction of COX-2 expression by lipopolysaccharides in RAW264.7 cells. These results suggest that overexpression of Hsp70 and IKKγ may be a strategic method for inhibition of NF-κB and related diseases.

폐암세포주에서 NF-κB 활성 억제를 통한 Proteasome 억제제 MG132의 TRAIL-유도성 Apoptosis 감작 효과

서필원 ( Pil Won Seo ),이계영 ( Kye Young Lee ) 대한결핵 및 호흡기학회 2008 Tuberculosis and Respiratory Diseases Vol.65 No.6

연구배경: 정상세포는 보호되고 종양세포에 독성을 보인다고 알려진 TNF유전자족으로 새로이 확인된 TRAIL이 폐암세포에서 보이는 아포프토시스 효과를 확인하고, 아포프토시스로부터 세포를 보호하는 전사인자 NF-κB가 TRAIL에 의하여 활성화 되는 정도를 평가하여 MG132의 NF-κB활성억제가 TRAIL 유도성 아포프토시스를 감작시키는지를 확인하기 위하여 본 연구를 시행하였다. 방법: A549(wt p53) 및 NCI-H1299(null p53) 폐암세포주를 사용하였다. 세포독성 검사는 MTT assay를 이용하였고 아포프토시스는 Annexin V assay와 FACS 분석을 이용하였다. NF-κB 전사활성은 luciferase reporter gene assay를 이용하였고 IκBα 분해는 western blot을 이용하였으며, TRAIL에 의해 활성화된 NF-κB와 DNA 결합은 electromobility shift assay와 anti-p65 antibody를 이용한 supershift assay로 확인하였다. 결과: 1) TRAIL 100 ng/ml 농도에서 wild-type p53인 A549 폐암세포는 34.4%, p53 null인 NCI-H1299 폐암세포는 26.4%의 세포사를 관찰하였다. 2) Luciferase reporter gene assay로서 TRAIL에 의한 NF-κB의 활성이 A549 IgGκB-luc세포에서 2.45배 증가하고 NCI-H1299 IgGκB-luc세포에서는 1.47배 증가함을 관찰하여 TRAIL에 의하여 NF-κB가 활성화됨을 확인하였다. 3) MG132의 전처치로 TRAIL에 의한 NF-κB의 활성이 A549 세포와 NCI-H1299 세포에서 각각 기저수준의 0.24, 0.21배로 강력히 억제되었다. 4) TRAIL단독으로 30% 전후의 세포독성이 MG132 전처치 후 TRAIL을 투여하면 두 세포주 모두에서 80% 이상의 세포독성이 관찰되어 MG132가 TRAIL유도성 아포프토시스에 감작효과가 있음을 확인하였다. 결론: 이상의 결과로 TRAIL에 상대적인 내성을 보이는 폐암세포주에서 MG132가 NF-κB 활성억제로서 TRAIL유도성 아포프토시스를 강화시키는 효과가 있음을 확인할 수 있었다. 따라서 본 연구는 향후 폐암치료에 있어서 TRAIL유도성 아포프토시스가 이용될 수 있는 가능성을 확인한 기초자료가 된다고 생각된다. Background: TRAIL (TNF-related apoptosis inducing ligand) is a newly identified member of the TNF gene family which appears to have tumor-selective cytotoxicity due to the distinct decoy receptor system. TRAIL has direct access to caspase machinery and induces apoptosis regardless of p53 phenotype. Therefore, TRAIL has a therapeutic potential in lung cancer which frequently harbors p53 mutation in more than 50% of cases. However, it was shown that TRAIL also could activates NF-κB in some cell lines which might inhibit TRAIL-induced apoptosis. This study was designed to investigate whether TRAIL can activate NF-κB in lung cancer cell lines relatively resistant to TRAIL-induced apoptosis and inhibition of NF-κB activation using proteasome inhibitor MG132 which blocks IκBα degradation can sensitize lung cancer cells to TRAIL-induced apoptosis. Methods: A549 (wt p53) and NCI-H1299 (null p53) lung cancer cells were used and cell viability test was done by MTT assay. Apoptosis was confirmed with Annexin V assay followed by FACS analysis. To study NF-κB-dependent transcriptional activation, a luciferase reporter gene assay was used after making A549 and NCI-H1299 cells stably transfected with IgGκ-NF-κB luciferase construct. To investigate DNA binding of NF-κB activated by TRAIL, electromobility shift assay was used and supershift assay was done using anti-p65 antibody. Western blot was done for the study of IκBα degradation. Results: A549 and NCI-H1299 cells were relatively resistant to TRAIL-induced apoptosis showing only 20~30% cell death even at the concentration 100 ng/ml, but MG132 (3μM) pre-treatment 1 hour prior to TRAIL addition greatly increased cell death more than 80%. Luciferase assay showed TRAIL-induced NF-κB transcriptional activity in both cell lines. Electromobility shift assay demonstrated DNA binding complex of NF-κB activated by TRAIL and supershift with p65 antibody. IκBα degradation was proven by western blot. MG132 completely blocked both TRAIL-induced NF-κB dependent luciferase activity and DNA binding of NF-κB. Conclusion: This results suggest that inhibition of NF-κB can be a potentially useful strategy to enhance TRAIL-induced tumor cell killing in lung cancer. (Tuberc Respir Dis 2008;65:476-486)