Activating Transcription Factor 3 (ATF3) represses inflammatory responses via binding to p65 subunit of NF-κB.
Activating Transcription Factor 3 (ATF3) represses inflammatory responses via binding to p65 subunit of NF-κB.
- 주제(키워드) ATF3 , p65 , NF-κB
- 발행기관 아주대학교
- 지도교수 최상돈
- 발행년도 2014
- 학위수여년월 2014. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000017775
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
ATF3 is a transcription factor that encodes a CREB / ATF family member and well known about stress inducible gene. ATF3 is induced under inflammatory responses or cell death, cytokines and oxidative stress condition. LPS-induced ATF3 is a role as negative regulator which represses generation of cytokine such as NO, IL-6, IL-12β and TNF-α in TLR4 signaling pathway. In this signaling, the key molecule which emerges inflammatory response is NF-κB. It is kwon that if p65, subunit of NF-κB, is translocated to nucleus, cytokines such as NO, IL-6, IL-12β and TNF-α are expressed. However, it has not been discovered interaction between p65 and ATF3. So we found directly interaction p65 and ATF3 using LPS-induced Raw 264.7 cell. First, we confirmed expression of NF-κB signaling after LPS treatment, and then observed that ATF3 and p65 were bound directly. In ATF3 deficient cells, NF-kB activity was up-regulated so we could guess ATF3 negative regulates p65 via binding with p65 and HDAC1. Likewise, we observed that inflammatory response genes which were induced by NF-κB activation were up-regulated in ATF3 deficient cells than control. In this result, we discovered that ATF3 plays as a negative regulator that inhibits activity of NF-κB via directly interaction with p65 subunit of NF-κB. ATF3 could bind promoter in the target cytokine genes and recruit with HDAC1 (Histone Deacetylase 1) so they deacetylased κB site in the promoter. But in this study, we discovered that ATF3 doesn’t inhibit target genes only, but also regulates NF-κB via binding to p65. Our results suggest that this is another regulation mechanism of ATF3 for negative regulator in TLR4 signaling pathway.
more목차
Acknowledgement.............................................................................................ⅰ
Abstract............................................................................................................ ⅱ
List of Figures.................................................................................................. ⅵ
Introduction.........................................................................................................1
Results..................................................................................................................4
LPS-induced inflammatory responses in Raw 264.7 Cells..............................4
ATF3 direct binds and regulates p65................................................................4
ATF3 inhibits LPS-induced nucleus translocation of NF-κB..........................5
Activation of NF-κB by ATF3 regulation........................................................6
ATF3 regulates NF-κB dependent gene expression.........................................8
Discussion...........................................................................................................21
Materials and Methods.....................................................................................24
Cell culture....................................................................................................24
RNA isolation and RT (Reverse Transcription) – PCR..................................24
Western blot analysis.....................................................................................25
Immunoprecipitation (IP) analysis................................................................26
IL-6 secretion analysis...................................................................................26
P65 Enzyme-linked immunosorbent assay (ELISA).....................................27
SEAP reporter assay......................................................................................27
NO (Nitric Oxide) assay................................................................................28
siRNA design and transfection......................................................................28
References..........................................................................................................30
국문요약.............................................................................................................35
