摘要
目的探索巨噬细胞中含bromodomain蛋白3(Brd3)对脂多糖(LPS)诱导的白细胞介素6(IL-6)产生的调控作用。方法利用CRISPR-Cas9技术筛选出Brd3敲除的细胞,以Brd3敲除细胞为实验组,正常表达Brd3的细胞为对照组。100 ng/m L LPS刺激后ELISA检测细胞培养上清中IL-6的水平;采用Western blot法检测核因子κB(NF-κB)和丝裂原激活蛋白激酶(MAPK)信号通路的表达活化情况;染色质免疫沉淀实验检测IL6基因启动子区乙酰基转移酶CREB结合蛋白(CBP)的募集和组蛋白H3乙酰化修饰水平。结果小鼠腹腔巨噬细胞中Brd3 mRNA和蛋白表达水平在LPS刺激后显著降低。与对照组相比,Brd3敲除细胞中LPS诱导产生的IL-6水平显著降低。NF-κB和MAPK信号通路相关分子的表达无差异;Brd3敲除细胞IL6启动子区乙酰基转移酶CBP的募集显著下降,组蛋白H3的乙酰化水平显著降低。结论 Brd3通过促进IL6启动子区乙酰基转移酶CBP的结合和组蛋白H3的乙酰化修饰,促进巨噬细胞中LPS触发的IL-6的产生。
Objective To investigate the role of bromodomain containing 3（ Brd3） in LPS-triggered interleukin-6（ IL-6）production in macrophages and the underlying mechanism. Methods CRISPR-Cas9 technology was used to screen an RAW264. 7 cell line with Brd3 knockout（ Brd3-/-）. The Brd3-/-cells were used as an experimental group,and the parential cells expressing wide-type Brd3 as a control group. The IL-6 level in cell culture supernatant was detected by ELISA after100 ng / m L LPS challenging. Effect of Brd3 knockout on the expression and activation of signal pathways involved in IL-6 expression,including the NF-κB and mitogen-activated protein kinase（ MAPK） pathways were examined by Western blot analysis.Chromatin immunoprecipitation（ Ch IP） assay was used to evaluate the recruitment of acetylase CREB-binding protein（ CBP） to IL6 gene promoter and the acetylation level of histone 3 within IL6 gene promoter. Results LPS treatment significantly downregulated Brd3 expression in mouse peritoneal macrophages. LPS-induced production of IL-6 was significantly inhibited in Brd3-/-macrophages. The expressions and activation of signal molecules within NF-κB and MAPK pathways were barely affected. Brd3 knockout significantly decreased the recruitment of acetylase CBP to IL6 gene promoter,and the acetylation level of histone3 within IL6 gene promoter was also repressed. Conclusion Brd3 promotes LPS-triggered IL-6 production via promoting the recruitment of CBP to IL6 promoter and enhancing the acetylation level of histone 3 within IL6 promoter.
作者
任文汇
孙东豪
王春梅
李楠
REN Wenhui SUN Donghao WANG Chunmei LI Nan(Department of Immunology, College of Basic Medicine, Second Military Medical University, Shanghai 200433 Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005, China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2016年第10期1301-1305,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81272279)
医学免疫学国家重点实验室开放课题(2015K01)
