Moon Eun-Yi, Park Hyunsung
Department of Bioscience and Biotechnology, Sejong University, Seoul 143-747, Republic of Korea.
Immunobiology. 2007;212(8):637-45. doi: 10.1016/j.imbio.2007.06.002. Epub 2007 Jul 26.
B-cell activating factor (BAFF) plays a critical role for mature B cell generation and maintenance. We have previously described that mouse BAFF (mBAFF) transcript expression was increased by toll-like receptor 4 (TLR4) agonist, lipopolysaccharide (LPS), through reactive oxygen species (ROS) production and NF-kappaB activation. Here, we investigated whether mBAFF expression could be regulated by promoter activation through the cooperation of NF-kappaB and p300, co-activator to various transcription factors. We cloned mBAFF promoter into luciferase-expressing pGL3-basic vector and computer-analyzed its NF-kappaB binding motif. Due to the existence of NF-kappaB binding motifs, activity in 2.0 kb mBAFF promoter was higher than that in 1.0 or 0.5 kb mBAFF promoter. When Raw 264.7 murine macrophages were stimulated with LPS, 2.0 kb mBAFF promoter activity was increased time dependently. Serum deprivation (0.5% FBS) producing ROS and exogenous H(2)O(2) treatment also enhanced mBAFF promoter activity, which was reduced by N-acetyl-l-cysteine (NAC), a well-known ROS scavenger. LPS and serum-starved ROS production increased NF-kappaB activation. mBAFF promoter activity was augmented by co-transfection with p65 and/or co-activator, p300. It was inhibited by dominant negative (DN) p300. Binding of p300 to BAFF promoter was detected by chromatin immunoprecipitation (ChIP) assay. Data suggest that mBAFF expression could be regulated by promoter activation through NF-kappaB activation, which might be dependent on the cooperation with co-activator, p300.
B细胞活化因子（BAFF）在成熟B细胞的生成和维持中起着关键作用。我们之前曾描述过，小鼠BAFF（mBAFF）转录本表达可通过Toll样受体4（TLR4）激动剂脂多糖（LPS），经由活性氧（ROS）生成和核因子κB（NF-κB）激活而增加。在此，我们研究了mBAFF表达是否可通过NF-κB与p300（多种转录因子的共激活因子）的协同作用而由启动子激活来调控。我们将mBAFF启动子克隆到表达荧光素酶的pGL3-基本载体中，并对其NF-κB结合基序进行计算机分析。由于存在NF-κB结合基序，2.0 kb mBAFF启动子的活性高于1.0 kb或0.5 kb mBAFF启动子。当用LPS刺激Raw 264.7小鼠巨噬细胞时，2.0 kb mBAFF启动子活性呈时间依赖性增加。产生ROS的血清剥夺（0.5%胎牛血清）和外源性H₂O₂处理也增强了mBAFF启动子活性，而这被著名的ROS清除剂N-乙酰-L-半胱氨酸（NAC）所降低。LPS和血清饥饿诱导的ROS生成增加了NF-κB激活。mBAFF启动子活性通过与p65和/或共激活因子p300共转染而增强。它被显性负性（DN）p300所抑制。通过染色质免疫沉淀（ChIP）分析检测到p300与BAFF启动子的结合。数据表明，mBAFF表达可通过NF-κB激活由启动子激活来调控，这可能依赖于与共激活因子p300的协同作用。
