Vietor I, Oliveira I C, Vilcek J
Department of Microbiology and Kaplan Cancer Center, New York University Medical Center, New York, New York 10016, USA.
J Biol Chem. 1996 Mar 8;271(10):5595-602. doi: 10.1074/jbc.271.10.5595.
A construct comprising three tandemly repeated copies of the kappaB element from the interleukin-8 gene linked to chloramphenicol acetyltransferase (CAT) (3xNF-kappaBCAT) was transcriptionally activated in normal human FS-4 fibroblasts by co-transfection with expression vectors for NF-kappaB p50, p65, or p52. Unexpectedly, a significant activation of 3xNF-kappaBCAT was also seen upon its co-transfection with the expression vector for CCAAT box enhancer binding protein alpha (C/EBP-alpha) (but not C/EBP-beta or C/EBP-delta). Stimulation by C/EBP-alpha required some other factor(s) present in FS-4 cells because no transcriptional activation of 3xNF-kappaBCAT was seen after co-transfection with C/EBP-alpha in F9 mouse embryonic carcinoma cells, known to be deficient in several transcription factors. To determine whether transcriptional activation was the result of interaction with one of the major NF-kappaB proteins, we co-transfected C/EBP-alpha with NF-kappaB p50, p65, p50 + p65, or p52 into F9 or FS-4 cells. No cooperative interaction was seen; in fact, C/EBP- alpha reduced p65-stimulated transcription, especially in F9 cells. Electrophoretic mobility shift assay with a kappaB probe revealed that the addition of recombinant C/EBP-alpha protein to nuclear extracts from untreated FS-4 cells resulted in the appearance of four bands. Only one of these bands was supershifted by antibody to p50, whereas antibodies to p65 or other NF-kappaB proteins had no effect. Our findings show that C/EBP-alpha may cause activation of some kappaB element-containing genes lacking C/EBP binding sites.
一种构建体，其包含来自白细胞介素-8基因的κB元件的三个串联重复拷贝，与氯霉素乙酰转移酶（CAT）相连（3xNF-κBCAT），通过与NF-κB p50、p65或p52的表达载体共转染，在正常人FS-4成纤维细胞中被转录激活。出乎意料的是，当3xNF-κBCAT与CCAAT盒增强子结合蛋白α（C/EBP-α）的表达载体共转染时（而不是与C/EBP-β或C/EBP-δ共转染），也观察到了3xNF-κBCAT的显著激活。C/EBP-α的刺激需要FS-4细胞中存在的一些其他因子，因为在已知缺乏几种转录因子的F9小鼠胚胎癌细胞中，与C/EBP-α共转染后未观察到3xNF-κBCAT的转录激活。为了确定转录激活是否是与主要NF-κB蛋白之一相互作用的结果，我们将C/EBP-α与NF-κB p50、p65、p50 + p65或p52共转染到F9或FS-4细胞中。未观察到协同相互作用；事实上，C/EBP-α降低了p65刺激的转录，尤其是在F9细胞中。用κB探针进行的电泳迁移率变动分析表明，将重组C/EBP-α蛋白添加到未处理的FS-4细胞核提取物中导致出现四条带。这些带中只有一条被p50抗体超迁移，而p65或其他NF-κB蛋白的抗体没有作用。我们的研究结果表明，C/EBP-α可能导致一些缺乏C/EBP结合位点的含κB元件基因的激活。
