Gray J G, Chandra G, Clay W C, Stinnett S W, Haneline S A, Lorenz J J, Patel I R, Wisely G B, Furdon P J, Taylor J D
Department of Molecular Biology, Glaxo Research Institute, Inc., Research Triangle Park, North Carolina 27709.
Mol Cell Biol. 1993 Nov;13(11):6678-89. doi: 10.1128/mcb.13.11.6678-6689.1993.
Transfection of U937 and THP-1 cells with a recombinant plasmid, pIL1(4.0kb)-CAT, containing 4 kb of the interleukin 1 beta (IL-1 beta) gene upstream regulatory sequence resulted in inducer-dependent expression of chloramphenicol acetyltransferase activity. Treatment of the transfected cells with various combinations of the inducers lipopolysaccharide, phorbol myristate acetate, and dibutyryl cyclic AMP upregulated the IL-1 beta promoter. In U937 and THP-1 cells, maximum stimulation of both the endogenous IL-1 beta gene and pIL1(4.0kb)-CAT transfectants was observed following treatment with the combination of inducing agents lipopolysaccharide-phorbol myristate acetate-dibutyryl cyclic AMP. This combination of inducing agents was used to identify and study, at the molecular level, some of the regulatory elements necessary for induction of the IL-1 beta gene. A series of 5' deletion derivatives of the upstream regulatory sequence were used in transient transfection assays to identify an 80-bp fragment located between -2720 and -2800 bp upstream of the mRNA start site that was required for induction. Exonuclease III mapping, electrophoretic mobility shift assays (EMSA), and DNA sequence analysis of this region were used to identify a transcription factor binding sequence which contained a potential cyclic AMP response element (CRE/ATF)- and NF-kappa B-like binding site. Site-directed mutagenesis of the CRE/ATF-like site resulted in the loss of binding of a specific factor or factors as determined by EMSA. The loss of binding activity directly correlated with a loss of approximately 75% of promoter activity as determined in transient transfection assays. As determined by EMSA, the factor binding to the CRE/ATF-like site was present in nuclear extracts prepared from both uninduced and induced THP-1 and U937 cells. However, the intensity of the band appeared to be increased when nuclear extracts from induced cells were used. In contrast to the CRE/ATF mutation, which resulted in the loss of promoter activity, mutation of the NF-kappa B-like site resulted in a moderate increase in activity in U937 cells. A similar increase in promoter activity was not observed in THP-1 cells. From these studies, we conclude that a CRE/ATF-like site and a factor or factors interacting with this site are essential for the maximum induction of the IL-1 beta gene in stimulated U937 and THP-1 cells.
用含有4kb白细胞介素1β（IL-1β）基因上游调控序列的重组质粒pIL1(4.0kb)-CAT转染U937和THP-1细胞，导致氯霉素乙酰转移酶活性呈诱导剂依赖性表达。用诱导剂脂多糖、佛波酯和二丁酰环磷酸腺苷的各种组合处理转染细胞，可上调IL-1β启动子。在U937和THP-1细胞中，在用脂多糖-佛波酯-二丁酰环磷酸腺苷诱导剂组合处理后，观察到内源性IL-1β基因和pIL1(4.0kb)-CAT转染子均受到最大刺激。这种诱导剂组合用于在分子水平上鉴定和研究诱导IL-1β基因所需的一些调控元件。上游调控序列的一系列5'缺失衍生物用于瞬时转染试验，以鉴定位于mRNA起始位点上游-2720至-2800bp之间的一个80bp片段，该片段是诱导所必需的。对该区域进行核酸外切酶III作图、电泳迁移率变动分析（EMSA）和DNA序列分析，以鉴定一个转录因子结合序列，该序列包含一个潜在的环磷酸腺苷反应元件（CRE/ATF）和NF-κB样结合位点。对CRE/ATF样位点进行定点诱变，导致如EMSA所确定的一种或多种特定因子的结合丧失。结合活性的丧失与瞬时转染试验中所确定的启动子活性约75%的丧失直接相关。如EMSA所确定的，与CRE/ATF样位点结合的因子存在于从未诱导和诱导的THP-1和U937细胞制备的核提取物中。然而，当使用诱导细胞的核提取物时，条带强度似乎增加。与导致启动子活性丧失的CRE/ATF突变相反，NF-κB样位点的突变导致U937细胞中活性适度增加。在THP-1细胞中未观察到类似的启动子活性增加。从这些研究中，我们得出结论，一个CRE/ATF样位点以及与该位点相互作用的一种或多种因子对于刺激的U937和THP-1细胞中IL-1β基因的最大诱导至关重要。
