Xia C, Cheshire J K, Patel H, Woo P
Department of Molecular Pathology, University College London, U.K.
Int J Biochem Cell Biol. 1997 Dec;29(12):1525-39. doi: 10.1016/s1357-2725(97)00083-6.
The study of the acute phase response has attracted substantial interest, not only for its medical implication, but also its provision as an excellent system with which to elucidate the molecular mechanisms involved in the modulation of gene expression. Our previous data suggest that the synergistic induction of the major acute phase reactant serum amyloid A2 (SAA2) expression by interleukin-1 (IL-1) and interleukin-6 (IL-6) is mediated by two families of transcription factors, namely NF-kappa B and C/EBP. To understand the molecular mechanisms of this synergy, we have undertaken a molecular dissection of the factors involved in the formation of the regulatory complex. Electrophoretic mobility shift analysis indicates that NF-kappa B p65 (RelA) and p50, but not p52 or c-Rel, bind specifically to the NF-kappa B site of the SAA2 promoter in response to IL-1 stimulation. In addition, C/EBP beta and C/EBP delta, but not C/EBP alpha, bind specifically to the C/EBP site of SAA2 in response to IL-6 stimulation. Transient co-transfection analysis indicates that co-operative association of NF-kappa B p65 with C/EBP beta and, in particular, with C/EBP delta, results in synergistic transcriptional activation of the SAA2 promoter. When incubated together, NF-kappa B p65 and C/EBP beta form a ternary complex by direct protein/protein interaction. Mutational analysis demonstrates that the C-terminus region of the Rel homology domain (RHD) and the C-terminus of the activation domain of p65 are important for its interaction with C/EBP beta. These results suggest the NF-kappa B and C/EBP may form a new complex of transcription factors that mediates the synergistic induction of SAA2 by IL-1 and IL-6.
急性期反应的研究不仅因其医学意义，还因其作为阐明基因表达调控分子机制的优秀系统而备受关注。我们之前的数据表明，白细胞介素-1（IL-1）和白细胞介素-6（IL-6）协同诱导主要急性期反应物血清淀粉样蛋白A2（SAA2）表达是由两类转录因子介导的，即核因子κB（NF-κB）和C/EBP。为了解这种协同作用的分子机制，我们对参与调控复合物形成的因子进行了分子剖析。电泳迁移率变动分析表明，NF-κB p65（RelA）和p50，而非p52或c-Rel，在IL-1刺激下特异性结合到SAA2启动子的NF-κB位点。此外，C/EBPβ和C/EBPδ，而非C/EBPα，在IL-6刺激下特异性结合到SAA2的C/EBP位点。瞬时共转染分析表明，NF-κB p65与C/EBPβ，特别是与C/EBPδ的协同结合，导致SAA2启动子的协同转录激活。当一起孵育时，NF-κB p65和C/EBPβ通过直接的蛋白质/蛋白质相互作用形成三元复合物。突变分析表明，Rel同源结构域（RHD）的C末端区域和p65激活结构域의C末端对其与C/EBPβ的相互作用很重要。这些结果表明，NF-κB和C/EBP可能形成一种新的转录因子复合物，介导IL-1和IL-6对SAA2的协同诱导。
