Differential functional roles for two AP1 binding sites of S100A7.

(A) The DNA sequence of S100A7 promoter (GenBank accession number: AF050167) is shown and two putative AP-1 binding sites are framed by gray shadow. (B) The schematic presentation of the luciferase reporters driven by S100A7 promoter containing different mutated AP-1 binding sites is indicated. (C) The luciferase reporter assays were performed using aforementioned individual construct in the absence or presence of either c-Jun/c-Fos or c-Jun/Fra-1 heterodimeric AP-1 complexes. Both the basal activity (lane 1, open bar) and AP-1-activated promoter activities (lanes 2 and 3, open bars) are inhibited by AP1-1 site mutation, indicating the essential role of this AP-1 binding site. On the other hand, the S100A7 promoter containing mutated AP1-2 site can still be activated by heterodimeric c-Jun/c-Fos instead of c-Jun/Fra-1 (compare lanes 2–3 to lane 1, dashed bars). The result indicated that the functional role of AP1-2 site is less than AP1-1 site for c-Jun/c-Fos; however, the two putative AP-1 binding are both important for c-Jun/Fra-1 to activate S100A7 promoter. Surprisingly, c-Jun/Fra-1 can activate the S100A7 promoter containing two mutated AP-1 binding sites (compare lanes 2–3 to lane 1, light grey bars), indicating that the S100A7 promoter may be activated by heterodimeric c-Jun/Fra-1 AP-1 complex with some unexplored mechanism. These data (C) are the average of three experiments (mean ± SD; n = 3). All p values less than 0.05 were considered statistically significant.(A) 已展示S100A7启动子的DNA序列（GenBank登录号：AF050167），两个预测的AP-1结合位点以灰色阴影框标注。 (B) 展示了由携带不同突变型AP-1结合位点的S100A7启动子驱动的荧光素酶报告基因的示意图。 (C) 采用上述单个重组载体，分别在无、或存在c-Jun/c-Fos、c-Jun/Fra-1异二聚体AP-1复合物的条件下开展荧光素酶报告基因实验。AP1-1位点突变可同时抑制基础转录活性（泳道1，空白条）与AP-1激活的启动子活性（泳道2、3，空白条），表明该AP-1结合位点发挥核心作用。另一方面，携带AP1-2位点突变的S100A7启动子仍可被c-Jun/c-Fos异二聚体激活，而非c-Jun/Fra-1（对比泳道2-3与泳道1，虚线条）。该结果表明，对于c-Jun/c-Fos而言，AP1-2位点的功能重要性弱于AP1-1位点；但对于c-Jun/Fra-1激活S100A7启动子而言，两个预测的AP-1结合位点均发挥关键作用。值得注意的是，c-Jun/Fra-1仍可激活携带双突变AP-1结合位点的S100A7启动子（对比泳道2-3与泳道1，浅灰色条），提示c-Jun/Fra-1异二聚体AP-1复合物可能通过某种未阐明的机制激活S100A7启动子。本部分数据为3次独立实验的平均值（均值±标准差；n=3）。所有p值小于0.05均被认为具有统计学显著性。