Zhang Jianhua, Wang Shuibang, Wesley Robert A, Danner Robert L
Critical Care Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 2003 Aug 1;278(31):29192-200. doi: 10.1074/jbc.M213043200. Epub 2003 May 19.
Nitric oxide (NO*) and cAMP-dependent protein kinase (PKA) inhibitors up-regulate tumor necrosis factor alpha (TNFalpha) by decreasing Sp1 binding to a proximal GC box element. Here, elements flanking GC boxes were tested for their role in determining whether Sp sites act as activators or repressors. Promoter studies in receptive human cell lines demonstrated that NO* down-regulated endothelial NO* synthase (eNOS) but up-regulated TNFalpha. Like TNFalpha, Sp1 binding to the eNOS promoter was decreased by NO* and a PKA inhibitor, H89, and increased by a PKA activator, dibutyryl cAMP (Bt2cAMP). For either promoter, mutation of Sp sites abolished NO* responses. In contrast, mutation of an upstream AP1 site in the TNFalpha promoter (not present in eNOS) maintained NO* responsiveness, but reversed the direction of NO* and cAMP effects. Using artificial constructs, NO* increased transcription when Sp and AP1 sites were both present (TNFalpha-like response), but decreased it when the adjacent AP1 site was disrupted (eNOS-like response). NO*, H89, and Bt2cAMP were found to produce reciprocal protein binding changes at contiguous AP1 and Sp sites (p < 0.0001 for an interaction). Chromatin immunoprecipitation assays demonstrated that Sp1 and to a lesser extent Sp3 bound to the GC box regions of eNOS and TNFalpha in intact cells. Thus, this NO*- and cAMP-responsive regulatory module has a Sp site sensor variably coupled to an adjacent element that determines response polarity. These results define a composite element that can utilize secondary inputs to convert off signals to on, thereby conferring complex functionalities to the same DNA binding motif.
一氧化氮（NO*）和环磷酸腺苷依赖性蛋白激酶（PKA）抑制剂通过减少Sp1与近端GC盒元件的结合来上调肿瘤坏死因子α（TNFα）。在此，对GC盒两侧的元件进行了测试，以确定其在决定Sp位点作为激活剂还是抑制剂方面的作用。在人感受态细胞系中的启动子研究表明，NO下调内皮型一氧化氮合酶（eNOS），但上调TNFα。与TNFα一样，NO和PKA抑制剂H89可降低Sp1与eNOS启动子的结合，而PKA激活剂二丁酰环磷酸腺苷（Bt2cAMP）可增加这种结合。对于任何一个启动子，Sp位点的突变都会消除NO反应。相反，TNFα启动子中上游AP1位点（eNOS中不存在）的突变保持了NO反应性，但逆转了NO和cAMP的作用方向。使用人工构建体，当Sp和AP1位点都存在时，NO增加转录（类似于TNFα的反应），但当相邻的AP1位点被破坏时，NO会降低转录（类似于eNOS的反应）。发现NO、H89和Bt2cAMP在相邻的AP1和Sp位点产生相互的蛋白质结合变化（相互作用 p < 0.0001）。染色质免疫沉淀分析表明，在完整细胞中，Sp1以及程度较轻的Sp3与eNOS和TNFα的GC盒区域结合。因此，这个对NO*和cAMP有反应的调节模块有一个Sp位点传感器，它与一个相邻元件可变地耦合，该相邻元件决定反应极性。这些结果定义了一个复合元件，它可以利用次级输入将关闭信号转换为开启信号，从而赋予相同的DNA结合基序复杂的功能。
