Binding of pleomorphic adenoma gene-like 2 to the tumor necrosis factor (TNF)-α-responsive region of the NCF2 promoter regulates p67phox expression and NADPH oxidase activity
Binding of pleomorphic adenoma gene-like 2 to the tumor necrosis factor (TNF)-α-responsive region of the NCF2 promoter regulates p67phox expression and NADPH oxidase activity
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DOI：
10.1074/jbc.m610618200
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发表时间：
2007-06-15
影响因子：
4.8
通讯作者：
Gauss, Katherine A.
中科院分区：
文献类型：
--
作者：
Ammons, Mary Cloud B.;Siemsen, Daniel W.;Gauss, Katherine A.
NCF2是编码NADPH氧化酶胞浆组分p67(Phox)的基因，它受到肿瘤坏死因子-α的上调，我们最近在NCF2启动子中定位了这种肿瘤坏死因子-α依赖的反应所必需的区域。由于这个肿瘤坏死因子-α反应区(TRR)缺乏可识别的转录因子结合元件，我们进行了研究，以确定通过TRR调控NCF2的因素。在酵母单杂交筛选中，以TRR序列为诱饵，我们确定锌指转录因子多形性腺瘤基因样2(PLAGL2)是NCF2表达的候选调控因子。产生PLAGL2特异性抗体，检测内源性PLAGL2的天然形式和SUMO1修饰形式。EMSA和DNA结合蛋白亲和纯化分析表明，体外翻译和内源性表达的PLAGL2与TRR具有特异性结合，染色质免疫沉淀实验表明，在肿瘤坏死因子-α处理后，体内内源性PLAGL2与TRR的结合增强。基因敲除PLAGL2蛋白可抑制肿瘤坏死因子-α对NCF2转录和p67Phox蛋白表达的上调，以及随后超氧化物的产生。此外，天然和SUMO1修饰的内源性PLAGL2蛋白的相对水平是以时间依赖的方式调节的，以响应肿瘤坏死因子-α处理。这些数据清楚地表明PLAGL2是NCF2基因表达和NADPH氧化酶活性的新调节因子，有助于更好地理解NCF2的转录调控。
NCF2, the gene encoding the NADPH oxidase cytosolic component p67(phox), is up-regulated by TNF-alpha, and we recently mapped a region in the NCF2 promoter that was required for this TNF-alpha-dependent response. Because this TNF-alpha-responsive region (TRR) lacked recognizable transcription factor binding elements, we performed studies to identify factors involved in regulating NCF2 via the TRR. Using the TRR sequence as bait in a yeast one-hybrid screen, we identified the zinc finger transcription factor Pleomorphic Adenoma Gene-Like 2 (PLAGL2) as a candidate regulator of NCF2 expression. PLAGL2-specific antibodies were generated that detected the native and SUMO1-modified forms of endogenous PLAGL2. EMSA and DNA-binding protein affinity purification analyses demonstrated specific binding of in vitro-translated as well as endogenously expressed PLAGL2 to the TRR, and chromatin immunoprecipitation assays demonstrated enhanced binding of endogenous PLAGL2 to the TRR in vivo with TNF-alpha treatment. Knockdown of PLAGL2 protein inhibited up-regulation of NCF2 transcript, p67phox protein expression, and subsequent superoxide production in response to TNF-alpha. Furthermore, relative levels of native and SUMO1-modified endogenous PLAGL2 protein were modulated in a time-dependant manner in response to TNF-alpha treatment. These data clearly identify PLAGL2 as a novel regulator of NCF2 gene expression as well as NADPH oxidase activity and contribute to a greater understanding of the transcriptional regulation of NCF2.