Cui Xiaolin, Zhang Jianhua, Ma Penglin, Myers Daniela E, Goldberg Ilana G, Sittler Kelly J, Barb Jennifer J, Munson Peter J, Cintron Ana del Pilar, McCoy J Philip, Wang Shuibang, Danner Robert L
Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
BMC Genomics. 2005 Nov 3;6:151. doi: 10.1186/1471-2164-6-151.
Regulatory functions of nitric oxide (NO*) that bypass the second messenger cGMP are incompletely understood. Here, cGMP-independent effects of NO* on gene expression were globally examined in U937 cells, a human monoblastoid line that constitutively lacks soluble guanylate cyclase. Differentiated U937 cells (>80% in G0/G1) were exposed to S-nitrosoglutathione, a NO* donor, or glutathione alone (control) for 6 h without or with dibutyryl-cAMP (Bt2cAMP), and then harvested to extract total RNA for microarray analysis. Bt2cAMP was used to block signaling attributable to NO*-induced decreases in cAMP.
NO* regulated 110 transcripts that annotated disproportionately to the cell cycle and cell proliferation (47/110, 43%) and more frequently than expected contained AU-rich, post-transcriptional regulatory elements (ARE). Bt2cAMP regulated 106 genes; cell cycle gene enrichment did not reach significance. Like NO*, Bt2cAMP was associated with ARE-containing transcripts. A comparison of NO* and Bt2cAMP effects showed that NO* regulation of cell cycle genes was independent of its ability to interfere with cAMP signaling. Cell cycle genes induced by NO* annotated to G1/S (7/8) and included E2F1 and p21/Waf1/Cip1; 6 of these 7 were E2F target genes involved in G1/S transition. Repressed genes were G2/M associated (24/27); 8 of 27 were known targets of p21. E2F1 mRNA and protein were increased by NO*, as was E2F1 binding to E2F promoter elements. NO* activated p38 MAPK, stabilizing p21 mRNA (an ARE-containing transcript) and increasing p21 protein; this increased protein binding to CDE/CHR promoter sites of p21 target genes, repressing key G2/M phase genes, and increasing the proportion of cells in G2/M.
NO* coordinates a highly integrated program of cell cycle arrest that regulates a large number of genes, but does not require signaling through cGMP. In humans, antiproliferative effects of NO* may rely substantially on cGMP-independent mechanisms. Stress kinase signaling and alterations in mRNA stability appear to be major pathways by which NO* regulates the transcriptome.
一氧化氮（NO*）绕过第二信使环磷酸鸟苷（cGMP）的调控功能尚未完全明确。在此，我们在U937细胞（一种组成性缺乏可溶性鸟苷酸环化酶的人单核细胞系）中全面研究了NO对基因表达的不依赖cGMP的作用。将分化的U937细胞（>80%处于G0/G1期）分别用NO供体亚硝基谷胱甘肽或单独的谷胱甘肽（对照）处理6小时，处理时有无二丁酰环磷酸腺苷（Bt2cAMP），然后收集细胞提取总RNA用于微阵列分析。Bt2cAMP用于阻断因NO*诱导的cAMP减少而产生的信号传导。
NO调控了110个转录本，这些转录本在细胞周期和细胞增殖方面的注释不成比例（47/110，43%），且比预期更频繁地含有富含AU的转录后调控元件（ARE）。Bt2cAMP调控了106个基因；细胞周期基因富集未达到显著水平。与NO一样，Bt2cAMP也与含有ARE的转录本相关。对NO和Bt2cAMP作用的比较表明，NO对细胞周期基因的调控独立于其干扰cAMP信号传导的能力。NO诱导的细胞周期基因注释为G1/S期（7/8），包括E2F1和p21/Waf1/Cip1；这7个基因中有6个是参与G1/S期转换的E2F靶基因。受抑制的基因与G2/M期相关（24/27）；27个基因中有8个是已知的p21靶基因。NO使E2F1 mRNA和蛋白增加，E2F1与E2F启动子元件的结合也增加。NO*激活p38丝裂原活化蛋白激酶（MAPK），稳定p21 mRNA（一种含有ARE的转录本）并增加p21蛋白；这增加了蛋白与p21靶基因的CDE/CHR启动子位点的结合，抑制关键的G2/M期基因，并增加G2/M期细胞的比例。
NO协调了一个高度整合的细胞周期停滞程序，该程序调控大量基因，但不需要通过cGMP进行信号传导。在人类中，NO的抗增殖作用可能很大程度上依赖于不依赖cGMP 的机制。应激激酶信号传导和mRNA稳定性的改变似乎是NO*调控转录组的主要途径。
