Research article
cGMP-independent nitric oxide signaling and regulation of the cell cycle
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* Corresponding author: Robert L Danner rdanner@mail.nih.gov
- † Equal contributors
1 Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA
2 Mathematical and Statistical Computing Laboratory, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, Maryland, USA
3 Flow Cytometry Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA
4 Intensive Care Unit of the Military 309th Hospital, Haidian District of Beijing, People's Republic of China
BMC Genomics 2005, 6:151 doi:10.1186/1471-2164-6-151
Published: 3 November 2005Abstract
Background
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.
Results
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.
Conclusion
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.