Abstract
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 AREcontaining
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.
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