Variation in gene expression profiles of human monocytic U937 cells exposed to various fluxes of nitric oxide

We examined early and late alterations in gene expression patterns and phosphorylation levels of key regulators of selected signaling pathways in U937 cells exposed to various ·NO fluxes. cDNA microarray analysis and real-time quantitative PCR identified 45 NO-sensitive genes (≥ 2-fold change), among which KLF2, KLF6, TSC22D3, DDIT4, MKP-5 (up-regulated), KIF23, histone H4, ARL6IP2, CLNS1A, SLC7A6, CDKN3, SRP19, and BCL11A (down-regulated) have not been reported before. For two selected genes, KLF2 and DDIT4, the sensitivity to ·NO was also proven at the protein level. Among the examined genes, only KLF2 had a higher sensitivity to slow release of NO (DETA-NO) than to high-dose, short-duration exposure (DPTA-NO), reaching an about 50-fold increase in mRNA level. Our study revealed that fast and slow NO donors activate similar signaling pathways and induce phosphorylation of MAP kinases and downstream transcription factors ATF2 and c-Jun. Inhibitory analysis of major signaling pathways showed that activity of p38 MAPK and tyrosine kinases is indispensable for gene induction in cells exposed to DPTA-NO, whereas G-protein Rho suppression caused superinduction of KLF2 in ·NO-stimulated cells. Finally, we showed that both ·NO donors caused a marked decrease in phosphorylation of p70S6K, an mTOR substrate and regulator of mRNA translation, and protein kinase Akt, an upstream positive regulator of mTOR.