Nitric oxide-derived nitrosating species and gene expression in human monocytic cells

In living cells, NO signaling is mediated by NO-derived metabolites and is therefore dependent on the rate of formation of these so-called reactive nitrogen intermediates (RNIs). We have examined the effects of NO-oxidizing agents, the nitronyl nitroxide PTIO and its less hydrophobic analogue carboxy-PTIO (CPTIO), on the expression of NO-sensitive genes in monocytic U937 and Mono Mac 6 cells. We have observed that pretreatment of cells with PTIO boosted expression of IL-8 and heme oxygenase 1 (HOX) genes to a high level in cells treated with the NO donor DPTA-NO. In contrast, pretreatment of cells with CPTIO significantly inhibited NO-dependent expression of IL-8 and hardly stimulated HOX gene expression by DPTA-NO. The effect of PTIO was abrogated by reduced glutathione, suggesting that upregulation of the IL-8 and HOX genes is dependent on RNI-mediated S-nitrosation of specific regulator(s). The concentration of PTIO required to enhance mRNA level was different for IL-8 and HOX genes. Analysis of 4,5-diaminofluorescein (DAF) nitrosation in the presence of PTIO and DPTA-NO showed that optimal PTIO concentrations required for maximal N2O3 synthesis and for highest IL-8 gene expression are similar. Furthermore, we have shown that, besides IL-8 and HOX, PTIO superactivates NO-dependent expression of TNF-alpha and p21/WAF1 genes. In contrast, the level of MIP-1alpha, c-jun, and c-fos genes was not changed by the presence of PTIO in U937 cells and was even reduced in Mono Mac 6 cells.