Nitric Oxide Regulation of Inflammatory Responses and Gene Expression

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Abstract

Human phagocytes, in particular neutrophils, were demonstrated to lack the capacity to endogenously produce NO under a number of ex vivo and in vivo conditions (J Immunol, 1994). Therefore, the ability of these cells to be regulated in a paracrine manner by exogenous sources of NO such as the endothelium was explored. In addition to upregulating TNFa production (J Immunol, 1994), NO was found to modulate IL-8 mRNA levels and IL-8 production in human neutrophil preparations (J Infect Dis, 1998). We confirmed that endogenously produced NO also up-regulates TNFa production using human U937 cells, a monoblastoid cell line, transfected to express murine iNOS (Blood, 1997). Investigation of TNFa regulation by NO resulted in the description of a cGMP-independent signaling pathway that utilizes cAMP downregulation as a signal transduction event (J Biol Chem, 1997). A NO-responsive Sp1 binding site was identified in the proximal TNFa promoter (J Biol Chem, 1999). NO-mediated decreases in cAMP reduced Sp1 binding to the TNF promoter with subsequent increases in TNFa transcription. NO was found to downregulate the eNOS promoter through effects on Sp1 identical to those that cause TNFa upregulation (J Biol Chem, 2003). For TNFa, an upstream AP1 site coupled to the Sp1 binding sequence serves as a molecular switch, the presence of which reverses the polarity of NO responses. Mutation of the AP1 site in the proximal TNFa promoter converts the NO effect from up to downregulation (eNOS-like). The IL-8 promoter lacks a canonical Sp1 site. Unlike TNFa, IL-8 regulation by NO is both cGMP and cAMP-independent. NO activation of p38 MAPK was shown to stabilize IL-8 mRNA via altered protein binding to AU-rich elements in its mRNA 3 UTR (J Leuk Biol, 2004). An oligonucleotide microarray analysis in differentiated U937 cells identified more than 100 additional NO regulated genes (BMC Genomics, 2005). NO coordinated a highly integrated program of cell cycle arrest through p38 MAPK stabilization of p21 mRNA, a master regulator of the cell cycle. Therefore, in humans the anti-proliferative effects of NO 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. Next, transcript stabilization by NO was investigated in human THP-1 cells using microarrays (Nucleic Acids Research, 2006). After LPS pre-stimulation, cells were treated with actinomycin D and then exposed to NO without or with the p38 MAPK inhibitor SB202190 (SB). Decay of 220 mRNAs was inhibited by NO, while their translation was simultaneously suppressed; both of these responses were associated with DICE-like, CU-rich elements (CURE) in target transcripts. NO activation of ERK1/2 was required, as was increased binding of hnRNP proteins to mRNA. This work demonstrated a novel mechanism of NO-mediated post-transcriptional regulation. In our previous study (BMC Genomic, 2005), NO was shown to up-regulate p21/Waf1, a master cell cycle regulator critical for cell cycle progression, and to down-regulate Polo-like kinase 1 (PLK1), an evolutionarily conserved serine/threonine kinase essential for cell mitosis. Both of these effects were independent of cGMP. Further investigation of this mechanism demonstrated that a NO-p38 MAPK-p21/Waf1 signal transduction pathway repressed PLK1 through a canonical CDE/CHR promoter element (J Biol Chem, 2007). Both NO and peroxisome proliferator-activated receptors (PPARs) protect the endothelium and regulate its function. Therefore, we tested for crosstalk between these signaling pathways using human umbilical vein and hybrid EA.hy926 endothelial cells. PPAR was activated by NO through a p38 MAPK dependent signal transduction pathway (FASEB J, 2007). This crosstalk mechanism may contribute to the anti-inflammatory and cytoprotective effects of NO in the vasculature and suggests new strategies for preventing and treating vascular dysfunction. CO effects on inflammation and gene regulation were evaluated and compared to NO. While NO up-regulated IL-1b and TNFa, CO was found to decrease the expression of both. Using microarrays, we identified early-immediate transcripts that were induced by LPS and suppressed by CO (PLoS One 2009). CO was found to block proximal events in NF-kB signal transduction, thereby broadly suppressing inflammation. In acute respiratory distress syndrome (ARDS) and other inflammatory lung conditions, combining clinically tolerated, low doses of inhaled NO and CO may have therapeutic advantages over either gas alone. O2 (70%) and NO (40 ppm), two gases used in the management of ARDS, were examined in air-liquid interface differentiated normal human bronchial epithelial cells (NHBECs) challenged with IFNg to simulate an influenza-like inflammatory response. In support of the concept above, global gene expression profiling demonstrated in the presence of IFNg that O2 and NO produced highly similar signatures of oxidant stress (American Thoracic Society, abstract 2011). NO activates MAPK pathways and enhances inflammatory responses, effects that might counteract the toxicity and immune suppression of anthrax lethal toxin (LeTx). Furthermore, NO has been shown to interact with and alter the activity of other zinc-containing proteins. The ability of NO to protect MAPK signaling pathways and the innate immune response from LF-mediated disruption was tested in both human monocyte-like THP-1 and murine macrophage-like RAW264.7 cells. Work demonstrating that NO/p38 MAPK activates PPARgamma was extended using PPRE reporter genes, NOS2 induction and p38 MAPK dominant negative mutant expression. The role of p38 MAPK in ligand/agonist activation of PPARgamma was shown in endothelial cells. Both NO and optimal PPARgamma ligand activations of PPARgamma were associated with PPARgamma acetylation. Current work is focused on identifying putative acetylation sites and participating acetyltransferases. Other preliminary experiments has indicated that CO may activate stress kinase pathways through a GPCR signaling pathway. Experiments are planned to further test this hypothesis.