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 has been explored. ? ? In addition to upregulating TNFalpha 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 have confirmed that endogenously produced NO also up-regulates TNFalpha production using human U937 cells, a monoblastoid cell line, transfected to express murine iNOS (Blood, 1997). Investigation of TNFalpha 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 TNFalpha promoter (J Biol Chem, 1999). NO-mediated decreases in cAMP leads to reduced Sp1 binding to the TNFalpha promoter with subsequent increases in TNFalpha transcription. Recent work has shown that NO downregulates the eNOS promoter through effects on Sp1 identical to those that cause alpha upregulation (J Biol Chem, 2003). For TNFalpha, an AP1 site upstream to Sp1 serves to reverse the direction of the NO response. Mutation of the AP1 site converts the effect of NO in TNFalpha from up to down regulation (eNOS-like).? ? The IL-8 promoter lacks a canonical Sp1 site. Unlike TNFalpha, IL-8 regulation by NO both cGMP and cAMP-independent. Further, NO-p38 MAPK-dependent effect on protein binding to regulation of IL-8 has been found to be post-transcriptional and mediated via AU-rich elements in its mRNA 3 UTR (J Leuk Biol, 2004). An oligonucleotide microarray analysis in differentiated U937 cells has identified more than 100 additional NO regulated genes (BMC Genomics, 2006). NO was found to coordinate a highly integrated program of cell cycle arrest that regulated a large number of genes, but did not require signaling through cGMP. Therefore, in humans the antiproliferative 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 affected. NO was shown to stabilize transcripts while suppressing their translation through DICE-like, CU-rich elements (CURE) in target transcripts. NO activation of Erk1/2 was required, as was an associated increase in the binding of hnRNP proteins to mRNA. This work demonstrated a novel mechanism of NO-mediated posttranscriptional regulation.? ? In our previous study (BMC Genomic, 2005), NO was shown to down-regulate Polo-like kinase 1 (PLK1), an evolutionarily conserved serine/threonine kinase essential for cell mitosis, and up-regulate p21/Waf1, a master cell cycle regulator critical for cell cycle progression. 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 (FASEB J, 2007). PPARgamma was activated by NO through a p38 MAPK dependent signal transduction pathway. 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. ? ? NO and carbon monoxide (CO), another low molecular weight endogenous messenger, have both overlapping and divergent effects on immunity and vascular health. Unlike NO, however, CO only weakly activates soluble guanylate cyclase and for many functions, the proximal targets of CO signaling are unknown. Therefore, we are comparing the effects of NO and CO on inflammation and gene regulation. While NO up-regulates IL-1beta and TNFalpha, 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. Notably, 16 percent were known transcription factors and most others were cytokines, chemokines and immune response genes. In silico analysis revealed that many CO-suppressed genes had NF-kappaB binding sites in their proximal promoters. CO was found to block proximal events in NF-kappaB 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, because NO has a stronger effect on V/Q mismatch-induced hypoxia, while CO has a better anti-inflammatory profile.

Agency
National Institute of Health (NIH)
Institute
Clinical Center (CLC)
Type
Intramural Research (Z01)
Project #
1Z01CL001158-10
Application #
7733547
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
10
Fiscal Year
2008
Total Cost
$123,672
Indirect Cost
Name
Clinical Center
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Ptasinska, Anetta; Wang, Shuibang; Zhang, Jianhua et al. (2007) Nitric oxide activation of peroxisome proliferator-activated receptor gamma through a p38 MAPK signaling pathway. FASEB J 21:950-61
Zhang, Jianhua; Wang, Shuibang; Kern, Steven et al. (2007) Nitric oxide down-regulates polo-like kinase 1 through a proximal promoter cell cycle gene homology region. J Biol Chem 282:1003-9
Wang, Shuibang; Zhang, Jianhua; Theel, Stephanie et al. (2006) Nitric oxide activation of Erk1/2 regulates the stability and translation of mRNA transcripts containing CU-rich elements. Nucleic Acids Res 34:3044-56
Cui, Xiaolin; Zhang, Jianhua; Ma, Penglin et al. (2005) cGMP-independent nitric oxide signaling and regulation of the cell cycle. BMC Genomics 6:151
Ma, Penglin; Cui, Xiaolin; Wang, Shuibang et al. (2004) Nitric oxide post-transcriptionally up-regulates LPS-induced IL-8 expression through p38 MAPK activation. J Leukoc Biol 76:278-87
Ma, Penglin; Danner, Robert L (2002) The many faces of sepsis-induced vascular failure. Crit Care Med 30:947-9