Introduction and Objective: Nitric oxide is an important inter- and intracellular messenger implicated in the pathogenesis of septic shock. Inhibition of nitric oxide synthase has been investigated as a treatment for hypotension in septic shock. Furthermore, inhaled nitric oxide is used clinically in pulmonary hypertension and respiratory distress syndrome. In addition to its vasodilating effects on the systemic and pulmonary vasculatures, nitric oxide also modulates immune responses and regulates gene expression. These latter attributes may have implications for the use of nitric oxide synthase inhibitors or nitric oxide itself for syndromes in which the host's inflammatory response plays a pathogenic role.? ? In this project, we are examining the role of nitric oxide as a modulator of inflammation and gene expression.? ? Proposed Course of Work:? Current work is focused on identifying all major pathways of nitric oxide gene regulation using microarray and molecular biology approaches. Parts of this effort are characterizing nitric oxide effects on the cell-cycle and the respective roles of p38 MAPK, proteasome inhibition, and mRNA stability on gene regulation by nitric oxide.? ? Progress:? Human phagocytes, in particular neutrophils, were demonstrated to lack the capacity to endogenously produce nitric oxide 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 endogenous sources of NO such as the endothelium has been explored.? ? In addition to upregulating TNFalpha production (J Immunol, 1994), nitric oxide 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 nitric oxide also up-regulates TNFalpha production using human U937 cells, a monoblastoid cell line, transfected to express murine inducible nitric oxide synthase (Blood, 1997).? ? Investigation of TNFalpha regulation by nitric oxide resulted in the description of a cGMP-independent signaling pathway that utilizes cAMP downregulation as a signal transduction event (J Biol Chem, 1997).? ? A nitric oxide-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.? ? Other work has shown that NO downregulates the eNOS promoter through effects on Sp1 identical to those that cause TNFalpha upregulation (J Biol Chem, 2003). For TNFalpha, an AP1 site upstream to Sp1 serves to reverse the direction of the nitric oxide response. Mutation of the AP1 site converts the effect of nitric oxide on TNFalpha from up to down regulation (eNOS-like).? ? The IL-8 promoter lacks a canonical Sp1 site. Unlike TNFalpha, IL-8 regulation by nitric oxide is both cGMP and cAMP-independent. Ultimately, nitric oxide activation of p38 MAPK was shown to stabilize IL-8 mRNA through AU-rich elements in its 3' untranslated region (3' UTR) (J Leuk Biol, 2004). A microarray approach has subsequently identified other genes including p21, a master regulator of the cell cycle, that are regulated by nitric oxide via this mechanism (BMC Genomics, 2005).? ? Recently the role of Erk MAPK in nitric oxide regulation of mRNA stability has been investigated. Using a microarray based approach, we globally identified a large number of transcripts stabilized by nitric oxide in endotoxin-stimulated THP1 cells, a human monocytic line. In addition to AU-rich elements, a large subset of these genes were found to contain CU-rich elements (CURE), a regulatory site analogous to the differentiation control element (DICE) originally described in lipoxygenase. Preliminary studies have shown that nitric oxide increases the half-life and decreases the translation of target genes containing this motiff through MAPK-dependent translocation of ribonucleoproteins to the cytoplasm.
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