Abstract
The expression of inducible nitric oxide synthase (iNOS) is a critical factor in both normal physiological functions and the pathogenesis of disease. This study was undertaken to determine the molecular mechanism by which nitric oxide (NO) exerts negative feedback regulation on iNOS gene expression. Isolated rat hepatocytes stimulated with cytokines exhibited a marked increase in NO production as well as iNOS mRNA and protein levels, which were significantly reduced by pretreatment of the NO donors S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and V-PYRRO/NO. This effect of SNAP was inhibited when NO was scavenged using red blood cells. Pretreatment with oxidized SNAP, 8-Br-cGMP, NO2-, or NO3- did not suppress the cytokine-induced NO production. Moreover, LPS/ IFN-gamma-stimulated RAW264.7 cells, which produce endogenous NO, expressed lower levels of iNOS, IL-1beta, IL-6 and TNF-alpha mRNAs, without changes in their mRNA half-lives, than those in the presence of the iNOS inhibitor NG-monomethyl-L-arginine. The iNOS gene transcription rate exhibited an 18-fold increase after cytokine stimulation, which was significantly inhibited by SNAP pretreatment. SNAP also blocked cytokine- induced increase in NF-kappaB activation, iNOS promoter activity, nuclear translocation of cytosolic NF-kappaB p65 subunit, and IkappaBalpha degradation, which correlated with its inhibitory effect on phosphorylation and ubiquitination of IkappaB. These data indicate that NO down-regulates iNOS gene expression and NO production by inhibiting the post-translational processes of IkappaBalpha thereby preventing NF-kappaB activation. These results identify a novel negative feedback mechanism whereby NO down-regulates iNOS gene expression.
Highlights
Nitric oxide (NO) is an important regulatory molecule involved in many homeostatic functions including neurotransmission, blood pressure control, and antimicrobial defense mechanisms (Nathan, 1992)
The low levels of NO produced by both endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) participate in cell signaling and regulate processes such as endothelium-dependent vascular relaxation and neurotransmission, respectively. inducible nitric oxide synthase (iNOS), the third isoform, while constitutively expressed in some tissues (Asano et al, 1994; Mannick et al, 1994), is expressed only after cells are exposed to lipopolysaccharide (LPS) and/or cytokines (Geller et al, 1995). iNOS expression results in sustained, high level of NO production which is responsible for the refractory hypotension associated
To investigate the effects of exogenous NO on cytokine mixture (CM)-induced NO production and iNOS expression, we examined the effects of the NO donor SNAP on NO production as well as iNOS mRNA and protein expression in CM-stimulated hepatocytes
Summary
Nitric oxide (NO) is an important regulatory molecule involved in many homeostatic functions including neurotransmission, blood pressure control, and antimicrobial defense mechanisms (Nathan, 1992). There are three known nitric oxide synthase (NOS) isoforms, each produced as a separate gene product Both the endothelial (eNOS) and neuronal (nNOS) isoforms are constitutively expressed and are predominantly regulated post-translationally by calcium and calmodulin (Nathan, 1992). NF-κB has been shown to be required for iNOS induction in many cell types including hepatocytes (Geller et al, 1995), rodent macrophages (Xie et al, 1994), and vascular smooth muscle cells (Spink et al, 1995) This transcription factor has been implicated in the induction of pro-inflammatory genes encoding TNF-α, IL-1β, IL-6, and COX-2 (Makarov, 2000). NO can inhibit iNOS gene expression and NO production in association with phosphorylation, ubiquitination, and subsequent proteolytic degradation of IκB upstream of NF-κB-DNA binding has not been clearly elucidated.
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