Uncoupling protein 2 plays an important role in nitric oxide production of lipopolysaccharide-stimulated macrophages

Abstract

The expression of uncoupling protein 2 (UCP2) was reduced in macrophages after stimulation with lipopolysaccharide (LPS). The physiological consequence and the regulatory mechanisms of the UCP2 down-regulation by LPS were investigated in a macrophage cell line, RAW264 cells. UCP2 overexpression in RAW264 cells transfected with eukaryotic expression vector containing ucp2 cDNA markedly reduced the production of intracellular reactive oxygen species. Furthermore, in the UCP2 transfectant, nitric oxide (NO) synthesis, inducible NO synthase (NOS II) protein, NOS II mRNA, and NOS II promoter activity were definitely decreased after LPS stimulation compared with those in parental RAW264 or RAW264 cells transfected with the vector alone. Reporter assays suggested that an enhancer element was located in the region of intron 2 of the UCP2 gene and that the UCP2 expression was down-regulated not by the 7.3-kb promoter region but by the 5 region of the UCP2 gene containing two introns. Deletion of intron 2 resulted in the low transcriptional activities and abolishment of the LPS-associated negative regulation. In addition, the mRNA expression of transfected UCP2 was suppressed in RAW264 cells transfected with expression vector containing UCP2 genomic DNA, but was markedly increased in cells transfected with the vector containing UCP2 intronless cDNA. These findings suggest that the LPS-stimulated signals suppress UCP2 expression by interrupting the function of intronic enhancer, leading to an up-regulation of intracellular reactive oxygen species, which activate the signal transduction cascade of NOS II expression, probably to ensure rapid and sufficient cellular responses to a microbial attack. U ncoupling protein 2 (UCP2) is a recently discovered member of the mitochondrial inner membrane carrier family with high homology to the brown adipose tissue-specific proton transporter, UCP1 (1-3). Because the gene ucp2 resides within a region of genetic linkage to obesity (1) and its product UCP2 uncouples respiration (4), a role in energy dissipation has been proposed. Mice lacking Ucp2 after targeted gene disruption, however, are not obese and have a normal response to cold exposure or high-fat diet (5). On the other hand, it has been proposed that UCP2 limits production of reactive oxygen species (ROS) by decreasing the mitochondrial membrane potential (6). Indeed, Ucp2 Ϫ/Ϫ mice are resistant to Toxoplasma gondii infection, and macrophages of the mutant mice have higher levels of ROS (5), which are associated with higher cytolytic activity (7). In addition, unlike UCP1, expression of UCP2 teems in spleen, lung, and isolated macrophages (1, 2, 8). These findings suggest a role for UCP2 in immunity or inflammatory responsiveness.Recognition of lipopolysaccharide (LPS) is crucial for host antimicrobial defense reactions (9, 10). Nitric oxide (NO) production by the inducible isoform of NO synthase (NOS II) after LPS stimulation plays a pivotal role in numerous and diverse biological functions, in particular, as a principal mediat...

Hydroperoxy Fatty Acid Cycling Mediated by Mitochondrial Uncoupling Protein UCP2

“…UCP2 has been implicated in reducing the size of atherosclerotic plaques (29) and in influencing the lipoperoxidation state of low density lipoproteins when co-incubated with endothelial cells in which UCP2-induced uncoupling has taken place (28). UCP2 has also been implicated in improving brain recovery after stroke and preventing neurodegeneration (30) and playing a role in ⅐ NO production by lipopolysaccharide-stimulated macrophages (27). These results support the importance of processes that regulate mitochondrial ROS production.…”

“…formation (7)(8)(9)(10)(11). These considerations led to the proposal that a physiological role of uncoupling protein-2 (UCP2), which is present in very low amounts in most tissues (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32), is to down-regulate mitochondrial ROS production (25,26). The ability to reduce ROS and/or lipoperoxidation not only locally in the mitochondria but within the cell or even in the extracellular space has also been ascribed to these novel UCPs (28,29).…”

Role of uncoupling protein UCP2 in cell-mediated immunity: How macrophage-mediated insulitis is accelerated in a model of autoimmune diabetes

“…Therefore, the development of strategies to induce UCP2 or inhibit its down-regulation in immune cells would be promising in autoimmunity. First, data are encouraging because in vitro UCP2 overexpression in macrophages diminished their migration capacity and NO production (10,11). More important, UCP2 overexpression in mice decreases their responsiveness to LPS in terms of cytokine (46) and provides protection in acute brain injury (37).…”

“…MAPK activation in Ucp2-KO macrophages is quicker and stronger than in WT macrophages (8), leading to increased nitric oxide (NO), cytokine production, and migration ability (8,9). Consistent with these findings, overexpression of UCP2 in macrophages is associated with diminished NO production (10) and decreased migration capacity (11).…”

Mitochondrial carriers in inflammation induced by bacterial endotoxin and cytokines

“…Metabolically the cellular use of glucose is increased, whereas glutamine oxidation is unchanged, and fatty acid utilisation is directed away from mitochondrial oxidation (Emre et al, 2007) (Figure 2). Decrease of UCP2 levels promotes not only the mitochondrial ROSdependent MAPK signalling, but also expression of inducible form of the NO synthase, NO production, and release of pro-inflammatory cytokines in macrophage (Arsenijevic et al, 2000;Kizaki et al, 2002;Lee et al, 2005;Rousset et al 2006;Emre et al, 2007).…”