Transcription of hypoxia-inducible genes is regulated by hypoxia response elements (HREs) located in either the promoter or enhancer regions. Analysis of these elements reveals the presence of one or more binding sites for hypoxia-inducible factor 1 (HIF-1). Hypoxia-inducible genes include vascular endothelial growth factor (VEGF), erythropoietin, and glycolytic enzyme genes. Site-directed mutational analysis of the VEGF gene promoter revealed that an HIF-1 binding site (HBS) and its downstream HIF-1 ancillary sequence (HAS) within the HRE are required as cis-elements for the transcriptional activation of VEGF by either hypoxia or nitric oxide (NO). The core sequences of the HBS and the HAS were determined as TACGTG and CAGGT, respectively. These elements form an imperfect inverted repeat, and the spacing between these motifs is crucial for activity of the promoter. Gel shift assays demonstrate that as yet unknown protein complexes constitutively bind to the HAS regardless of the presence of these stimuli in several cell lines, in contrast with hypoxia-or NO-induced activation of HIF-1 binding to the HBS. A common structure of the HRE, which consists of the HBS and the HAS, is seen among several hypoxia-inducible genes, suggesting the presence of a novel mechanism mediated by the HAS for the regulation of these genes.

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...