| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2000: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
It has been proposed that uncoupling protein 2 (UCP-2) limits production of reactive oxygen species (ROS) by decreasing the mitochondrial membrane potential. Although reactive oxygen species are involved in cellular signaling for inducible nitric oxide synthase (NOS II) gene expression following lipopolysaccharide (LPS) stimulation in macrophages, the role of UCP-2 in the regulation of the response to LPS has not been elucidated. The expression of UCP2 was reduced in macrophages following stimulation with LPS. The physiological consequence and the regulatory mechanisms of the UCP-2 down-regulation by LPS were investigated in a macrophage cell line, RAW264 cells. UCP-2 overexpression in RAW264 cells transfected with eukaryotic expression vector containing ucp-2 cDNA markedly reduced the production of ROS, Furthermore, in the UCP-2 transfectant, NO synthesis, NOS II protein, NOS II mRNA, and NOS II promoter activity were definitely decreased following LPS stimulation compared with those
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in parental RAW264 or RAW264 cells transfected with the vector alone. Reporter assays suggested that an enhancer element was located in the region of the second intron of UCP-2 gene and that the UCP-2 expression was down-regulated not by the 7.3 kb promoter region but by the 5'-region of UCP-2 gene containing two introns. Deletion of the second intron resulted in the low transcriptional activities and abolishment of the LPS-associated negative regulation. In addition, the mRNA expression of transfected UCP-2 was suppressed in RAW264 cells transfected with expression vector containing UCP-2 genomic DNA, but was markedly increased in cells transfected with the vector containing UCP-2 intronless cDNA. These findings suggest that the LPS stimulated signals suppress UCP-2 expression by interrupting the function of intronic enhancer, leading to an up-regulation of intracellular ROS which activate the signal transduction cascade of NOS II expression, probably to ensure rapid and sufficient cellular responses to a microbial attack. Less
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