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

doi:10.1073/pnas.142206299

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

^*Department of Molecular Predictive Medicine and Sport Science, Kyorin University, School of Medicine, Mitaka 181-8611, Japan; ^‡Department of Biochemistry, Osaka University Medical School, Suita 565-0871, Japan; ^§Department of Traumatology and Critical Care Medicine, National Defense Medical College, Tokorozawa 359-8513, Japan; ^¶Watanabe Clinic, Shizuoka 422-8067, Japan; ^‖Division of Immunobiology, Research Section of Pathophysiology, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan; and ^**Department of Pediatrics, University of South Florida/All Children's Hospital, St. Petersburg, FL 33701

Edited by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA, and approved May 20, 2002 (received for review April 5, 2002)

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.

Footnotes

↵ † To whom reprint requests should be addressed. E-mail: kizaki{at}kyorin-u.ac.jp.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

UCP2,

uncoupling protein 2;

LPS,

lipopolysaccharide;

ROS,

reactive oxygen species;

NOS II,

NO synthase II;

uORF,

upstream open reading flame;

RT,

reverse transcription