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.

Figure 1

UCP2 expression after LPS stimulation. Total cellular RNA and mitochondrial protein were prepared from RAW264 cells stimulated with LPS for 0, 6, and 24 h. (A) UCP2 mRNA expression was analyzed by Northern blotting analysis. (B) Amount of UCP2 protein in mitochondria was analyzed by Western blotting analysis.

Figure 2

Intrinsic or transfected UCP2 expression in RAW264 cells and RAW264 cells transfected with ucp2 construct or with vector alone. Total cellular RNA and mitochondrial protein were prepared from these cells after they were cultured with or without LPS for 24 h. (A) The expression of intrinsic and transfected UCP2 mRNA was analyzed by RT-PCR analysis. For normalization, β-actin was used. (B) The expression of transfected ucp2 products was analyzed by Western blotting analysis with anti-myc antibodies.

Figure 3

Effects of UCP2 overexpression on intracellular ROS. Cells were cultured with or without LPS for 1 h, and intracellular ROS was analyzed by flow cytometry.

Figure 4

Effects of UCP2 overexpression on NO production and NOS II expression. (A) Cells were stimulated with LPS for 48 h, and accumulation of nitrite in the supernatants was measured with Griess reagent. (B) Cells were stimulated with LPS for 0, 6, and 24 h. Amount of NOS II protein was analyzed by Western blotting analysis. (C) Cells were stimulated with LPS for 0, 6, and 24 h. NOS II mRNA expression was analyzed by RT-PCR analysis. (D) Cells were transiently transfected with luciferase reporter construct containing NOS II promoter. After LPS stimulation for 24 h, luciferase activities were determined. (A and D) Results are expressed as the mean ± SEM from quadruplicate cultures.

Figure 5

Reporter gene assay of promoter and 5′ region of UCP2. RAW 264 cells were transiently transfected with various reporter constructs. Cells were cultured without (□) or with (■) LPS for 24 h, and luciferase activities were determined. Results are expressed as the mean ± SEM from quadruplicate cultures. (A) Reporter constructs containing the UCP2 promoter regions. (B) Reporter constructs containing promoter region and 5′ region upstream from the translation initiation site of UCP2. (C) Reporter constructs containing the UCP2 5′ region with the indicated mutations.

Figure 6

Reporter gene assay of the 5′ region of UCP2 with deletion of intronic region. RAW 264 cells were transiently transfected with various reporter constructs. Cells were cultured without (□) or with (■) LPS for 24 h, and luciferase activities were determined. Results are expressed as the mean ± SEM from quadruplicate cultures. (A) Reporter constructs containing the 5′ region of UCP2 with deletion of intronic region. (B) Reporter constructs containing the 5′ region of UCP2 with deletion of AvrII or SacI fragment.

Figure 7

Transfected UCP2 expression in RAWucp2/m (A) and RAWucp2/hn (B) cells. Total cellular RNA was prepared from these cells after they were cultured with or without LPS for 0, 1, 3, 6, and 24 h. The expression of transfected UCP2 mRNA was analyzed by RT-PCR analysis. For normalization, 18S was used.