| Summary: | 碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 92 === From our previous studies, levels of resistin mRNA expression were significantly increased in both epididymal white adipose tissue and white blood cells after intraperitioneal injection of lipopolysaccharide (LPS). Furthermore, we found that resistin mRNA was expressed in human peripheral blood mononuclear cells and THP-1 cells ,and mRNA level was up-regulated by LPS treatment. We also showed expression of resistin mRNA in RAW264.7, a mouse macrophage-like cell line, and its expression was induced by LPS as in human cells. These results suggesting that resistin might be involved in the inflammatory process.
Transfection of resistin-Luciferase reporter gene into RAW264.7 showed that Luciferase activity was significantly up-regulated by LPS treatment. Two DNA fragments, from –1037 to –969 and –968 to –660, were found to be essential for LPS induction of Luciferase activity in 5’ deletion analysis. In this study, we aimed to identify the regulatory elements within the nt –1037 to -969 and nt –968 to –660 of resistin promoter that involved in LPS-induced expression of resistin in RAW264.7 cells.
We constructed various internal deletion (��-968~-881, ��-968~-734, ��-968~-659) from Resistin(-1037/+61)-Luc and transfected into RAW 264.7 macrophages, and stimulated the cells with 10 ng/ml LPS for 4 hours. The result showed that the Luciferase activity induced by LPS of these constructs decreased greatly, indicating that there are regulatory element(s) located within this region. Sequencing analysis exhibited one octamer element (ATTTGCAT) within -968 to -882 region of resistin promoter. Mutation of the octamer in Resistin(-1037/+61)-Luc resulted in significant decrease of inducibility of Luciferase activity. Electrophoretic mobility shift assays (EMSA) using an oligonucleotide probe containing the octamer have identified a DNA/Protein complex. LPS induced increase of Luciferase activity of cells transfected with Resistin(-968/+61)-Luc can be partially blocked by Ly294002, a PI3K inhibitor, and U0126, a MEK inhibitor, but was unaffected by SB203580, a p38 inhibitor. Indicating the octamer element is activated by LPS through the MEK/ERK and PI3K pathways, but not p38 pathway. Several putative transcription factor binding sites, including C/EBP(CCAAT), TATA box (TATAA) and Ets (TTCC) were identified with nt –1037 to –997 of the promoter. However, mutation of these sites in Resistin(-1037/+61)-Luc did not change the induction of Luciferase activity by LPS treatment.
These data suggest that, a 28bps fragment (–996 to –969) and an octamer element are both essential for LPS induction of mouse resistin gene expression in RAW264.7 cells. Maximal activity of the promoter is obtained through the synergistic effect of these two elements. Moreover, MEK/ERK and PI3K pathways are involved in LPS activation of the –968/+61 resistin promoter.
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