SB203580 Enhances LPS-Induced G-CSF Production in Macrophages by Increasing G-CSF mRNA Stability

• Main Authors: Wen-Ju Tsai, 蔡雯茹
• Other Authors: Shao-Chun Lu
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/51485355276471635542

碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 98 === The granulocyte colony-stimulating factor (G-CSF) is a member of the glycoprotein growth factor family that controls the proliferation and differentiation of the neutrophilic granulocyte lineage. G-CSF is produced by endothelium, macrophages, and a number of other immune cells. Expression of G-CSF is induced by several inflammatory stimuli such as lipopolysaccharide (LPS), interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) through transcriptional and post-transcriptional mechanisms. The G-CSF mRNA contains adenosine uridine-rich elements (AREs) and stem loop destabilizing element (SLDE) in the 3’ untranslated region (3’UTR) which have been associated with mRNA instability. AREs are found in the 3’UTRs of many inflammatory cytokines. Numerous reports demonstrated that AREs act as mRNA destabilization determinants but also confer LPS-induced stabilization of the mRNA through p38 MAPK dependent pathway. The p38α has been found to regulate cytokine biosynthesis in both translation and mRNA stability. Pyridinyl imidazole compounds, such as SB203580, inhibit p38α and β and block production of several major inflammatory cytokines, such as TNF-α, IL-1β, IL-6 and granulocyte macrophage-colony stimulating factor (GM-CSF), by decreasing their mRNA half-life. However, the effects of SB203580 on G-CSF expression in macrophages are unclear. In this study, we investigated the effects of SB203580 on G-CSF expression in LPSstimulated macrophages. Surprisingly, we found that SB203580 increases both G-CSF mRNA and protein levels in LPS-stimulated RAW264.7 macrophages and mouse bone marrow derived macrophages (BMDM). In contrast, levels of IL-1β mRNA were lower in SB203580/LPS treated cells compared to that in LPS treated cells. To test if increase of G-CSF mRNA in the SB203580/LPS treated cells is resulted from the increase of transcription, we investigated the effect of SB203580 and LPS on G-CSF promoter activity by luciferase reporter assay and evaluated transcription rate by nuclear run on assay. The results showed that luciferase activity and transcription rate are both upregulated by LPS, while SB203580 decreased luciferase activity without affecting transcription rate. We then tested if G-CSF mRNA stability is enhanced by SB203580, LPS or SB203580/LPS treated cells were further treated with actinomycin D, mRNA were isolated at 0, 30, 60, 90 and 120 min after actinomycin D treatment, and the levels of G-CSF mRNA were determined by Northern blotting. The results show that G-CSF mRNA is more stable in SB203580/LPS treated cells than that in cells treated with LPS only. To investigate the mechanisms that mediate the increase of G-CSF mRNA stability in response to SB203580/LPS treatment, the G-CSF 5’UTR, 3’UTR and the G-CSF coding regions were cloned into the 5’or 3’ end of luciferase gene in the pGL3-control vector to produce pGL3-G-CSF 5’UTR-Luc, pGL3-Luc-G-CSF 3’UTR, pGL3-Luc-G-CSF(+35/+372) and pGL3-Luc-G-CSF(+340/+720) plasmids. These plasmids were then transfected into RAW264.7 cells and the luciferase activities were determined after treating with SB203580 and/or LPS. We found that the cells transfected with pGL3-Luc-G-CSF 3’UTR and pGL3-G-CSF 5’UTR-Luc plasmid expressed very low luciferase activity, and the treatment with SB203580 did not increase the luciferase activity in LPS-stimulated cells. However, cells transfected with pGL3-Luc-G-CSF(+35/+372) or pGL3-Luc-G-CSF(+340/+720) express luciferase activities similar to that of cells transfected with pGL3-control, and the treatment of SB203580/LPS resulted in slightly higher luciferase activities. The data suggest that 3’UTR and 5’UTR mediated mRNA de-stabilization while the coding region may contain sequences that increase mRNA stability. However, in siRNA mediated p38α knockdown cells, LPS-stimulation did not result in the increase of G-CSF mRNA compared with control siRNA transfected cells. Taken together, our data show that p38 inhibitor SB203580 induced expression of both G-CSF mRNA and protein, which is resulted from the increase, at least in part, of G-CSF mRNA stability; however, the effect may not due to the inhibition of p38 in macrophages.