NF-κB及Oct-2在脂多醣透過MEK/ERK訊息傳遞路徑活化巨噬細胞表現G-CSF之過程中所扮演的角色

Abstract

Granulocyte-colony stimulating factor (G-CSF) is a 19kD glycoprotein. It is the major cytokine regulator of neutrophilic granulocyte because G-CSF was discovered could stimulate differentiation and proliferation of neutrophil and macrophage which are essential cell types for the clearance of bacterial pathogens. G-CSF could be dramatically and rapidly upregulated by several inflammatory stimuli such as LPS, TNF-α and IL-1β. Thus, the high level of G-CSF can help our body to defend the infection and inflammation. In these years, G-CSF was also shown to improve survival and recovery of cardiomyocyte and neuron, and to promote the mobilization of hemaopoietic stem cells from bone marrow to peripheral blood. These information indicate that G-CSF plays multiple and important physiological roles in human body. However, not much is known about regulation of G-CSF expression in macrophages. In the present study, we used U0126 to inhibit the MEK1/2 activity induced by LPS and found that the LPS-induced G-CSF promoter activity, and G-CSF expression levels at mRNA and protein were dramatically blocked. There are two elements, a CSF-box and a octamer within G-CSF promoter are essential for its promoter activity. The CSF-box is a potential NF-κB binding site, and the octamer was bound by Oct-2 (unpublished result). Therefore, it is important to ask whether both Oct-2 and NF-κB participate the MEK/ERK dependent G-CSF expression. G-CSFp-(-289~+25)-Luc, CSF-box-deleted G-CSFp- (-145~+25)-Luc, or octamer-mutated Oct-mut-G-CSFp-(-289~+25)-Luc reporter plasmids were used to investigate the importance of CSF-box and octamer in response to LPS. I found that the CSF-box and the octamer were both indispensible for basal and LPS-induced G-CSF promoter activity, and the functions of these two elements were both inhibited by U0126. However, the LPS-induced activation of NF-κB was not affected by U0126 demonstrated by NF-κB reporter assay and Western blotting of p50 and p65 in nuclear extract. The results indicate that NF-κB transactivity is not diminished by U0126. In addition, the LPS-induced Oct-2 mRNA and protein expression were not inhibited by U0126. However, chromatin immunoprecipitation (ChIP) assay revealed that the LPS-induced Oct-2 binding to G-CSF promoter was strikingly reduced by pretreatment of U10126. Taken together, our data showed that both the CSF-box and the octamer are important for the LPS-induced G-CSF promoter activity, moreover, the activation of MEK/ERK signaling pathway is indispensible for LPS-induced G-CSF expression in RAW264.7 macrophage.. It seems that LPS-induced activation of MEK/ERK pathway does not directly regulates NF-κB transactivity, but is essential for LPS-induced Oct-2 binding to the octmaer within G-CSF promoter and transcription activation of the G-CSF expression. Moreover, since both the CSF-box and the octamer are indispensible for LPS-induced G-CSF promoter activity, there might be a coactivation mechanism composed of Oct-2 and NF-κB for G-CSF transcription. However, the mechanism is not yet understood and deserves further investigations.