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

顆粒性白血球群落性促進因子（Granulocyte-colony simulating factor，G-CSF）為一19kD的醣蛋白，其具有促進嗜中性球以及巨噬細胞分化增生的能力，並且能促使嗜中性球的前驅細胞自骨髓移動進周邊血液循環，往發炎患處聚集以吞噬病原體。當人體接受到細菌的LPS，或是因發炎所大量產生的細胞激素如TNF-α及IL-1等因子刺激時，會使血液中的G-CSF濃度大幅增加，以促進身體對感染做出必要的免疫反應。除了對血球增生及分化作用有正向影響之外，近年來G-CSF亦被發現具有更多樣化的生理功能如：保護神經、促使骨髓幹細胞進入血液循環、降低移植體對抗宿主疾病（GVHD）等等，都相當具有其生理上的應用價值。但目前的研究卻尚未清楚闡明G-CSF在巨噬細胞中被活化的機制為何，因此這個研究主要是探討在巨噬細胞內的MEK/ERK訊息傳遞路徑如何調控G-CSF之表現。 當以U0126抑制MEK1/2活性而阻斷LPS所活化的MEK/ERK訊息傳遞路徑時，發現LPS所引發的G-CSF表現量顯著地下降下來。在G-CSF的啟動子上有兩段已被發現對其活性相當重要的序列：CSF-box及octamer，根據過去研究及本實驗室未發表的實驗結果，此兩段序列分別由NF-κB以及Oct-2所結合；於是我們推測MEK/ERK訊息傳遞路徑的下游可能透過NF-κB及Oct-2來調控G-CSF表現。Luciferase reporter assay實驗結果顯示，CSF-box及octamer兩段序列對LPS活化G-CSF啟動子是缺一不可的，且U0126會抑制此兩段序列對G-CSF啟動子之正向調控。然而藉由西方墨點法及reporter assay卻發現U0126不影響NF-κB的transactivity，而EMSA及西方墨點法實驗結果也顯示，U0126同樣不影響Oct-2之表現量以及在in vitro條件下對G-CSF啟動子之結合；但另一方面透過chromatin immunoprecipitation（ChIP）assay卻顯示出在in vivo中LPS的確會促進Oct-2結合上G-CSF啟動子，且U0126會顯著地將此結合現象抑制下來。 透過這些結果，我們認為MEK/ERK pathway的確參與LPS活化G-CSF表現的過程，雖然MEK/ERK pathway都影響了CSF-box以及octamer對G-CSF啟動子所貢獻的活性，但似乎MEK/ERK pathway的下游並非直接影響NF-κB調控基因轉錄的活性，而主要是影響了Oct-2對G-CSF啟動子的調控。此外，既然CSF-box以及octamer對G-CSF的啟動子活性缺一不可，那麼Oct-2和NF-κB之間可能存在著某種共同調控G-CSF表現的機制，尚待進一步釐清。

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.