探討受 LPS 刺激的小鼠巨噬細胞中 Oct-2 在 G-CSF 表現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)為一具有促進嗜中性球及巨噬細胞分化增生能力的醣蛋白，可促使嗜中性球的前驅細胞自骨髓進入周邊血液循環，往發炎反應發生處聚集以吞噬病原體。當人體接受外來刺激例如格蘭氏陰性菌的脂多醣 (Lipopolysaccharide, LPS)，或促發炎細胞激素如 TNF-α (Tumor necrosis factor)、IL-1β (Interleukin-1 beta) 等因子刺激時，會使血液中的 G-CSF 濃度增加，促使身體對感染產生免疫反應。近年來G-CSF 亦被發現具有更多樣的生理功能如：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反應(GVHD) 等，皆具生理上的應用價值。本實驗室過去的研究發現在小鼠的巨噬細胞 Raw264.7 中，G-CSF 啟動子受到 LPS 刺激時，會被 Oct-2、C/EBPβ 等轉錄因子所結合。Oct-2 在神經細胞內及 B 淋巴球中是非常重要的轉錄因子，且其表現均需要 NF-κB 的參與，但Oct-2 在巨噬細胞內的表現和功能仍尚未明瞭，故我們想知道在LPS 誘導巨噬細胞表現 G-CSF時，Oct-2 與 C/EBPβ 是透過什麼機制誘導 G-CSF 表現的。此外，本實驗室過去發表的研究指出， LPS 可誘導巨噬細胞表現 Oct-2，故其在巨噬細胞內之功能可能與免疫反應發生有關，但其詳細機制仍需進一步探討。 我們利用 G-CSF Luciferase reporter assay 探討轉錄因子 Oct-2 與 C/EBPβ間的關係，發現同時表現 Oct-2 與 C/EBPβ 時可誘導 G-CSF promoter Luciferase的活性，且其具有協同作用。我們更進一步使用 GST-Oct-2 pull-down assay 與共免疫沉澱的方式分別探討體外與體內的機制，結果皆顯示 Oct-2 可與 C/EBPβ 產生交互作用。若將位於 C/EBPβ C 端的白胺酸拉鏈 (leucine zipper) 區域去除後，此交互作用則會消失，可推測白胺酸拉鏈對於 C/EBPβ 與 Oct-2 的交互作用是很重要的。為了探討這個交互作用的功能性影響，我們在進行 G-CSF Luciferasereporter assay 時使用白胺酸拉鏈區域被去除而無法與 Oct-2 產生交互作用的C/EBPβ 與完整的C/EBPβ 競爭，發現若加入愈多無法與 Oct-2 產生交互作用的C/EBPβ，則其誘導 G-CSF promoter Luciferase 活性的效果就愈低，故可推測C/EBPβ 與 Oct-2 的交互作可提高其促進轉錄的效果。此外，若進行 GSTpull-down 時加入 Oct-1 或是 Oct-2 進行競爭，加入的 Oct-1 或是 Oct-2 愈多，可被 pull-down 的 C/EBPβ 愈少，這可能代表了 Oct-2 與 Oct-1 在結構上具有一定程度的相似性。然而在小鼠巨噬細胞中 LPS 並不會誘導 Oct-1 的表現，但可顯著誘導 Oct-2 使其表現量增加。 另外有關 LPS 在小鼠巨噬細胞中可正向調控 Oct-2 表現的部份，我們根據Oct-2 promoter Luciferase reporter assay 的結果顯示，在 Oct-2 promoter 的-205/-82 之間具有對其基礎表現量重要的序列；在小鼠巨噬細胞中以 LPS 刺激後使 Oct-2 表現量增加，則與 -349/-205 間的序列有關。然而以 NF-κB 的抑制劑Bay11-7082 進行實驗，發現在 LPS 誘導 Oct-2 promoter Luciferase 活性、Oct-2mRNA 及 Oct-2 蛋白質的過程中，Bay11-7082 皆無顯著抑制效果，故可推測 LPS刺激小鼠巨噬細胞後使 Oct-2 表現量增加，其訊息傳遞路徑並不經過 NF-κB。

G-CSF (granulocyte-colony simulating factor) is a glycoprotein which stimulates the differentiation and proliferation of neutrophils and macrophages, it promotes promyelocytes to migrate from bone marrow to peripheral blood, and gather at the inflammation area to clear out the pathogens. When the human body is stimulated by LPS (lipopolysaccharide) of Gram negative bacteria or pro-inflammatory cytokines such as TNF-α (tumor necrosis factor) and IL-1β (interlukin-1 beta), the G-CSF in blood increases dramatically to promote the immune response against infections. G-CSF has also been found to have multiple physiological roles, such as neural protection, promotion of bone marrow stem cells to migrate to the peripheral blood and decrease the GVHD (graft-versus-host disease) reaction. According to the unpublished results in our lab, we found that when murine macrophage Raw264.7 is stimulated by LPS, the G-CSF promoter is then bound by transcription factors such as Oct-2, C/EBPβ and p65. However, the functions of Oct-2 in LPS-induced G-CSF up-regulation in macrophages are still unclear. Thus in this study we investigated the role of Oct-2 and C/EBPβ in LPS stimulated Raw264.7. In addition, one previous data showed that Oct-2 expression was induced by LPS in Raw264.7, therefore the mechanism of LPS-induced Oct-2 expression in macrophages is also studied. First, the G-CSF Luciferase reporter assay was used to investigate the relationships between Oct-2, C/EBPβ and p50. We found that co-expression of Oct-2 and C/EBPβ induced G-CSF promoter Luciferase activity synergistically. We then used GST-Oct-2 pull-down assay and co-immunoprecipitation assay to investigate the protein-protein interaction of Oct-2 and C/EBPβ, and we found that Oct-2 interacted with three forms of C/EBPβ including LAP*, LAP and LIP in either in vitro or in vivo conditions. To map the interacting domain of C/EBPβ with Oct-2, we deleted the leucine zipper domain of C/EBPβ and found that the C/EBPβ-D-ZIP did not interact with Oct-2 in the GST pull-down assay, the result suggests that the leucine zipper domain of C/EBPβ is indispensible to this interaction. To study the function of this interaction, the C/EBPβ-D-ZIP was used to compete the intact C/EBPβ in a G-CSF Luciferase reporter assay, and we found that the G-CSF promoter Luciferase activity decreased gradually with increasing amount of C/EBPβ. Although Oct-1 also compete the interaction between Oct-2 C/EBPβ, the expression of Oct-1 is not induced by LPS in Raw264.7, but the Oct-2 expression level can be induced significantly by LPS. In the investigation of Oct-2 promoter by Luciferase reporter assay, we found that the sequence between -205/-82 of Oct-2 promoter is crucial for Oct-2 basal expression level, and the sequence between -349/-205 is important for LPS induced Oct-2 expression. We further used the NF-κB inhibitor Bay11-7082 to investigate the signaling pathway of LPS induced Oct-2 expression, and found that Bay11-7082 cannot inhibit the LPS induced Oct-2 promoter luciferase activity, Oct-2 mRNA and protein expressions. The results suggest that LPS-induced Oct-2 expression may through a NF-κB independent pathway in macrophages.