MEK-ERK-C/EBPbeta在巨噬細胞中對脂多醣誘導G-CSF表現的必要角色

Essential Role of MEK-ERK-C/EBPbeta in LPS-induced G-CSF Expression in Macrophages

楊惠晴(Hui-Ching Yang)

指導教授 ： 呂紹俊

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10882

摘要

顆粒性白血球群落刺激因子(Granulocyte-colony stimulating factor, G-CSF)是一個造血性醣蛋白，分子量約19.6 kDa。G-CSF最主要的功能，是刺激嗜中性白血球前驅細胞的分化，並促進嗜中性白血球的產生，往發炎部位聚集以吞噬外來病菌，以及降低嗜中性白血球細胞凋亡促進其存活。G-CSF在正常狀況下表現量極低，但當生物體接受外物如：LPS (lipopolysaccharide)、PMA (phorbol 12-myristate 13-acetate)或是其他發炎反應細胞激素TNF-alpha、IL-1beta和IFN-gamma等刺激後，會大幅提高其表現。近年研究顯示，G-CSF也具有其他生理功能，例如：保護神經、調節T淋巴球的耐受性、促進造血幹細胞由骨髓移動至周邊血液循環系統等。除此之外，近年一些研究顯示G-CSF在慢性發炎疾病中也扮演重要角色。ERK已知可調控許多發炎反應細胞激素的表現，如：TNF-alpha、IL-1beta、IL-6、IL-8、IFN-gamma誘導基因，並影響免疫細胞的分化，由此可知ERK對免疫系統的調節非常重要。本實驗室過去研究發現LPS可誘導小鼠巨噬細胞RAW264.7 G-CSF表現，事先處理MEK/ERK抑制劑U0126和PD98059會抑制LPS誘導的G-CSF表現，但ERK調節G-CSF表現的機制目前仍不清楚。因此，我們想更進一步探討ERK如何參與LPS誘導小鼠巨噬細胞表現G-CSF的機制。
　　我們的研究發現，不論是在LPS刺激細胞前，或在LPS刺激細胞三到四小時後給予U0126，皆可抑制RAW264.7細胞G-CSF的表現，相同的現象也可在小鼠骨髓分離出的初代巨噬細胞和人類單核球細胞株中觀察到。我們更利用luciferase reporter分析探討ERK是否活化G-CSF啟動子來調節G-CSF表現，結果顯示當細胞表現了constitutive active-ERK (CA-ERK)時，可活化G-CSF啟動子活性，而kinase dead-ERK (KD-ERK)則無此活性，由此可知ERK參與調控G-CSF的表現。文獻指出Oct-2、NF-kappaB和C/EBPbeta在調控G-CSF表現扮演重要角色，因此我們分析ERK是透過活化那一個轉錄因子增強G-CSF啟動子的。由G-CSF promoter luciferase活性分析顯示，同時轉染CA-ERK與C/EBPbeta時，G-CSF啟動子活性明顯提高，且兩者有協同作用；但同時轉染KD-ERK與C/EBPbeta時則無此現象。由於NF-kB具有p50和p65兩個subunits，我們也在細胞中同時表現p65，觀察其對CA-ERK與C/EBPbeta調控G-CSF啟動子活性的影響發現，p65存在時對G-CSF啟動子活性提升並無明顯影響。此外在細胞中表現CA-MEK也可活化G-CSF啟動子活性，而同時表現CA-MEK與C/EBPbeta時可更加提升G-CSF啟動子活性。這些結果顯示MEK/ERK signaling pathway在調控G-CSF表現扮演重要角色。
　　我們進一步將C/EBPbeta中高度保留的MAPK磷酸化位置(Thr188)進行突變後，由G-CSF promoter luciferase活性分析發現，ERK可藉由磷酸化C/EBPbeta Thr188來影響G-CSF啟動子活性。接著分析細胞受LPS刺激後增加ERK與C/EBPbeta磷酸化，U0126則會減少C/EBPbeta Thr188磷酸化。此外DNA親和力免疫沉澱分析(DAPA)的結果顯示ERK存在於G-CSF啟動子的transcription complex中。然而，若將C/EBPbeta的結合阻斷時，並不影響ERK存在於G-CSF啟動子transcription complex中，因此ERK並不是透過C/EBPbeta存在於transcription complex中。綜合以上結果，ERK確實參與LPS誘導的G-CSF基因表現調控，然而ERK活化G-CSF啟動子活性有部分是透過活化C/EBPbeta。因此，關於ERK在G-CSF啟動子的轉錄活化與G-CSF基因表現所扮演的角色仍需更進一步的研究。

關鍵字

顆粒性白血球群落刺激因子 ； 巨噬細胞 ； ERK ； C/EBPbeta ； 基因轉錄調控 ； MEK/ERK抑制劑U0126和PD98059

並列摘要

Granulocyte colony-stimulating factor (G-CSF) is a 19.6 kDa hematopoietic glycoprotein growth factor. The main functions of G-CSF are to stimulate differentiation of neutrophilic precursors and promote neutrophil production, migration, and survival. The expression level of G-CSF is very low in healthy individuals, but it is elevated substantially after stimulation with LPS, PMA, or pro-inflammatory cytokines such as TNF-alpha, IL-1beta, and IFN-gamma. Expression of G-CSF after stimulation will promote immune response and is important for host defense during infection. Studies also showed that G-CSF has other physiological roles, such as neuroprotection, regulation of T cell tolerance, and promotion of hematopoietic stem cell migration to peripheral tissues. On the other hand, recent studies also showed that G-CSF plays an important role in chronic inflammatory diseases. ERK1/2 is one of the mitogen activated protein kinases (MAPK). It involves in the expression of many pro-inflammatory cytokines, such as TNF-alpha, IL-1beta, IL-6, IL-8, and IFN-gamma-stimulated gene. Studies also showed that ERK1/2 affect the differentiation of immune cell such as B and T lymphocytes. Above information indicates that ERK plays an important role in regulation of immune system. Our previous studies showed that the expression of G-CSF is upregulated by LPS in RAW264.7 cells; while pretreatment with MEK inhibitors (U0126 and PD98059) inhibited the LPS-induced G-CSF expression. However, it is still unclear how ERK may regulate G-CSF expression in LPS-induced cells. Therefore, the goal of this study is to explore the involvement of ERK1/2 in LPS induced G-CSF expression.
First, U0126 and PD98059 were used to inhibit MEK1/2 activity induced by LPS. The expression of G-CSF was down-regulated by ERK inhibitors at 3-4h after the cells were stimulated by LPS in Raw264.7 cells. Similar phenomena were observed in mouse bone-marrow derived macrophages and in human monocytic cell line THP-1. We then studied how ERK interacts with transcription factors that regulate G-CSF expression by co-transfecting a G-CSF promoter-Luciferase reporter with a constitutive active- or kinase dead-ERK expression plasmid in Raw264.7 cells. Results show that G-CSF promoter activity is activated when forced expression of CA-ERK, but is not activated when forced expression of KD-ERK. These results support that ERK is involved in the regulation of G-CSF expression. We then studied which transcription factor interacts with ERK by transfection of NF-kappaB p50, p65, Oct-2, C/EBPbeta and/or CA-ERK with the G-CSF promoter-Luciferase construct. Results show that G-CSF promoter activity is induced synergistically only when C/EBPbeta and CA-ERK were co-transfected. Transfection of CA-MEK also results in elevation of G-CSF promoter activity. Moreover, co-transfection of C/EBPbeta and CA-MEK further elevates G-CSF promoter activity. These results further support that activation of MEK/ERK signaling pathway is essential for the expression of G-CSF in macrophages.
To further study whether ERK activates G-CSF promoter activity through promoting C/EBPbeta phosphorylation, we constructed two C/EBPbeta mutants (C/EBPbeta T188A and S64A) and transfected cells with these mutants with CA-ERK separately. Results show that transfection with C/EBPbeta T188A resulted in about 50 % decrease of G-CSF promoter activity, but transfection with C/EBPbeta S64A has no effect on promoter activity. Western blot analyses also show that C/EBPbeta Thr188 phosphorylation is partially inhibited by U0126 in LPS-treated cells. Moreover, ERK can be precipitated by a DNA affinity precipitation assay. These results suggest that ERK is in the transcriptional complex that binds to G-CSF promoter. However, ERK still bound to G-CSF promoter when the binding of C/EBPbeta was competed out by C/EBPbeta consensus sequence. These results suggest that ERK does not bind to the transcriptional complex through interaction with C/EBPbeta. Taken together, our results show that activation of ERK is essential for G-CSF expression induced by LPS; however, the functional role of ERK in the transcriptional activation of G-CSF is still unclear and requires further investigation.

並列關鍵字

C/EBPbeta ； ERK ； G-CSF ； gene transcription regulation ； Macrophage ； PD98059 ； U0126

參考文獻

李曜宏（2008）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。國立台灣大學醫學院生物化學暨分子生物學研究所碩士論文

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