ERK2在LPS誘導巨噬細胞G-CSF表現的角色

Roles of ERK2 in LPS-induced G-CSF expression in macrophage

林詩珊(Shih-Shan Lin)

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(2012年)

https://doi.org/10.6342/NTU.2012.02854

摘要

顆粒性白血球群落刺激因子(granulocyte-colony stimulating factor, G-CSF)是一個可調控嗜中性白血球與巨噬細胞生長與分化的分泌型細胞激素，可在血管內皮細胞與巨噬細胞等受到免疫刺激（如LPS）時分泌。類風濕性關節炎患者具高度內生性的G-CSF表現，而過量的G-CSF表現可能是引起發炎部位的白血球侵入的原因。並且在一些較會轉移的癌細胞中G-CSF的表現也比不易轉移的癌細胞以及正常細胞高，這些研究都顯示瞭解G-CSF的調控對相關疾病的治療策略是相當重要。
我們實驗室先前發現在人類單核球細胞與小鼠巨噬細胞，被LPS所誘導的G-CSF皆可被MEK1/2抑制劑（U0126及PD98059）所抑制，顯示MEK/ERK 訊息傳遞在調控G-CSF表現扮演重要角色。因為U0126會同時抑制ERK1與ERK2的活性，因此我利用帶有shRNA的慢病毒專一性地knockdown THP-1細胞的 ERK1或ERK2，我們發現由LPS刺激G-CSF的表現，會於knockdown ERK2時下降；而knockdown ERK1則沒太大的影響。並且在帶有G-CSF啟動子調控的螢火蟲冷光酶報導基因的細胞中同時轉染能表現持續活化的ERK2 (CA-ERK2) 的質體時，可增加冷光酶活性； 此現象會在將G-CSF啟動子上C/EBPβ的結合位置做突變時減少， 顯示ERK2和C/EBPβ對於G-CSF調控扮演重要的角色。
然而C/EBPβ和ERK2的參與並不足以解釋U0126對G-CSF基因表達的影響。我們發現將C/EBPβ可磷酸化的位置Thr 188突變成Ala，會降低G-CSF啟動子活性約三成，這與預處理U0126使G-CSF mRNA幾乎完全被抑制的情形不同。因此我們推測應該還有其他因子參與其中。過去研究發現MEK/ERK訊息傳遞可改變染色質結構，以染色質免疫沉澱法分析，我們發現在U0126預處理下，結合到G-CSF啟動子上的轉錄因子 (如C/EBPβ、NF-κB及p65等) 大幅減少，故我們推測ERK2可能是透過影響染色質結構來調控G-CSF的表達。因此我們利用脫氧核糖核酸酶 (DNase I) 敏感度實驗評估染色質結構的變化，在LPS刺激下，G-CSF啟動子受到DNase I切割的程度於預處理U0126及knockdown ERK2時明顯下降，顯示ERK2的活化與G-CSF啟動子的染色質結構變化有關。但ERK2是如何調控G-CSF啟動子的染色質結構變化仍不清楚，有待後續實驗探討。由於G-CSF的表現與一些發炎疾病有關，這個研究的發現將有助於研發抑制G-CSF的藥物用於治療相關的疾病。

關鍵字

C/EBPβ ； DNase I敏感度實驗 ； ERK1 ； ERK2 ； 顆粒性白血球群落刺激因子 ； 巨噬細胞 ； MEK1/2抑制劑U0126

並列摘要

Granulocyte colony-stimulating factor (G-CSF) is an extracellular cytokine that controls the production and differentiation of granulocytes and macrophages. When endothelial cells and macrophages expose to inflammatory stimuli (such as lipopolysaccharide, LPS), they secret significant amout of G-CSF. Moreover, high expression level of G-CSF has been found in rheumatoid arthritis (RA) and in most malignant cancers. In addition, G-CSF expression is reported to be associated with recruitment of leukocytes into the inflammatory sites, and possibily associated with cancer cell invasion. These results suggested that understanding the regulation of G-CSF expression is important.
Our previous studies showed that the expression of G-CSF in LPS-induced macrophages could be downregulated by MEK1/2 inhibitors (U0126 and PD98059). These results suggest that LPS-induced G-CSF expression through a MEK/ERK dependent pathway in macrophages. Because U0126 inhibits both ERK1 and ERK2, thus it is unclean which ERK is involved in G-CSF expression. In this study, we investigated the functional roles of ERKs in THP-1 macrophage cell line by specifically knocking down ERK1 or ERK2 with lentivirus carrying shRNA. ERK2 knockdown, but not ERK1, resulted in reduction of LPS-induced G-CSF mRNA expression. Furthermore, co-transfect G-CSF promoter-Luciferase reporter plasmid with constitutive active-ERK2 (CA-ERK2) resulted in an increase in luciferase activity. This effect was abolished when the C/EBPβ binding site on the G-CSF promoter was mutated. These results suggest that both ERK2 and C/EBPβ play important roles in G-CSF regulation.
However, the interaction between ERK2 and C/EBPβ is not enough to explain the inhibitory effects of U0126 on G-CSF gene expression. We found that G-CSF promoter activity was reduced by 30%-40% with C/EBPβ T188A mutant. However, pretreatment with U0126 results in over 95% inhibition on LPS-induced G-CSF mRNA. Therefore, we considered that other factors may involve in the regulation of G-CSF expression in the downstream of ERK2. Recent studies showed that MEK/ERK signal pathway is involved in chromatin remodeling. In chromatin immunoprecipition (ChIP) assay, we found that U0126 prevented binding of transcription factors (C/EBPβ, NF-κB and p65, etc) onto the G-CSF promoter in response to LPS stimulation. Thus, we speculate that ERK2 may regulate LPS-induced G-CSF expression through changing of chromatin structure. By using DNase I accessibility assay, we found that LPS-induced accessibility of G-CSF promoter to DNase I digestion is reduced in ERK2 knockdown cells than in the control and ERK1 knockdown cells. These results suggest that activation of ERK2 may change chromaten remodeling. However, it is unclear how ERK2 regulates chromatin remodeling in G-CSF promoter. Because the expression of G-CSF has been associated with inflammatory diseases, the finding in this study may help development of new drugs to inhibit G-CSF expression in related diseases.

並列關鍵字

C/EBPβ ； DNase I accessibility assay ； ERK1 ； ERK2 ； G-CSF ； Macrophage ； U0126

參考文獻

林偲涵 (2011) ERK2調控G-CSF在侵略性癌細胞中的表現。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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