p38α/β MAPK在SB203580誘導下增加RAW264.7細胞中G-CSF表現的角色

Abstract

顆粒性白血球群落刺激因子 (Granulocyte colony-stimulating factor, G-CSF)是造血性醣蛋白生長因子的成員之一，具有促進白血球增生、分化、成熟及移動的功能。主要由巨噬細胞、內皮細胞所分泌，當LPS、IL-1β 或TNF-α 刺激時，透過轉錄及後轉錄增加 G-CSF 表現。G-CSF mRNA 穩定度主要由三端非轉譯區 (3’ untranslated region, 3’UTR) 的 ARE (AU-rich element) 及 SLDE (stem-loop destabilizing element)所調控。多篇文獻報導在 p38 MAPK 訊息路徑活化下，給予 SB203580 會降低 3’UTR 帶有 ARE 細胞激素的表現。然而，實驗室先前的研究結果發現，在有或沒有LPS刺激下，於RAW264.7細胞中給予SB203580可透過3’UTR的SLDE序列穩定G-CSF mRNA進而增加其表現。顯示 SB203580 增加G-CSF表現可能不是透過p38α/β MAPK所導致的。本研究目的首先要確認 SLDE 及p38α/β MAPK 在 SB203580 增加 G-CSF mRNA 穩定度的作用中扮演的角色；接著用microarray 分析探討是否有其他基因也會受到 SB203580 所誘導，並分析這些被誘導的基因其 3’UTR 是否帶有類似 SLDE 的序列。最後探討目前已知與發炎反應相關的miRNA是否參與 SB203580 增加G-CSF mRNA 穩定度的作用。以site-direct mutagenesis 改變SLDE的序列發現 SB203580 是透過 SLDE序列穩定G-CSF mRNA ，與莖-環結構較無關。將RAW264.7細胞中p38α或p38β MAPK knockdown後發現雖然 LPS 誘導的G-CSF 表現會受到p38α MAPK knockdown 的影響而下降，然而，在有或沒有 LPS 刺激下，SB203580 可在p38α 或p38β MAPK knockdown 細胞增加 G-CSF mRNA 穩定度及蛋白質表現。Microarray實驗結果找到在有或沒有 LPS 刺激下 SB203580 都能增加其表現量共 21個基因，其中 Gnpda1 3’UTR也帶有類似 SLDE 序列。最後利用miRNA PCR array實驗發現目前已知與發炎反應調節相關的 miRNA中有 7個會受 SB203580 所調節，由 miRWalk 預測軟體分析發現G-CSF 3’UTR 中並沒有這 7個 miRNA的結合位子，顯示 SB203580 可能不是透過這 7 個 miRNA 直接調控 G-CSF 的表現，但我們也不能完全排除這些miRNA間接調控的可能。綜合以上實驗結果，SB203580透過G-CSF 3’UTR的SLDE序列穩定mRNA進而增加其表現的作用機制與 p38α/β MAPK無關，可能是透過其他機制來調節。

Granulocyte-colony stimulating factor (G-CSF) is a member of the glycoprotein growth factor family that regulates granulocyte production, neutrophil maturation and mobilization. The main sources of G-CSF are fibroblast cells, endothelial cells and macrophages. The production of G-CSF is induced by inflammatory stimuli, such as LPS, TNF-α and IL-1β. The expression of G-CSF is regulated at both transcriptional and posttranscriptional levels. In the 3’-UTR of G-CSF mRNA, there are two destabilizing elements, AU-rich elements (ARE) and stem-loop destabilizing element (SLDE), these elements have been identified conserved among different species. Our previous results showed that SB203580, a pyridinyl imidazole inhibitor of p38α/β MAPK, enhances G-CSF production by increasing mRNA stability in the presence or absence of LPS. Moreover, the sequence of SLDE in G-CSF 3’UTR is identified essential for SB203580-induced increase of mRNA using a luciferase-G-CSF 3’UTR reporter system. In this study, our first aim is to further confirm the role of SLDE in SB203580-induced increase of G-CSF mRNA stability; and to investigate the involvement of p38α/β MAPK in SB203580-induced G-CSF expression. Second aim is to determine if SB203580 can up-regulate other transcripts and if 3’-UTR of these transcripts also contain SLDE sequence. Third aim is to explore whether miRNA involves in SB203580-induced G-CSF expression. Using site-direct mutagenesis mutated one or two bases on SLDE, we further confirmed that SLDE in G-CSF 3’UTR is critical for SB203580-induced increase of G-CSF mRNA. The p38α and p38β MAPK were knockdown separately in RAW264.7 by infected with lentivirus carrying specific shRNA. We found that SB203580 induces increase of G-CSF mRNA in p38α or p38β knockdown cells in the presence or absence of LPS. The results indicate that SB203580-induced increase of G-CSF expression is independent of p38α or p38β MAPK. In microarray analysis, 21 genes were identified to be up-regulated by SB203580 in the presence or absence of LPS. Only one of 21 genes, Gnpda1, contains a SLDE-like sequence in the 3’UTR. Moreover, we investigate the involvement of inflammatory-related miRNAs in this mechanism by miRNA PCR array. Seven miRNAs were found to be regulated by SB203580, but 3’UTR of G-CSF does not contain binding site for these miRNAs. The results indicate that these inflammatory-related miRNA may not directly involve in SB203580-induced increase of G-CSF expression. But the indirect involvements of these miRNAs cannot be exclude. Taken together, our data show that SB203580 increases both G-CSF mRNA and protein levels by stabilizing G-CSF mRNA through the conserved SLDE sequence in the 3’UTR; however, the effect is independent of p38α/β MAPK.