小鼠巨噬細胞RAW264.7在脂多醣刺激下Zfp36l2訊息核醣核酸受Tristetraprolin蛋白負調節之研究及Zfp36l2蛋白之功能分析

Abstract

轉錄後調控主要是藉由mRNA穩定性或轉譯效能層面來調控。其中訊息傳遞所造成基因表現的改變有40-50%是由mRNA的穩定性所調控，其中藉由降解相關因子結合到mRNA三端未轉譯區(3’untranslated region,3’UTR)的多腺嘌呤-尿嘧啶序列(AU-rich element,ARE)所參與的mRNA降解就是其中一種機轉。這些mRNA通常是負責編碼即時反應的基因，像是細胞素,發炎因子,原致癌基因,轉錄因子。TTP (tristetraprolin)家族蛋白可以藉由鋅指結構結合在目標mRNA的ARE上促進mRNA的降解,進而影響到下游蛋白質的表現。TTP家族蛋白包含四個成員: TTP (ZFP36)、ZFP36L1、ZFP36L2、ZFP36L3，其中ZFP36L3只出現在齧齒類動物的胎盤跟胚外組織。目前對於ZFP36L2的基因調控及功能特徵尚未清楚，先前我們觀察到受脂多醣 (lipopolyssacharide,LPS)刺激的小鼠巨噬細胞RAW264.7中，Zfp36l2 mRNA的表現降低，在RNA pull-down assay中證實了藉由NFкB路徑誘發Ttp的表現並結合到Zfp36l2的ARE而導致在LPS刺激下Zfp36l2 mRNA的不穩定。此外我們也利用deletion construct研究ZFP36L2蛋白質上的功能分析，發現全長的蛋白質(WT Zfp36l2)均勻分布於細胞質中，去掉C端( Zfp36l2-N-TZF)或去掉N端(Zfp36l2-TZF-C)的蛋白會改變其在細胞內的位置，Zfp36l2-TZF-C偏向累積在核膜外圍，而Zfp36l2-N-TZF會在處理小體 (processing body)形成聚集的點狀物。用luciferase assay則發現Zfp36l2的N端是主要降低基因表現功能域。另外，我們利用lentivirus-mediated RNAi發現在小鼠巨噬細胞內MKP-1 (MAPK phosphatase-1)訊息RNA是ZFP36L2的目標之一，MKP-1在受到脂多醣 (LPS)刺激時會被誘導進而去抑制MAPK pathway而降低innate immune response，我們觀察到在短時間內細胞再受到LPS第二次刺激時Zfp36l2的表現量下降，而MKP-1的表現量更提升，可能使得下游inflammatory cytokine無法表現造成endotoxin tolerance。我們的結果顯示在脂多醣刺激的巨噬細胞中，Zfp36l2的表現量受到縝密的調控，以調節細胞的發炎反應。

Post-transcriptional control comprises regulation of the stability or translational efficiency of mRNA. Microarray analyses have revealed that 40-50% of changes in gene expression in response to cellular signals occur at the level of mRNA stability. Adenine and uridine -rich element mediated decay is one of several mechanisms the degrade mRNAs in the post-transcriptional regulations. AU-rich elements are always found in 3’UTR of a variety of transcripts that encode immediate response genes such as cytokines and inflammatory mediators, like proto-oncogenes and transcription factors. TTP family proteins bind to ARE of target mRNA by zinc finger domain and promote degradation. There are four members belong to TTP family: TTP (ZFP36), ZFP36L1, ZFP36L2 and ZFP36L3 which is specifically expressed in the placenta and extraembryonic tissues. Currently, the gene regulation and functional characterization of Zfp36l2 are unclear. We observed that Zfp36l2 mRNA expression was decreased in LPS-stimulated RAW264.7 cells. RNA pull down assay have improved that the downregulation of Zfp36l2 mRNA was caused by TTP binding to its ARE, which was induced by NFкB signaling pathway in LPS-stimulated RAW264.7 cells. On the other hand, we have identified two mRNA targets of Zfp36l2, Cox2 and Mkp-1 mRNA, in resting RAW264.7 cells. To investigate the molecular mechanism of Zfp36l2-specific Cox2 mRNA downregulation, we characterized the functional domains of Zfp36l2 by using some deletion constructs. In Immunofluorescence staining, WT- Zfp36l2 dispersed in the cytoplasm evenly, whereas Zfp36l2-N-TZF formed distinct foci and located at processing body, and Zfp36l2 –TZF-C tended to gather at the nuclear membranes. The luciferase reporter analysis showed that N-terminus of Zfp36l2 is required for its mRNA downregulation activity. In addition, we observed that the LPS treatment would lead to a lasting decrease of Zfp36l2 expression for more than 16 h, which might cause a higher induction of anti-inflammatory Mkp-1and IL-10 mRNA and a lower induction of pro-inflammatory cytokine TNFα in response to the second LPS treatment. Our results indicate that the expression levels of Zfp36l2 are tightly controlled to regulate intracellular inflammatory response in LPS-stimulated macrophages.