Tristetraprolin家族蛋白在小鼠巨噬細胞功能之研究

Functional Characterization of Tristetraprolin Family Proteins in Mouse Macrophages

王冠婷(Kuan-Ting Wang)

指導教授 ： 張瀞仁

國立臺灣大學/生命科學院/生化科學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.02576

摘要

先天免疫反應的基因表達受到嚴密調控。絲裂原活化蛋白激酶（MAPK）在先天免疫反應中扮演關鍵腳色，它透過磷酸化下游轉錄因子和RNA結合蛋白，可活化炎性細胞因子（proinflammatory cytokines）的合成。MAPK磷酸酶（MKP）藉由去磷酸化作用使MAPK失去活性。目前研究指出Mkp-1是一個重要的負調控因子，它可以關閉炎性細胞因子的生產。我們已經證明Tristetraprolin（Ttp）可以藉由與具有特殊的多腺嘌呤-尿嘧啶序列（AU-rich element, ARE）的Mkp-1 mRNA結合，並透過後轉錄機制將Mkp-1 mRNA降解。TTP家族包含三個主要成員，Ttp，Zfp36l1和Zfp36l2。本論文的目標即在了解其他TTP家族蛋白在先天免疫反應中扮演的角色。首先我們觀察TTP家族蛋白的mRNA與蛋白質表達。在受到脂多醣（Lipopolyssacharide, LPS）刺激的老鼠巨噬細胞RAW264.7中，TTP的mRNA和蛋白質被高度誘導，但Zfp36l1和Zfp36l2的mRNA表現降低，而蛋白質表現則保持一致。利用Zfp36l1和Zfp36l2的knockdown分析，我們發現兩個受到Zfp36l1和Zfp36l2調控的目標mRNA：Mkp-1和Cox-2。減少Zfp36l1和Zfp36l2的表現，可延長目標mRNA的半衰期，進而使目標mRNA增加。當細胞內Mkp-1的表達增加時，會抑制p38 MAPK的活性，使老鼠巨噬細胞對LPS刺激的敏感度下降。此外，我們還發現高度磷酸化的Zfp36l1會與支架蛋白14-3-3結合，並使Mkp-1 mRNA不被降解。綜合上述，我們的研究結果顯示Zfp36l1和Zfp36l2的表現與磷酸化修飾，可調控老鼠巨噬細胞RAW264.7受脂多醣刺激時，Mkp-1 mRNA的表現，這也說明Zfp36l1和Zfp36l2是先天免疫反應中重要的調控因子。

關鍵字

mRNA 穩定性 ； Tristetraprolin ； 先天免疫反應 ； 磷酸化 ； 絲裂原活化蛋白激酶

並列摘要

Gene expressions are tightly controlled in the innate immune response. Mitogen-activated protein kinases (MAPKs) play critical roles in the innate immune response through phosphorylating downstream transcription factors and RNA binding proteins to elicit the biosynthesis of proinflammatory cytokines. Inactivation of MAPKs is done by MAPK phosphatases (MKPs) through dephosphorylation. The previous studies strongly suggested that Mkp-1 was a critical negative regulator for switching off the production of proinflammatory cytokines. We had demonstrated that Mkp-1 mRNA containing AU-rich element (ARE) was post-transcriptionally regulated by an ARE-binding protein Tristetraprolin (Ttp). The TTP family contains three major members, Ttp, Zfp36l1 and Zfp36l2. To examine whether other family proteins also play roles in the innate immune response, their expression profiles were determined. The mRNA and protein of Ttp were highly induced by Lipopolyssacharide (LPS) in mouse macrophage RAW264.7 cells, whereas the mRNAs of Zfp36l1 and Zfp36l2 were down-regulated and their proteins were maintained in the consistent levels in the period of LPS-stimulation. By knockdown analysis, we found that Mkp-1 and Cyclooxygenase-2 (Cox-2) were the mRNA targets of Zfp36l1 and Zfp36l2 in the resting condition. Knockdown of Zfp36l1 and Zfp36l2 increased the basal levels of target mRNAs by prolonging their half-lives. Increasing the expression of Mkp-1 repressed the activity of p38 MAPK, and the sensitivity to LPS-stimulation was decreased. Furthermore, we found that hyper-phosphorylation of Zfp36l1 stabilized Mkp-1 expression by forming a complex with adapter protein 14-3-3. Our findings imply the expression and phosphorylation of Zfp36l1 and Zfp36l2 might play roles in modulating the mRNA level of Mkp-1 to control p38 MAPK activity in LPS-stimulation, and both Zfp36l1 and Zfp36l2 are important regulators in the innate immune response.

並列關鍵字

mRNA stability ； Tristetraprolin ； Innate immune response ； Phosphorylation ； Mitogen-activated protein kinase

參考文獻

[1] B. Varnum, Q. Ma, T. Chi, and B. Fletcher, “The TIS11 primary response gene is a member of a gene family that encodes proteins with a highly conserved sequence containing an unusual Cys-His repeat.,” Mol Cell Biol., vol. 11, no. 3, pp. 1754-1758, 1991.

[4] P. Blackshear, R. Phillips, and S. Ghosh, “Zfp36l3, a rodent X chromosome gene encoding a placenta-specific member of the Tristetraprolin family of CCCH tandem zinc finger proteins.,” Biol Reprod., vol. 73, no. 2, pp. 297-307, Aug. 2005.

[5] G. Taylor, E. Carballo, D. Lee, and W. Lai, “A pathogenetic role for TNF alpha in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency.,” J Immunol., vol. 4, no. 5, pp. 445-454, 1996.

[6] E. Carballo et al., “Bone marrow transplantation reproduces the tristetraprolin-deficiency syndrome in recombination activating gene-2 (-/-) mice. Evidence that monocyte/macrophage progenitors may be responsible for TNFalpha overproduction.,” J Clin Invest., vol. 100, no. 5, pp. 986-995, 1997.

[7] E. Carballo, “Feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin.,” Science., vol. 281, no. 5379, pp. 1001-1005, Aug. 1998.