第一部分：Tristetraprolin 藉由與PABPN1 的交互作用抑制細胞核內帶 有多AU 序列的訊息核醣核酸的聚腺苷酸化 第二部分：小鼠巨噬細胞 RAW264.7 中以脂多醣誘發Mkp-1 表現的轉 錄調控

Abstract

第一部分： 已知TTP藉由與多AU序列的結合導致成熟訊息核醣核酸的不穩定，本實驗室先前利用phage-display尋找與TTP交互作用的蛋白質來進一步瞭解TTP的作用機制。phage-display數據顯示PABPN1與TTP有交互作用。PABPN1可以幫助訊息核醣核酸的聚腺苷酸化 (polyadenylation)並增加聚腺苷酸化酶 [Poly(A) polymerase]對聚腺苷酸的親和性。聚腺苷酸化在訊息核醣核酸由細胞核到細胞質的運輸、本身穩定度以及轉譯效率的維持都扮演重要角色。本論文使用共同免疫沉澱法確認TTP與PABPN1的交互作用，雖然TTP與PABPN1都存在於細胞質與細胞核，但兩者的交互作用只在細胞核內。若在細胞內表現侷限在細胞核內的突變TTP，可以觀察到此突變TTP具有降低帶有腫瘤壞死因子的多AU序列的報導基因的表現蛋白酵素效力降低。有趣的是，在試管中TTP被發現藉由與PABPN1交互作用造成帶有多AU序列的訊息核醣核酸的聚腺苷酸化程度降低。當在小鼠巨噬細胞RAW264.7中以脂多醣誘導腫瘤壞死因子的表現，可以觀察到在細胞核內被誘導出腫瘤壞死因子的訊息核醣核酸的聚腺苷酸化程度的降低與被誘導表現增加的TTP具有關連性。結論是，TTP除了已知在細胞質內造成帶有多AU序列的訊息核醣核酸的降解功能，TTP在細胞核內藉由與PABPN1的交互作用來調節帶有多AU序列的訊息核醣核酸的表現。 第二部分： 免疫反應仰賴於MAPK訊息傳遞，特別是p38與Jnk。這些激酶會經由原發炎促進劑刺激細胞而受到活化，並誘發許多免疫調節基因的表現進而引起發炎或免疫反應。p38與Jnk會經由DUSP的其中一員, Mkp-1, 將其去磷酸化而喪失活性。近年報導提出Mkp-1的確在免疫反應中的有重要的調節功能。本論文中發現在以脂多醣刺激的小鼠巨噬細胞RAW264.7中Mkp-1的訊息核醣核酸的表現在三十至六十分鐘被快速活化，接著在刺激兩小時後又降低不到誘發最多量的一半。在此Mkp-1的訊息核醣核酸的增多與減少是受到何種轉綠因子與訊息傳遞路徑調控將在本論文中被探討。發現屬於Atf/Creb家族蛋白的Creb1與Atf3會與Mkp-1基因的啟動子 (promoter)結合並會增加帶有Mkp-1啟動子的報導基因的產物酵素的活性。若在RAW264.7中造成Atf3的基因靜默，可以觀察到Mkp-1訊息核醣核酸表現降低。因此，Creb1與Atf3會正向調節Mkp-1的表現。另外，在脂多醣刺激兩小時後的RAW264.7中可以觀察到PI3K訊息傳遞路徑的活化，已知該訊息途徑會負向調節TLR4的訊息途徑。當細胞內存在PI3K訊息途徑的抑制子，Wortmannin時，Mkp-1的訊息核醣核酸量會提升。藉由微陣列矩陣分析與即時聚合酶反應，Id3被鑑定為PI3K下游的轉錄因子來負向調節Mkp-1核醣核酸的表現。未來將需要更多實驗證明在脂多醣刺激的RAW264.7中Id3調節Mkp-1核醣核酸表現的分子機制。

Part I Tristetraprolin (TTP) binds mRNA AU-rich elements (ARE) and thereby facilitates the destabilization of mature mRNA. To understand how TTP mechanistically functions, previously our lab biopanned with a phage-display library for proteins that interact with TTP and retrieved, among others, poly(A) binding protein nuclear 1 (PABPN1). PABPN1 assists in the 3’-polyadenylation of mRNA by binding to an immature poly(A) tail and increasing the affinity of poly(A) polymerase, which is directly responsible for polyadenylation. The poly(A) tail is important for mRNA export from the nucleus, mRNA stabilization, and translation efficiency. The TTP/PABPN1 interaction was characterized using co-immunoprecipitation assays. Although TTP and PABPN1 are located in both the cytoplasm and the nucleus, they interacted in vivo only in the nucleus. Expression of a TTP mutant restricted to the nucleus resulted in the downregulation of a TNFα ARE-containing luciferase activity .Interestingly, we found that TTP binds PABPN1 and thereby inhibits polyadenylation of ARE-containing mRNA in vitro. When TNFα mRNA was induced in mouse RAW264.7 cells by lipopolysaccharide treatment, synthesis of a shorter poly(A) tail in nuclear TNFα mRNA and an increased expression of TTP occurred. Consequently, in addition to its known cytosolic mRNA-degrading function, TTP inhibits poly(A) tail synthesis by interacting with PABPN1 in the nucleus to regulate expression of ARE-containing mRNA. Part II Innate immune responses rely on the MAPK signaling pathway, especially p38 and Jnk. These kinases are activated by pro-inflammatory agonists, and lead to inflammatory or innate immune responses by regulating the expression of several effector genes. Both p38 and Jnk are negatively regulated through their dephosphorylation by MAPK phosphatase 1 (Mkp-1), a member of dual specificity protein phosphatase (DUSP). Recent reports supported that Mkp-1 is a critical regulator of innate immunity. Previously, we have demonstrated that the mRNA of Mkp-1 was rapidly elevated around 30 to 60 minutes and soon decreased less than half after LPS stimulation for 2 h in RAW264.7 cells. Here we concerned with the molecular mechanism in control of the growth and decline of Mkp-1 mRNA at transcriptional level. Creb1 and Atf3, members of the Atf/Creb family protein, were shown to associate with the Mkp-1 promoter and increase the activity of luciferase reporter that carrying the Mkp-1 promoter. Knockdown of Atf3 in RAW264.7 cells resulted in Mkp-1 mRNA downregulation. Therefore, Creb1 and Atf3 regulate Mkp-1 expression positively. The PI3K signaling pathway, which was activated after LPS stimulation for 2 h, was suggested to play an opposite role to restrict the TLR4 signaling. The Mkp-1 mRNA level was increased in the presence of the PI3K signaling inhibitor, Wortmannin. Using the microarray analysis and real time PCR, the Id3 was identified and confirmed for the candidates that downstream of PI3K signaling to regulate Mkp-1 expression. Further studies are needed to examine the function of Id3 and the molecular mechanism of regulating Mkp-1 expression in LPS-stimulated RAW264.7 cells.