巨噬細胞中RNA結合蛋白HuR和TRISTETRAPROLINE對細胞激素RNA穩定性的調控之研究

Abstract

訊息RNA的穩定性是真核細胞調控基因表現的一個主要機制，因而影響細胞的生長和分化。AU-rich elements (AREs)位在許多原致癌基因、細胞激素以及一些生長因子的mRNA之3’untranslated regions當中，可能是使它們自己的mRNAs快速降解的目標序列。HuR是RNA結合蛋白當中ELAV family的一個成員，它的表現非常廣泛；在短暫轉殖的細胞，HuR會結合到AREs上，並且使含有ARE的mRNAs變的穩定。Tristetraproline（TTP）是一個immediate-early gene，它可以結合到AREs引起RNA的不穩定。為了要研究ARE-containing mRNAs穩定性的調控機制，因此我們在LPS刺激的巨噬細胞中，完成了TNFα和IL-1β mRNAs半衰期的表現和調控機制的實驗。泳動阻滯電泳分析（EMSA）顯示由LPS誘導的去穩定因子TTP可以以較高的親和力結合到TNFα ARE上，而對IL-1β ARE的親和力則較低。HuR被發現可以和TNFα ARE相互作用來穩定它的RNA。有趣的是，由LPS誘導的IL-1β mRNA穩定性是p38 signaling pathway-dependent，而TNFα mRNA的穩定性則是p38 signaling pathway-independent。p38 pathway的活化導致TTP磷酸化並且降低它和RNA結合的活性。相對於TNFα的ARE，p38的訊息可以取消TTP在IL-1β ARE上的抑制作用。我們的結果指出TTP可以反應p38的訊號來調節IL-1β等ARE-containing mRNA的表現。

Messenger RNA stability is one of the key mechanisms that eukaryotic cells regulate gene expression and influence cell growth and differentiation. AU-rich elements (AREs) present in the 3’ untranslated regions of mRNAs from many protooncogenes, cytokines, and growth factors may be targets for rapid degradation. HuR, a ubiquitous expressed member of the ELAV family of RNA binding proteins, selectively binds to AREs and stabililizes ARE-containing mRNAs in transiently transfected cells. Tristetraproline (TTP) is an immediate-early gene that could bind to AREs and trigger RNA destabilization. To investigate the regulation of stability of ARE-containing mRNAs, we performed experiments on the expression and regulation of half-life of TNFα and IL-1β mRNAs in LPS-stimulated macrophages. Electrophoretic mobility shift assays showed that the LPS-induced destabilization factor TTP could bind to TNFα ARE much better than that of IL-1β ARE. HuR was found to interact with TNFα ARE to stabilize its RNA. Interestingly, LPS-induced stability of IL-1β mRNA was p38 signaling pathway-dependent while that of TNFα mRNA was p38 signaling pathway-independent. Activation of p38 pathway resulted in the phosphorylation of TTP and decrease of its RNA-binding activity. Contrary to ARE of TNFα, p38 signal could reverse the inhibitory activity of TTP on IL-1β ARE. Our results indicate that TTP could respond to p38 signal to modulate the expression of specific ARE-containing mRNAs such as IL-1β.