腫瘤壞死因子TNFα誘發基因表現之機轉 Part I: 組蛋白去乙醯化酵素抑制劑trichostatin A對環氧化酵素COX-2基因表現的抑制作用 Part II: RNA結合蛋白tristetraprolin的後轉錄調節

The mechanism of TNFα-induced gene expression Part I: HDAC inhibitor trichostatin A-mediated inhibition of cyclooxygenase 2 gene expression Part II: The post-transcriptional regulation of RNA-binding protein tristetraprolin

江玉雯(Yu-Wun Jiang)

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(2009年)

https://doi.org/10.6342/NTU.2009.02101

摘要

腫瘤壞死因子(Tumor necrosis factor-α, TNF-α)是一個免疫系統中的細胞激素，此細胞激素也能夠刺激急性反應的發生。我們研究了二個被TNF-α所誘導表現的基因的調控機制：環氧化酵素(cyclooxygenase-2, COX-2)以及Tristetraprolin (TTP)。我們觀察到在NIH3T3細胞中，COX-2 mRNA可以被TNF-α所誘導表現，並且TNF-α的誘導效果能夠被組蛋白去乙醯化酵素抑制劑TSA所抑制。除此之外，NFκB抑制劑BAY也具有和TSA相同的抑制效果，因此我們推測NFκB signaling pathway可能對COX-2的調控有重要影響。但是實驗結果卻發現NFκB 在細胞核與質的分佈或者與DNA的結合能力都不受TSA影響。最後藉由chromatin immunoprecipitation的方法知道TSA是作用在抑制polymerase II進行cox-2基因轉錄的elongation階段，至於詳細的分子機制仍需進一步的探討。另一個研究的主題是TTP mRNA穩定性的調控機制:當細胞受到TNF-α刺激時，TTP mRNA的半衰期會短暫地增長使mRNA能夠累積表現，進一步的研究後知道這個變化是在post-transcriptional level受到3’ untranslated region上AU-rich element的調控。我們會再進一步了解這個調控方式是透過何種訊息傳導來達成。

關鍵字

腫瘤壞死因子 ； 環氧化酵素 ； 核酸結合蛋白TTP ； 組蛋白去乙醯化酵素抑制劑 ； AU富含區

並列摘要

Tumor necrosis factor (TNF)-α is a cytokine involved in systemic inflammation, and is a member of a group of cytokines that stimulate the acute phase reaction. In this study, we focus on the regulatory mechanism of two TNF-α induced genes, cyclooxygenase-2 (COX-2) and Tristetraprolin (TTP). Using NIH3T3 cell line as a model, we found that COX-2 mRNA was activated by TNF-α treatment, and histone deacetylase inhibitor (HDACi) TSA could significantly block COX-2 activation. In addition, TNFα induced COX-2 expression could be inhibited by NFκB inhibitor BAY to a similar level as TSA. This indicated that NFκB signaling pathway may play an important role in modulating COX-2 expression. However, effects of TSA were not on NFκB nucleocytoplasmic distribution or DNA-binding ability. Results of chromatin immunoprecipitation (ChIP) assay revealed that TSA impaired COX-2 mRNA production by suppressing polymerase II elongation on the cox-2 gene. Further investigation on the molecular mechanism of this action would help to understand how HDACi suppressed gene expression. Another focus of this study is about the transient stabilization of TTP mRNA in response to TNF-α stimulation. We investigated the role of 3’untranslated region (UTR) in the regulation mechanism of TTP, and we found that the AU-rich element (ARE) was crucial for TTP expression modulation in the post-transcriptional level. Nevertheless, related works are still ongoing to explore the signaling cascade involved in transient TTP mRNA accumulation.

並列關鍵字

TNF-α ； cyclooxygenase-2 ； tristetraprolin ； histone deacetylase inhibitor ； AU-rich element

參考文獻

1. Chen, G. and D.V. Goeddel, TNF-R1 signaling: a beautiful pathway. Science, 2002. 296(5573): p. 1634-5.

2. Wajant, H., K. Pfizenmaier, and P. Scheurich, Tumor necrosis factor signaling. Cell Death Differ, 2003. 10(1): p. 45-65.

3. Dean, J.L., et al., The involvement of AU-rich element-binding proteins in p38 mitogen-activated protein kinase pathway-mediated mRNA stabilisation. Cell Signal, 2004. 16(10): p. 1113-21.

4. Ghosh, S. and M. Karin, Missing pieces in the NF-kappaB puzzle. Cell, 2002. 109 Suppl: p. S81-96.

5. Clark, A.R., J.L. Dean, and J. Saklatvala, Post-transcriptional regulation of gene expression by mitogen-activated protein kinase p38. FEBS Lett, 2003. 546(1): p. 37-44.