張瀞仁臺灣大學:生化科學研究所江玉雯Jiang, Yu-WunYu-WunJiang2010-05-042018-07-062010-05-042018-07-062009U0001-2001200902301200http://ntur.lib.ntu.edu.tw//handle/246246/178858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。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.口試委員會審定書謝文摘要文摘要. INTRODUCTIONumor necrosis factor signaling...1FκB...3DAC inhibitors...5yclooxygenase-2 (COX-2)...6U-rich element (ARE)-mediated mRNA stability regulation...7ristetraprolin (TTP)...9I. MATERIALS AND METHODSell culture...13eagents for cell treatment...13lasmids and constructs...14ite-directed mutagenesis...15NA isolation...16eal-time PCR...17ransient transfection, luciferase and galactosidase assays...17reparation of cytosolic and nuclear extracts and Western blotting assay...18lectrophoretic mobility shift assay (EMSA)...19hromatin-immunoprecipitation (ChIP)...20NA pull-down assay...22II. RESULTSart ISA suppressed TNF-α induced COX-2 mRNA expression in a cell type-specific manner...23oth HDAC inhibitors, TSA and sodium butyrate impaired COX-2 production...24FκB inhibitor had a similar effect as TSA in suppressing COX-2 mRNA expression...25ox-2 promoter contained a functional NFκB element...26SA did not alter NFκB expression and nucleocytoplasmic shuttling during TNF-α activation...27SA did not alter NFκB DNA binding ability during TNF-α activation...28SA suppressed COX-2 activation by inhibiting polymerase II enlongation...29uppression of polymerase elongation by TSA was not due to alternation of the interaction between NFκB and P-TEFb...31art IIhe expression profile of TTP during TNF-α stimulation of RAW264.7...32apid change of TTP mRNA stability in the course of TNF-α stimulation...33RE as an essential element on ttp 3’UTR in regulating TTP expression...34118N mutant was created to monitor the possibility of TTP negative autoregulation...36TP AREs mediated the reduction of reporter activity...38etection of TTP ARE-binding proteins...39V. DISCUSSIONart Ieduction of COX-2 expression was due to TSA-induced pre-mature transcription...40SA may modulate signaling transduction pathways...41SA suppressed TNF-α induced COX-2 mRNA expression in a cell type-specific manner...42SA-induced initiation of cox-2 transcription is cell type-specific...43SA did not alter NFκB activity in TNF-α stimulated NIH3T3 cells...44nhibition of COX-2 basal level expression by TSA...46mpaired COX-2 production by TSA and sodium butyrate but not by Apicidin or Scriptaid...46art IIhe stability of TTP mRNA changes rapidly in response to TNF-α stimulation...48TP ARE as a sufficient regulatory target of mRNA stabilization...48ossible signaling transduction pathways involved in the stability of TTP mRNA...49etection of destabilizing or stabilizing proteins associated with TTP AREs...51. FIGURESigure 1. TNF-α and COX-2 mRNA expression profile in response to TNF-α and TSA...52igure 2. Effects of various HDAC inhibitors on TNF-α-induced COX-2 mRNA expression in NIH3T3 cells...54igure 3. Effects of BAY on TNF-α-induced COX-2 mRNA expression in NIH3T3 cells...55igure 4. Analysis of cox-2 promoter activity in TSA-treated and TNF-α-stimulated NIH3T3 cells...56igure 5. NFκB expression and subcellular localization of TSA treated and TNF-α-stimulated NIH3T3 cells...57igure 6. DNA binding activity of NFκB on murine cox-2 promoter...58igure 7. Association of pol II on exon 1, 2, and 10 of the cox-2 gene...60igure 8. NFκB and P-TEFb association in TSA-treated and TNF-α-stimulated NIH3T3 cells...61igure 9. TTP mRNA and protein expression of TNF-α stimulated RAW264.7...62igure 10. TTP mRNA stability in TNF-α-stimulated RAW264.7 cells...64igure 11. Ectopic expression of TTP constructs...65igure 12. RNA pull-down assay...69igure 13. Functional characterization of TTP AREs...70igure 14. Identification of TTP ARE-associated protein by RNA pull-down...71I. SUPPLEMENTAL DATA AND TABLESupplemental data 1. Subcellular localization of HuR...72upplemental data 2. COX-2 mRNA stability in response to TSA in NIH3T3 cells...73able 1. Histone deacetylase inhibitors...74II. REFERENCES...75III. ABBREVIAION AND CHEMICAL SYMBOLS...82application/pdf3249737 bytesapplication/pdfen-US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TNF-αcyclooxygenase-2tristetraprolinhistone deacetylase inhibitorAU-rich elementhttp://ntur.lib.ntu.edu.tw/bitstream/246246/178858/1/ntu-98-R95b46036-1.pdf