呂勝春 博士臺灣大學：生化科學研究所陳俞伶Chen, Yu-LingYu-LingChen2007-11-262018-07-062007-11-262018-07-062007http://ntur.lib.ntu.edu.tw//handle/246246/52774Tristetraprolin (TTP)是一個可以結合到一些發炎相關基因的mRNA 上，而使之降解的蛋白質。其中最重要同時也被廣泛討論的是它可以造成TNF-α的mRNA 不穩定而使得發炎反應在細胞中得到適度的緩解。然而如何調控對於如此重要的發炎負調節者TTP 的轉錄作用及其表現的相關研究卻付之闕如。因此，本論文利用抑制不同訊息傳導之抑制劑來探討TTP 以及其下游基因TNF-α如何被調控。發現利用BAY 抑制NF-κB、MG132 抑制proteasome、SB203580 抑制p38 pathway 等效果皆會抑制由脂多醣引發之TTP mRNA 以及蛋白質表現。藉由ChIP 實驗證實，在脂多醣刺激巨噬細胞過程中NF-κB 這個轉錄因子可以結合到TTP 基因位於-1838~1859 的啟動子位置上。 而TTP 的啟動子活性也可被SB203580 抑制，表示p38 pathway 參與TTP 的轉錄控制。 此外在脂多醣不同時間刺激下，發現p38 pathway 對影響TTP mRNA 的穩定性有所差異。因此這些結果表示p38 pathway 能夠藉由調節轉錄以及後轉錄(post-transcription)等層面來調節TTP 的表現。另一方面，在LPS 刺激巨噬細胞中分析各種抑制劑的效果中發現TSA 這個藥物能夠很劇烈的減低TNF-α mRNA 的表現。TSA 帶有抑制HDAC酵素活性的特性，使得chromatin 結構變的鬆散而有助於轉錄作用進行。然而本論文藉由luciferase reporter 的實驗結果顯示，TSA 卻能抑制TNF-α啟動子之活性。因此進一步分析調節TNF-α啟動子上的重要轉錄因子NF-κB 之活性來深入探討是否參與其中之機制。有趣的發現是處理TSA 能夠抑制NF-κB 結合到TNF-α的啟動子上，卻不會影響NF-κB 進入到細胞核內、也不影響TNF-α mRNA 的穩定性。綜合上述，我們的實驗結果提供更多的證據來了解NF-κB 及p38 pathway 利用轉錄以及後轉錄等不同層面來調控在脂多醣刺激巨噬細胞中誘發之TTP 以及TNF-α表現。Tristetraprolin (TTP) is an mRNA-destabilizing protein that negatively regulates the expression of proinflammatory mediators such as TNF-α. However, the mechanism of transcriptional regulation of TTP remains ill defined. Here we investigate the regulation of TTP as well as TNF-a expression in the mouse macrophage cell line RAW264.7. We found that pharmacological inhibition of NF-κB (BAY), proteasome (MG132) and p38 pathway (SB203580) resulted in downregulation of LPS-induced TTP mRNA and protein expression. A novel NF-κB binding element located within -1838 to -1859 relative to TTP transcription start site was identified and confirmed by ChIP experiments. Functional analysis using luciferase reporter assay demonstrated that TTP promoter activity was suppressed by SB203580 treatment. The half-life of TTP mRNA was also decreased by SB203580 treatment. These data suggest that p38 pathway regulates TTP expression at both transcriptional and post-transcriptional levels. Furthermore, the HDAC inhibitor, TSA, decreases the LPS-stimulated TNF-α mRNA expression. TSA is a general inhibitor of HDAC enzyme activity that results in chromatin structure loosening and transcriptional upregulation. In contrary, our data showed that TSA reduced the TNF-α promoter activity by luciferase reporter analysis. Interestingly, TSA disrupts NF-κB recruitment to TNF-α promoter without affecting NF-κB nuclear localization. Taken together, these results suggest that TTP mRNA transcription may be regulated by NF-κB while p38 signaling could modulate both TTP and TNF-α expression at both transcriptional and post-transcriptional levels during LPS stimulation.口試委員會審定書 中文摘要 英文摘要 I. INTRODUCTION 1 1.1 TTP.2 1.2 TNF-alpha .5 1.3 NF-κB .5 II. MATERIALS AND METHODS 2.1 Plasmids constructs.8 2.2 Cell culture.8 2.3 Preparation of antibodies.9 2.4 RNA isolation and RT-PCR.9 2.5 Real-time PCR.10 2.6 Preparation of cytoplasmic and nuclear extracts and Western blotting assay.10 2.7 Chromatin-immunoprecipitation (ChIP) ssay.11 2.8 Transfection, luciferase, and beta-galactosidase assay.12 2.9 Incorporation of biotin into RNA .13 2.10 RNA pull-down assay.14 2.11 In-gel digestion of gel-separated proteins, Peptide identification by mass spectrometry and bioinformatics analysis.15 III. RESULTS 3.1 The expression profile of TTP and TNF-α mRNA during LPS stimulation.16 3.2 The mRNA levels of TTP were suppressed by treatment with MG132, BAY and SB203580 .16 3.3 The expression of TNF-α mRNA were inhibited by TSA .17 3.4 The protein levels of TTP were suppressed by MG132, BAY and SB203580.17 3.5 The time frame of the MG132 effect on the production of TTP and mRNA accumulation in LPS-stimulated RAW264.7 macrophages .18 3.6 The mRNA stability of TTP in LPS-stimulated macrophages in the absence and Presence of MG132 .18 3.7 The effects of NF-κB activation on TTP mRNA accumulation in LPS-stimulated RAW264.7 cells.19 3.8 Identification of the putative NF-κB binding element in the TTP promoter ..19 3.9 The effects of p38 signaling pathway on TTP gene expression.21 3.10 SB203580 modulates TTP and its downstream target, TNF-α mRNA stability.21 3.11 Transcriptional regulation of TTP promoter by the p38 pathway .22 3.12 Activation of TTP expression by arsenite treatment.22 3.13 Identification of TTP-ARE binding proteins .23 3.14 Both HDAC inhibitors, TSA and sodium butyrate impair TNF-α production .24 3.15 Neither TSA nor sodium butyrate affect TNF-α mRNA stability..25 3.16 TSA treatment does not alter NF-κB expression and subcellular localization during LPS stimulation.25 3.17 TSA treatment reduced TNF-α -promoter activity driven by LPS stimulation.26 3.18 TSA treatment disrupts the in vivo association of NF-κB with the promoter of TNF-α in LPS-stimulated RAW264.7 cells .26 IV. DISCUSSION 4.1 Differential regulation of TTP expression by the NF-κB and p38 signaling pathways during LPS induction.28 4.2 A novel NF-κB binding site within 5’ promoter region of the ttp gene was identified .29 4.3 TTP expression was modulated by the p38 signaling pathway at both transcriptional and post-transcriptional levels .30 4.4 Heat shock proteins were associated with TTP-ARE ..31 4.5 Reduction of TNF-α expression by TSA can be attributed to down-regulated NF-κB binding to promoter .33 4.6 TTP and TNF-α expression was coordinated by NF-κB and p38 signaling pathway at both transcriptional and post-transcriptional levels ………….…….37 V. REFERENCES .38 VI. FIGURES Figure 1. Effects of various inhibitors on LPS-induced TTP mRNA expression in macrophages..47 Figure 2. Effects of various inhibitors on LPS-induced TNF-α mRNA expression in RAW264.7 cells.49 Figure 3. Effects of various inhibitors on TTP protein expression in LPS stimulated macrophages.50 Figure 4. The effect of MG132 on kinetics of TTP protein and mRNA expression in LPS stimulated macrophages.51 Figure 5. MG132 does not alter TTP mRNA stability in LPS-stimulated macrophages.52 Figure 6. Induction of TTP mRNA expression by recombinant TNF-α.52 Figure 7. NF-κB family members were recruited to TTP promoter by LPS treatment of RAW264.7 cells..53 Figure 8. Expression of multiple, phosphorylated forms of TTP could be inhibited by adding p38 inhibitor..54 Figure 9. TTP mRNA half-life were reduced by SB203580 treatment in LPS -stimulated macrophages.55 Figure 10. SB203580 treatment suppresses TTP promoter activity in LPS -stimulated macrophages.56 Figure 11. P38 MAPK signaling pathway modulates TTP expression in RAW264.7 macrophages.56 Figure 12. TTP-ARE binding protein identification.58 Figure 13. The time frame of the effect of TSA and sodium butyrate on TNF-α mRNA expression in LPS-stimulated macrophages.59 Figure 14. Effects of TSA and sodium butyrate on the turnover of TNF-α mRNA in macrophages stimulated with LPS..60 Figure 15. NF-κB expression and subcellular localization were not influenced by TSA treatment in LPS-stimulated macrophages.61 Figure 16. TSA treatment reduces LPS-stimulated TNF-α -promoter activity..62 Figure 17. TSA disrupts the interaction between p50 and the TNF-α promoter in LPS-stimulated RAW264.7 cells.63 Figure 18. Coordinative expression of TTP and TNF-α in transcriptional and post-transcriptional processes .64 VII. TABLES Table 1. Analysis of mouse TTP promoter sequence .65 Table 2. Sequence of murine TNF-α gene promoter..66 Table 3. Inhibitors of certain signaling pathways on LPS-induced TTP expression.67 Table 4. Identification of TTP-ARE binding proteins by mass spectrometry analysis .68 VIII. ABBREVIATION AND CHEMICAL SYMBOLS .701994703 bytesapplication/pdfen-US脂多醣巨噬細胞lipopolysaccharidemacrophageTTPTNF-alphaotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52774/1/ntu-96-D89242001-1.pdf