呂勝春臺灣大學：分子醫學研究所蘇文琪Su, Wen-ChiWen-ChiSu2007-11-262018-07-092007-11-262018-07-092004http://ntur.lib.ntu.edu.tw//handle/246246/51375在發炎反應所誘發基因表現中，C/EBPβ是參與轉錄調控的重要因子之一。NF-κB 是另一個大家熟知，控制發炎細胞激素基因表現的主要因子。這兩個蛋白質均能在轉錄層級上協同地調控IL-6基因的表現。然而，正確調控IL-6基因表現的機制仍待釐清。本論文利用NF-κB抑制劑或減弱C/EBPβ內生性的表現量，我們證實這兩個蛋白質可能在IL-6基因調控上，會隨著細胞的差異而有不同的角色。在LPS的刺激下，NF-κB在RAW264.7細胞中扮演主要活化IL-6基因的角色，但在P388D1（IL1）細胞中，C/EBPβ則顯的較為重要。 C/EBPβ有三個isoforms，其中有兩個是轉錄活化因子（LAP* 和LAP），有一個是轉錄抑制因子（LIP）。我們除了說明C/EBPβ在誘發IL-6基因表現的角色；我們還提供證據支持細胞中的氧化還原狀態可能會控制其中一個C/EBPβisoform （LAP*）的轉錄活性。在還原狀態下，LAP*的DNA結合能力和轉錄活性可被引發。LAP*第11個氨基酸(即半胱氨酸)可能的功能是作為氧化還原狀態的分子探針並調控LAP*活化它的標的基因。C/EBPβ另兩個isoforms ：LAP和LIP，由於各別缺少前21和151個氨基酸而不能經由相似的氧化還原法來調節。總結我們所發現的證據提供在P388D1（IL1）細胞裡，主要是由C/EBPβ其中一個isoform： LAP*經氧化還原轉換來調控由LPS所誘 發的IL-6基因表現。C/EBPb is one of the key transcription factors responsible for the induction of genes involved in inflammatory response. NF-kB is another well-known crucial factor essential for controlling inflammatory cytokine genes expression. These two proteins have been reported to regulate IL-6 gene expression synergistically at the transcription level. However, the exact mechanism through which IL-6 expression is regulated remains uncharacterized. By treating cells with specific inhibitors of NF-kB or knock down the endogenous level of C/EBPb, we have demonstrated that these two proteins may have regulatory roles in IL-6 gene induction during inflammation in cell type-specific manner. In RAW264.7 cells, NF-kB plays predominant roles in activating IL-6 gene upon treatment with LPS while in p388D1(IL1) cells, C/EBPb is apparently more important than NF-kB. C/EBPb has three isoforms, including two transcription activators (LAP* and LAP) and one transcription repressor (LIP). In addition to demonstrating the role of C/EBPb in IL-6 gene induction, we provide evidence that the transcriptional activities of one of the C/EBPb isoforms, LAP*, may be governed by the redox state of the cell. The DNA binding activity and transcriptional activation of LAP* are induced under reducing condition. The 11th amino acid (Cys-11) of LAP* may function as a molecular sensor of redox state that regulates the activity of LAP* toward activating its target genes. The LAP and LIP isoforms of C/EBPb, lacking 21 and 151 amino acids, respectively, are not modulated in a similar redox-responsive manner. Taken together, our observations provide evidence that LAP* is the primary isoform of C/EBPb that regulates, through a redox switch, the LPS-induced expression of the IL-6 gene in P388D1 cells.Abbreviations and Chemical symbols……………………………..…..3 摘要………………………………………………………………………5 Abstract………………………………………………………………….7 I. Introduction…………………...……………………...………….……9 II. Materials and Methods Plasmids………………………………………………………………………..….16 Preparation of recombinant proteins and antibodies…………………………...….16 Cell culture, transfection and chloramphenicol acetyltransferase assay…………..17 Nuclear extract preparation and Western blot……………………………….…….19 Electrophoretic mobility shift assay…………………………………………….…19 Chromatin immunoprecipitation assay…………………………………………….20 RT-PCR analysis…………………………………………………………………...22 Indirect immunofluorescence staining…………………...………………………..22 Disulfide bond determination by liquid chromatography electrospray ionization mass spectrometry…………………………………..……………………………..23 III. Results Induction of C/EBPb and NF-kB in RAW264.7 cells stimulated with LPS…...…25 C/EBPb and NF-kB acquire DNA binding activity after LPS stimulation…..…...26 Specific recruitment of C/EBPb and NF-kB to IL-6 gene promoter region by LPS treatment of RAW264.7 and P388D1(IL1) cells…….…..………………..………27 Knockdown expression of endogenous C/EBPb by antisense RNA leads to reduced IL-6 gene expression in LPS-stimulated P388D1(IL1) cells……………...…… ..28 The functional roles of NF-kB for IL-6 gene induction in LPS-stimulated RAW264.7 cells………………………………………………….…………….….29 Redox modulation of intramolecular disulfide bonds of LAP* and its activation………………………………………………………………….………30 Trx/TxR system induces the DNA-binding activity of LAP*………..…….……..32 Antioxidant treatment could stimulate the transcriptional activity of LAP*…...…33 Identification of the intramolecular disulfide bonds of LAP*……….…..………..34 C11S and C33S mutants have higher DNA-binding and transcriptional activities than other cysteine mutants or wild-type LAP*……………………….……...…...35 IV. Discussion…………………..…………………………...………………….…37 V. References……………………………………………………………………...43 VI. Figures………………………………………………………………………...50en-US脂多醣 間白素-6 基因轉錄調控C/EBPb isoforms redox lipopolysaccharide Interleukother