轉錄因子Oct-2參與在脂多醣誘發巨噬細胞中iNOS啟動子活化的研究

Oct-2 transcription factor mediates in LPS-induced increase of iNOS promoter activity in macrophage

吳宗諭(Tsung-Yu Wu)

指導教授 ： 呂紹俊

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.00479

摘要

Inducible nitrogen synthase (iNOS) 是一個能被誘發產生一氧化氮的酵素，在免疫反應中，一氧化氮有幫助殺死外來微生物以及調控免疫反應等作用。在LPS誘導iNOS表現的過程中，需要其啟動子上-90 ~ +150區域中NF-κB及Octamer這兩個轉錄因子的結合區域。之前的研究主要對於NF-κB的參與有較深入探討，但對結合Octamer結合區的轉錄因子探討則較少。但根據 (Xie, 1997) 的研究，當這兩個區域其中任一被突變後，LPS只能誘發啟動子這兩個結合區未被突變時2~3%的活性，顯示結合到Octamer的轉錄因子在LPS誘發iNOS表現是不可或缺的，因此本論文的研究是探討在iNOS啟動子上與Octamer結合的轉錄因子。
與Octamer結合的轉錄因子有Oct-1及Oct-2；Oct-1是普遍存在於細胞中的，而Oct-2則被認為主要在B細胞及神經細胞中存在，但也有一些研究認為Oct-2可能在macrophage也有表現。我們以LPS誘發Raw264.7細胞發炎反應，發現Oct-2會隨著LPS的劑量以及作用時間增長而增加其表現量，但是Oct-1的表現量則略有下降，另外iNOS的mRNA以及蛋白質也都會被LPS作用而增加表現。從Balb/c小鼠的腹腔巨噬細胞分析也發現到Oct-2的表現，而且其表現量也會受LPS刺激而增加。將iNOS的啟動子接到會表現Luciferase的表現載體上，並且分別與Oct-1和Oct-2表現載體一起轉染到細胞中，LPS促進iNOS啟動子活化的作用都明顯的提高。另外利用293T細胞，以上述具有啟動子的載體加上NF-κB的次單元體，分別與Oct-1和Oct-2一起轉染，可以看到Oct-2的效果會比Oct-1來的強，顯示Oct-2可能參與iNOS的基因轉錄，而且在轉染p65時的活化效果就已達到p50和p65一起轉染時的程度。為了進一步探討Oct-1或是Oct-2在LPS促進iNOS啟動子的活性所扮演的角色，使用RNAi的方式分別降低Oct-2及Oct-1的表現，在LPS刺激之後觀察iNOS的表現。結果發現，當Oct-2被降低表現之後，LPS促進iNOS mRNA的表現明顯的減少了許多，降低Oct-1表現時iNOS的表現沒有受到影響，此結果與啟動子活性分析結果不同的原因，可能是因為Oct-2才是在細胞核中能與染色質體結合的轉錄因子。為了證實Oct-2能結合到iNOS啟動子DNA上，在DNA affinity precipitation assay以及Chip (chromatin immunoprecipitation) assay顯示在LPS刺激之後，可以偵測到較多的Oct-2以及NF-κB次單元體結合到iNOS啟動子上，顯示Oct-2確實能結合到iNOS啟動子DNA上。所以綜合以上結果，在LPS誘導iNOS表現時，是需要Oct-2，而不是Oct-1，參與在其啟動子活化過程中。

關鍵字

巨噬細胞 ； 轉錄因子 ； iNOS ； Oct-2

並列摘要

iNOS is responsible for nitric oxide (NO) production under various condition, and engaged in inflammation and killing extrinsic microorganism. Previous studies have demonstrated that iNOS promoter -90bp ~ +150bp region containing a NF-κB response and an Octamer cis-elements is required for LPS-induced iNOS expression. When either one of the two cis-elements is mutated, LPS-induced promoter activity reduced to only 2 to 3% as much activity as in wild type. Most studies have focused on NF-κB mediated signaling, but less discussed in the importance of Octamer cis-element. The main purpose of this thesis is to investigate which Octamer binding protein is involved in activation of iNOS promoter.
There are two Octamer binding transcription factors, Oct-1 and Oct-2. Oct-1 is ubiquitously expressed in most cells, while Oct-2 express primarily in B-cell and neuron cell. Several studies suggest that Oct-2 is expressed in macrophage. When RAW264.7 cells were treated with LPS, mRNA levels of Oct-2 and iNOS increased in a dose- and time-dependent manner, while Oct-1 mRNA decrease slightly. We also show that both mRNA and protein of Oct-2 increased in peritoneal macrophage from Balb/c mice. Cotransfection of iNOS (-90bp ~ +150bp)-Luc with Oct-1 or Oct-2 expression plasmid into RAW264.7 cells showing that both Oct-1 and Oct-2 were able to activate iNOS promoter. However, knockdown of Oct-2 by RNAi in RAW264.7 cells prevented LPS-induced iNOS mRNA expression, whereas knockdown of Oct-1 in RAW264.7 cells had no effect on LPS-induced iNOS mRNA expression. The RNAi results was different from promoter assay. It is possible that Oct-2 can bind to chromatin structure, but not Oct-1. DAPA and ChIP assay showing that LPS-treatment increases binding of Oct-2 and NF-κB subunits to iNOS promoter. Taken together, our results suggest that Oct-2, but not Oct-1, is involved in iNOS promoter activation in response to LPS treatment.

並列關鍵字

iNOS ； LPS ； Oct-2 ； macrophage

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