在類成骨細胞MC3T3-E1細胞中Staurosporine誘導第二型環氧化酵素基因表現之機轉研究

Abstract

中文摘要 Staurosporine是從鏈黴菌萃取出的一種高效能磷酸激酵素抑制物。它被發現會引起細胞凋亡（Apoptosis）及細胞循環停滯（G1-arrest）等生理機制。近期我們發現staurosporine會激發環氧化酵素-2（COX-2）在類成骨細胞中的表現，此一新發現促使我們想進一步探究staurosporine激發COX-2轉錄的作用機轉。 首先我們將環氧化酵素-2（COX-2）的啟動子部份片段-188~+70bp接入載有螢光蟲酵素表現基因的載體上，pGL2-Basic，此一啟動子片段上載有CRE、NF-IL6、AP-2等三種轉錄因子的結合片段。我們並以自製的微脂粒將此報導質體轉殖入小鼠類骨細胞中，並測試其轉殖效益，我們發現DNA與微脂粒的混合比例到達1μg DNA/12 nmol微脂粒時具有較佳的轉殖效果。當我們以不同濃度的staurosporine (0~200nM) 來研究螢光蟲酵素的表現時，發現當細胞以20nM 的staurosporine處理時，酵素活性值可表現的最好，當濃度高於20nM後酵素活性值便急速下滑。而反應時間的測試中，則發現staurosporine刺激處理達八小時可使螢光蟲酵素活性值達最高。接著我們在pGLB/18-W的啟動子片段中做定點突變，分別去掉轉錄因子NF-IL6、AP-2及CRE的接合片段，形成質體pGLB/18-AC、pGLB/18-NC、pGLB/18-NA及pGLB/18-N，將其轉殖入MC3T3-E1細胞中，比較staurosporine刺激處理後的螢光蟲酵素活性值，發現均有程度上的活化表現，而其中以pGLB/18-N的活性表現最為明顯，相對地pGLB/18-AC的活性表現則較低落，因此我們初步認為NF-IL6應是參與此訊息傳遞路徑的主要轉錄因子。

Abstract Staurosporine, an alkaloid isolated from streptomyces culture, is a potent protein kinase inhibitor with a broad spectrum of activity. Staurosporine could induce programmed cell death and to inhibit cell cycle progression in a variety of cell lines as suggested by several reports. In previous study, we have found that staurosporine could increase the mRNA as well as protein level of cyclooxygenase-2 (COX-2). This finding prompted us to further explore the molecular mechanism of staurosporine-mediated COX-2 gene expression. In this study, we first looked for the transcription factor(s) that might be activated by staurosporine. The luciferase reporter plasmids, containing specific segment (-188 to +70 bp) of promoter region of COX-2 gene were constructed. This segment harbors three known responding elements, termed CRE, NF-IL6 and AP-2, respectively. These reporter plasmids were then transfected into osteoblast cell line, MC3T3-E1, by lipofection. The result showed that the transcription efficiency reached maximum under the condition of DNA to liposome ratio at 1μg DNA:12 nmol liposome and incubated at 37℃ for 6 hrs. The dose-response study suggested that staurosporine induced the expression of COX-2-promote bearing luciferase gene in a dose-dependent manner (0-200 nM). The luciferase activity of reporter plasmid-transfected cells reached maximum when treated with 20 nM staurosporine. The luciferase activity rapidly decreased with staurosporine concentration higher than 20 nM. The luciferase activity of pGLB/18-transfected cells also increased in a time-dependent manner (4-24 hrs) in the presence of 50 nM staurosporine. The luciferase activity reached maximum 8 hrs post-staurosporine treatment. To further identify the transcription factor(s) that may involve in the staurosporine-mediated COX-2 induction, we mutated the promoter region of pGLB/18 at AP-2, NF-IL6, and CRE site, respectively. Accordingly, four luciferase reporter plasmids, i.e., pGLB/18-NC, pGLB/18-AC, pGLB/18-AN and pGLB/18-N, were generated and transfected into MC3T3-E1 cells through lipofection. We found that the luciferase activity of cells transfected with pGLB/18-N was higher than that of the cells transfected with other mutated plasmids. In conclusion, we have shown for the first time that staurosporine could activate NF-IL6 in osteoblast-like cell line, MC3T3-E1, and induce the expression of Cyclooxygenase-2.