鼠類血管平滑肌細胞中Beraprost透過增加CBP共活化因子入核作用誘導PPARδ依賴性p21及p27增加而造成抗增生的作用

PPARδ-dependent p21 and p27 induction via increased translocation of CREB-binding protein coactivator in the beraprost-induced antiproliferation of murine vascular smooth muscle cells

鍾智鵬(Chih-Peng Chung)

指導教授 ： 阮淑慧

共同指導教授 ： 蔡郁惠(Yu-Hui Tsai)

臺北醫學大學/醫學院/細胞及分子生物研究所/碩士(2008年)

摘要

最近有許多的研究指出，過氧化體增生活化受體δ(peroxisome proliferators-activated receptor-delta, PPARδ)的促效劑:貝前列素鈉(beraprost)，有著抑制血小板的凝集、降低血管動脈平滑肌細胞的增生和促進血管的舒張的效果，而這些影響都能夠很有效的對抗動脈粥狀硬化的形成。在我們實驗室之前的研究中，在血管壁受損的動物模組中，貝前列素鈉讓過氧化體增生活化受體δ增加表現的同時，也會接連著誘導出誘導型一氧化氮生成酶(inducible nitric oxide synthase , iNOS)去抑制平滑肌細胞的增生。在此，我們描繪出一個貝前列素鈉的藥效所產生抗增生的分子機制和細胞週期抑制蛋白p21/p27增加之間的關係。貝前列素鈉會有濃度遞增性的促進cyclin依賴性激酶的抑制蛋白:p21/p27的啟動子活性，而此現象會被過氧化體增生活化受體δ的拮抗劑所抑制。此外，我們使用MOTIF搜尋分析p21、p27的啟動子位置找到有環化單磷酸腺苷酸反應元件(cyclic AMP response element, CRE)存在於其上。然而，在過氧化體增生活化受體δ的訊息傳遞路徑中，貝前列素鈉會增加一種叫環化單磷酸腺苷酸反應元件結合蛋白(cAMP response element binding protein, CREB)的共活化因子:環化單磷酸腺苷酸反應元件結合蛋白的結合蛋白(CREB-binding protein, CBP)進入核內的量，但是卻不是透過環化單磷酸腺苷酸反應元件結合蛋白的增加。我們實驗將細胞送入了啟動子上含有三重複的CRE序列的pGL2載體，發現貝前列素鈉會促進有CRE序列的啟動子活性，並且此活性會因為受到過氧化體增生活化受體δ的拮抗劑的影響或是核酸干擾技術使CBP減少而被抑制。此外，細胞的CBP降低也會減少因貝前列素鈉所誘導出的p21/p27蛋白質的量。綜合以上的實驗，結果顯示出過氧化體增生活化受體δ會受到貝前列素鈉的誘導，使細胞週期抑制蛋白:p21/p27的轉錄反應增加是透過CBP進入核內的量增加之影響。

關鍵字

鼠類,平滑肌,抗增生,細胞週期抑制蛋白,入核

並列摘要

A numerous studies have shown that a peroxisome proliferators-activated receptor-delta (PPARδ) agonist, beroprost, inhibits platelet aggregation, suppresses smooth muscle cell proliferation, and promotes vasolation, which are effective against atherosclerosis. We previously showed that increased PPARδ together with subsequence induction of iNOS by beroprost inhibited smooth muscle cell proliferation in the balloon injuried animal model. Herein, we delineate the molecular mechanisms of antiproliferative effects of beraprost related to the induction of cell cycle inhibition protein; p21/p27. BPS concentration-dependently induced promoter activities of cyclin-dependent kinase inhibitor p21/p27, which were significantly inhibited by PPARδ antagonists. Additionally, putative CRE in the p27 promoter region was observed in the MOTIF search analysis. However, BPS increased nuclear translocation of CREB-binding protein (CBP), a cAMP response element binding protein (CREB) co-activator, but not CREB through a PPARδ-dependent pathway. Three repeats of the consensus CRE constructed in the pGL2-promoter vector was assessed for its activity; BPS increased CRE activity, whereas the activity was suppressed by PPARδ antagonists, or in cells with PPARδ or CBP knockdown by siRNAs. Furthermore, cells with CBP knockdown decreased p21/p27 protein level by BPS. Taken together, the results suggest that PPARδ induced by BPS enhances transcriptional activation of cell cycle inhibition protein p21/p27 by an increased translocation of CBP into nucleus

並列關鍵字

PPARδ,p21,p27,beraprost,CBP,CREB,antiproliferation

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