- 學位論文
β-Catenin 藉由增加AR 進入細胞核內的量 與DNA 的結合來促進其轉錄活性
β-Catenin potentates androgen receptor-mediated-transcription activity through effects on nuclear translocation and DNA binding in androgen-independent fashion
摘要
β-Catenin在Wnt/Tcf4訊息傳遞中可與轉錄因子Tcf4結合以促進下游基因 轉錄，功能上被定義為coactivator。此外,有一些研究中也發現β-catenin可促 進AR所調控的轉錄活性，然而β-catenin在其中所扮演的功能調控腳色仍不清 楚。為了了解β-catenin如何調控AR的功能，我們建構帶有正常以及突變 （constitutive active T41A, S45A mutation form）的β-catenin基因的殖體， 將其過度表現在具有大量AR的MDA-MB-453細胞株。利用報導基因的分析，我們發 現表現T41A,S45A突變株的細胞，其轉錄活性與對照組相比高出2.5倍，在加入 androgen活化AR後，細胞轉錄活性更為明顯。此外，在有無androgen存在的情況 下，β-catenin促進AR轉錄活性上升的比率都相同，而且過度表現正常以及突變 （T41A, S45A）的β-catenin細胞與對照組相比對androgen的作用都呈現相同的 EC50，顯示β-catenin對於AR的調控並不需要androgen的參與相反的，在系統中 加入AR抑制劑－bicalutamide可抑制β-catenin所促進的轉錄活性，顯示β -catenin對於轉錄活性的調控的確是經由AR的訊息傳遞。最後我們利用西方點墨 法以及電泳位移法來探討β-catenin如何促進AR所調控的轉錄活性，實驗結果發 現β-catenin可促進AR進入細胞核內的量並增加AR與ARE的結合。有趣的是，我 們同時也發現除了AR之外，β-catenin也會促進GAGATA結合蛋白以及TCF-4進入 細胞核的量並增加其與DNA的結合。由以上的實驗證實β-catenin可藉由增加AR 進入細胞核內的量來達到促進AR所調控的轉錄活性，而且β-catenin的功能可能 也在其他的轉錄因子中扮演類似的腳色。
並列摘要
β-Catenin works as a coactivator in Wnt/Tcf4 signal transduction pathway, and can also increase androgen receptor (AR)-mediated transcription activity in transient transfection assay. In Wnt/Tcf4 pathways, β-catenin interacts with Tcf4-DNA complex to recruit CBP for activation of their target genes. In contrast, the role of β-catenin in AR-mediated transcription activity remains unclear. To delineate how β-catenin promotes AR-mediated transcription activity, we enforced the expression of wild type and mutant form of β-catenin (T41A, S45A) in MDA-MB-453 cell lines that contain a wild type form of AR. In pSEAP-PSA reporter gene assay, β-catenin (T41A, S45A) could enhance transcription activity about 2.5 fold than that of wild type β-catenin and empty vector control in the presence and absence of androgen. The same induction fold with/without androgen suggested that enhancing effect of mutated β-catenin on pSEAP-PSA reporter gene activity is androgen-independent. About the equal EC50 of dihydrotestosterone for the respective wild type, mutated β-catenin and control confirmed this androgen-independent outcome. The antagonist of AR, bicalutamide, could reduce the enhancing-effect of β-catenin (T41A, S45A) on this reporter gene activity with or without androgen, suggesting this effect through AR. We further performed Western blotting and EMSA to explore how β-catenin potentates AR-mediated functions. The results indicated that β-catenin (T41A, S45A) promotes nuclear translocation of AR, resulting in the increase of AR-ARE interaction. Interestingly, the increasing DNA-protein interaction also appeared in GAGATA binding protein and TCF4 in the presence of β-catenin (T41A, S45A), and it substantially promotes nuclear translocation of TCF4. Thus, β-catenin might enhance nuclear translocation for a broad spectrum of transcription factors, consequently increasing their DNA-protein interactions and transcription activities.