- 學位論文
維他命D3調控基因轉錄機制之探討
A Novel Mechanism for 1,25-(OH)2-D3-Mediated Gene Transcription
摘要
維他命D3除了是人體必需的養分外，它的活化態代謝物1,25-(OH)2-D3經由結合它的接受體－Vitamin D3 Receptor (VDR) ，發揮在生理功能上的作用。VDR是一個轉錄因子，受到1,25-(OH)2-D3活化之後，調控的基因不僅在鈣磷離子的恆定性上扮演重要的角色，亦影響細胞的生長與分化。傳統上1,25-(OH)2-D3的調控機制，主要經由其接受體結合至標的基因的啟動子上的維他命D3反應結合位 (VDRE) ，並啟動基因的轉錄機制。但是維他命D3除了經由傳統上的調控機制來影響基因的表現外，本論文證實它也可以經由另一個方式調控基因的表現。此機制不需要VDR直接結合至其標的基因啟動子上的VDRE，取而代之的方式是利用蛋白質與蛋白質的結合，也就是VDR結合另一個轉錄因子蛋白質Sp1，以調控基因的表現。由於維他命D3具有抑制人類前列腺癌細胞生長的能力，所以於本論文中我們使用人類前列腺癌細胞－LNCaP，作為我們探討維他命D3抑制癌細胞時，參與基因的調控機制的探討。我們探討維他命D3活化p27Kip1與抑制p45Skp2基因的分子機制。利用reporter assay、DNA親和免疫沉澱分析、染色質免疫沉澱分析等實驗結果證明維他命D3處理下，VDR與Sp1形成複合體後，再經由啟動子上Sp1的結合位，進而活化p27Kip1與抑制p45Skp2基因的表現，同時我們也發現部分參與在活化或抑制基因轉錄的協同因子。本論文也提供一個全新的基因調控機制觀念，即經由VDR與Sp1形成複合體的方式影響基因的轉錄，而非是直接由VDR結合上VDRE的方式。相信本篇的研究成果，對於未來利用維他命D3治療前列腺癌上能夠提供一個新契機。
並列摘要
Vitamin D3 is a conditionally required nutrient traditionally thought to exert its physiological activity via the binding of its active metabolite, 1,25-(OH)2-D3, to the vitamin D receptor (VDR) .VDR is a ligand-activated transcription factor that plays a central role in calcium/phosphorus homeostasis and has been implicated in regulating diverse biological functions, including cellular proliferation and differentiation. The classical pathway for VDR signaling is mediated by receptor binding to vitamin D response elements (VDREs) located within the promoters of vitamin D3- responsive genes. In this study, we demenstrate that VDR can alter gene transcription via a novel mechanism that does not require direct VDR binding to the VDRE. Interaction between the VDR and the transcription factor Sp1, leads to changes in the transcription of target genes. Vitamin D3 may suppress proliferation of human prostate cancer cells. Therefore, we used the human prostate cancer cell line LNCaP to examine the effect of vitamin D3 on gene expression. In this study, we elucidate the underlying mechanism by which vitamin D3 activates p27Kip1 and inhibits p45Skp2. Reporter assay, DNA affinity precipitation assay (DAPA) and chromatin immunoprecipitation (ChIP) assay reveal that VDR and Sp1 form a complex and bind to the promoter of VDRE-lacking genes. The VDR/Sp1 complex acts via Sp1 binding site to regulate gene transcription. Thus, our study elucidates the molecular mechanism by which VDR regulates gene activation or suppression via interaction with Sp1. We also reveal the particular cofactors that involved in gene activation or repression. These results may be of important clinical significance and provide a new strategy for the treatment of prostate cancer.