Zn2+/MTF1在多巴胺活化PC12細胞中自噬相關基因之表現調控

Abstract

金屬調控轉錄因子1 (metal-regulatory transcription factor 1, MTF1) 與鋅離子 (Zn2+)結合後，可與基因增強子區域的MRE結合，進而調控基因表現。多巴胺（dopamine, DA）是一種神經遞質，與G蛋白偶聯受體結合活化相關訊息傳導途徑。我們之前的報導顯示DA可提高胞內鋅離子濃度（[Zn2+]i），更是DA誘導PC12細胞產生細胞自噬的先決條件，然而相關機制並不清楚。綜合以上訊息，我們認為可能是透過活化MTF1，促進細胞自噬相關基因的表現。因此我們將MTF1表現在PC12細胞，並探討DA誘導的基因表現與自噬通量的變化。結果顯示MTF1過表現，可顯著增強DA處理所造成的PC12細胞存活率，由95±3 %降到57±3 %。免疫細胞化學（immunocytochemistry）測定顯示，DA可促使MTF1由細胞質中易位至細胞核中 (共定位相關係數為0.4)，而Zn2+螯合劑可逆轉此比例為0.1；顯示[Zn2+]i 的變化，是MTF1進入細胞核的重要因素。而當在PC12細胞中同時表現MTF1與細胞自噬體(autophagosome)的標記蛋白EGFP-LC3，以計算DA處理所造成的自噬體數目，結果顯示，MTF1會顯著增加DA處理所增加的自噬體數目，由7增加為9.5。而為瞭解MTF1是否會影響細胞自噬相關基因表現，我們設計了9組主要自噬相關基因（Atg基因）的引子，進行qPCR試驗，結果顯示MTF1會影響DA處理所造成的幾種主要Atg基因的表現，顯示MTF1具有調控Atg基因的能力。以生物信息學分析顯示，幾個Atg基因的增強子區域，含有多個MRE結合位點，這支持MTF1可能與這些Atg基因結合。因此我們的結果說明，DA透過DA受體-Zn2+-MTF1-Atg途徑，誘導自噬基因表現，以調節自噬通量。本研究結果有助於深入了解，Zn2+恆定如何調控細胞自噬和隨後的細胞死亡，並提供更多關於神經退化的資訊。

MTF1 (metal-regulatory transcription factor 1) senses zinc ion (Zn2+) and binds to MRE (metal response elements) at the enhancer regions of various genes. Dopamine (DA) is a neurotransmitter and functions when binding with G protein-coupled receptors. Our previous reports show that DA treatment elevates the intracellular zinc concentration ([Zn2+]i), which is a prerequisite for DA-induced autophagic flux and cell death in PC12 cells. However, it is not clear the roles MTF1 play in DA-induced autophagy. In this report, we overexpressed MTF1 in PC12 cells and examine the DA-induced changes in gene expression and autophagic flux. Our results showed that MTF1 overexpression enhanced the cell death in DA-treated PC12 cells from 95 ± 3 % to 57 ± 3 % comparing to control groups. The immunocytochemistry assay showed that MTF1 translocated from cytosol to nucleus in DA-treated cells by 0.4 (colocalization coefficient) which could be reversed by Zn2+ chelator to 0.1, similar to control group, indicating that DA-induced elevation in [Zn2+]i can trigger the translocation of overexpressed MTF-1 to the nucleus. MTF1 overexpression enhanced the formation DA-induced autophagosome puncta, represented by the co-expressed EGFP-LC3, from 7 ± 0.5 of control group to 9.5 ± 0.5 per cell. To examine the changes in gene expression profile, we used qPCR to analyze the expression of major autophagy-related genes (Atg genes). The results show that MTF1 overexpression changed the expression profile of several major Atg genes, implying that MTF1 has a potential to regulate Atg genes. Our bioinformatics analysis revealed that the enhancer regions of several Atg genes contain MRE binding sites, suggesting the possible binding of MTF1 to these Atg genes. In conclusion, our results demonstrate that DA-induced autophagy is via the DA receptor-Zn2+-MTF1-Atg pathway to modulate the autophagic flux. This study provides insights into the importance of Zn2+ homeostasis in modulating the DA-induced autophagy and subsequent cell death, especially their roles in neurodegenerative disorders.