評估五類轉錄因子參與抗利尿激素誘導第二型水通道蛋白質基因表現的機制

Evaluation of Five Transcription Factor Families Involved in Vasopressin-Induced Aquaporin-2 Gene Expression

林樹廷(Shu-Ting Lin)

指導教授 ： 余明俊

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201703542

摘要

抗利尿激素調控腎臟集尿管區域中第二型水通道蛋白質(AQP2)的基因表現，當這項調控機制失調時，會造成一些和身體水分平衡相關的疾病。然而抗利尿激素是透過什麼樣的分子機制去調控AQP2基因的表現，仍然是不清楚的。在本篇研究中我們使用小鼠集尿管細胞株-mpkCCD作為研究平台，評估五個可能參與在抗利尿激素所誘導的AQP2基因表現的轉錄因子結合位並研究其相關的轉錄因子，這五個轉錄因子結合位分別是NF-κB、 Ets、Sp1、CRE及GATA。我們發現突變的Sp1轉錄因子結合位不會影響AQP2啟動子的活性。突變的GATA轉錄因子結合位會提升AQP2基礎的啟動子活性但降低AQP2啟動子對抗利尿激素的反應。突變的CRE轉錄因子結合位會完全抑制AQP2啟動子對抗利尿激素的反應，這項結果也與過去所認知的CREB1轉錄因子在抗利尿激素所誘導的AQP2基因表現中所扮演的角色一致。但讓我們驚訝的是，當我們個別抑制七種表現在mpkCCD細胞內的CREB轉錄因子的基因表現後，發現沒有一個CREB轉錄因子會影響抗利尿激素所誘導的AQP2的基因表現。因此我們尚未找到一個CREB轉錄因子是負責調控抗利尿激素所誘導的AQP2的基因表現。突變的NF-κB或Ets會降低基礎及抗利尿激素所誘導的AQP2啟動子的活性，但並不會影響AQP2啟動子對抗利尿激素的反應，這個結果與過去抑制轉錄因子Elf3所導致現象類似。有研究指出轉錄因子Elf3會與組蛋白乙醯基轉移酶CBP/p300結合並活化乙醯化的功能，所以轉錄因子Elf3可能會透過調節組蛋白的乙醯化來調控AQP2的基因表現。然而在我們的實驗中，H3組蛋白的乙醯化程度並沒有因為抑制轉錄因子Elf3的基因表現而有所改變。總結本篇研究，我們使用了與原生集尿管主要細胞最為相近的細胞株作為研究平台來探討五個轉錄因子結合位及其相關的轉錄因子在抗利尿激素所誘導的AQP2的基因表現中所扮演的角色。我們的結果顯示這項調控機制非常複雜，很可能包含了數個轉錄因子結合位及其轉錄因子來共同調控抗利尿激素所誘導的AQP2的基因表現，其中也可能包含表徵遺傳方面的調控。

關鍵字

抗利尿激素 ； 第二型水通道蛋白質 ； 轉錄因子

並列摘要

Vasopressin regulates aquaporin-2 (AQP2) gene expression in the kidney collecting ducts. Dysregulation of vasopressin-induced AQP2 gene expression is associated with a number of water balance disorder. However, the regulatory mechanisms for AQP2 gene expression are still not clear. Here, we evaluated roles of five conserved TFBSs potentially involved in vasopressin-induced AQP2 gene expression i.e. NF-κB, Ets, Sp1, CRE, and GATA. Mutation in Sp1-binding element did not affect AQP2 promoter activity. Mutation in GATA-binding element increased basal AQP2 promoter activity and reduced vasopressin responses. Mutation in CRE abolished vasopressin responses, consistent with a role CREB1 in mediating vasopressin-induced AQP2 gene expression. To our surprise, knockdown of seven CREBs identified in mpkCCD cells did not affect vasopressin-induced AQP2 gene expression. Thus, CRE-binding factors participating in vasopressin-induced AQP2 gene expression remains unidentified. Mutation in NF-κB or Ets-binding element reduced both basal and vasopressin-induced AQP2 promoter activity without affecting vasopressin responses, similar to the effects of Elf3 knockdown found in our previous study. Because Elf3 binds and activates the CBP/p300 acetylation complex, the above observations suggest an epigenetic mode of AQP2 gene regulation via chromosomal remodeling, although Elf3 knockdown did not affect the acetylation levels of histone H3 in our experiments. In summary, we have evaluated five TFBSs and their associated TFs in a collecting duct cell model that most resembles the native colleting duct principal cells. Our analysis shows that the regulatory mechanism for AQP2 gene expression is very complex, likely involving multiple TFBSs and TFs as well as proteins involved in epigenetic regulations.

並列關鍵字

Vasopressin ； Aquaporin-2 ； Transcription factor

參考文獻

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