人類心臟血管收縮素轉化酶II基因調控序列的探討

Abstract

腎素-血管收縮素系統（renin-angiotensin system; RAS)是人體內負責調節血壓與體液平衡的系統，並且與許多心臟血管疾病的發生有很大的相關。血管收縮素II（angiotensin II; Ang II）為RAS系統中重要的調控胜肽，其能引起血管收縮而使得血壓升高。血管收縮素轉化酶II（angiotensinconverting enzyme II; ACE2）可將Ang II水解成具有保護血管效用的另一胜肽血管收縮素-(1-7)（angiotensin-(1-7); Ang-(1-7)），因而具有益於心血管功能的功用，並成為可調節心血管疾病的潛力標的。近年來，越來越多研究結果顯示ACE2扮演益於心血管功能的角色，然而ace2基因表現的調控機制仍尚待探討。在本研究中，我們探討ace2基因中的表現調控序列。我們選殖了人類ace2基因之自-2,069至+20的基因組5’端DNA片段，藉以建構其一系列不同5’端基因調控序列長度與特定序列剔除或置換的DNA片段，並將其構築入帶有冷光基因（luciferase）做為報導基因的載體，這些表現質體分別被轉染入人類心纖維母細胞（human cardiofibroblast; HCF）中，經由冷光強度分析探討ace2基因調控序列的表現調節。實驗結果顯示，在ace2基因有一ACE2表現抑制區段，其位於-627至-516；另有一ACE2表現增強區段，位於-516至-481，在全長（-2,069/+20）的ace2基因調控序列中剔除-516/-481序列片段會造成ace2基因表現顯著地降低。同時，electrophoresis mobility shift assay也顯示核蛋白內有蛋白質與-516/-481序列進行結合。我們亦測定血管收縮素胜肽Ang II 和Ang-(1-7)，以及促發炎因子（pro-inflammatory factors）中轉形生長因子-beta□1（transforming growth factor-beat 1; TGF-beta□1）和腫瘤壞死因子-alpha（tumor necrosis factor-alpha;TNF-alpha）對ace2基因表現調控之影響。研究結果顯示，在HCF細胞中Ang II與Ang-(1-7)的處理皆可提高ace2基因的表現，而其主要調控區域位於ace2基因的-516/+20間。然而在HCF細胞中，ace2的基因表現並未受到TGF-beta□1和TNF-alpha□處理的影響。我們的實驗結果指出ace2基因的5’端表現調控序列中的-516/-481區域可能為一尚未被辨識出的調控因子之結合位，而其結合後可活化ace2的表現，而此調控可能與血管收縮素胜肽的訊息傳遞途徑有關。

The renin-angiotensin system (RAS) is a key regulatory axis on blood pressure and fluid homeostasis and critically involves in cardiovascular pathophysiology. Angiotensin- converting enzyme 2 (ACE2) has been proposed as a potential cardioprotective target in regulating cardiovascular functions owing to its key role in the formation of vasoprotective peptides angiotensin-(1-7) [Ang-(1-7)] from angiotensin II (Ang II), an effector peptide of RAS with vasoconstrictive, pro-inflammatory and pro-fibrotic properties. However, the regulatory mechanism of human ace2 expression remains to be explored. In this study, we investigated the human ace2 promoter to identify regulatory elements of the gene. The human ace2 gene promoter from position -2,069 to +20 was cloned and series 5’ deletion mutants were constructed in a luciferase reporter vector. The reporter expressions were analyzed by transient transfection of the constructants in human cardiofibroblast (HCF) cells. The results indicate one activating region at -516/-481 and one repressing region at -627/-516 in HCF cells. Furthermore, deletion of domain (-516/-481) within the ace2 gene construct (-2,069/+20) resulted in a significant decrease in luciferase activity. Mobility shift electrophoresis with double stranded oligonucleotides (-516/-481) resulted in a DNA-protein complex. The effects of angiotensin peptides, Ang II and Ang-(1-7), and pro-inflammatory factors, TGF-beta□1 and TNF-alpha, on the transcriptional activity of the ace2 promoter variants in HCF cells were investigated. The experimental results show that the relative luciferase activities of the (-516/+20) construct increase significantly in response to Ang II and Ang-(1-7) indicating that the angiotensin peptides can upregulate transcription of ace2 gene. However, the transcriptional activity of the ace2 promoter in HCF cells did not seem to be affected by TGF-beta□1 and TNF-alpha treatments□ In conclusion, our results suggest that domain (-516/-481) of ace2 gene may be a binding domain for as yet unidentified regulatory factors that can activate ace2 expression and the activation is associated with angiotensin peptides signaling.