血管收縮素對心臟細胞的第二型血管收縮素轉化酶表現調節與第二型基質金屬蛋白酶表現之影響

Abstract

腎素-血管收縮素系統（renin-angiotensin system, RAS）為人體最重要的調節系統之一，不正常的RAS與心血管疾病的病程機制有很大的關聯性。血管收縮素II（Angiotensin II, Ang II）、血管收縮素1-7（angiotensin 1-7, Ang 1-7）、血管收縮素轉化酶（angiotensin converting enzyme），以及第二型血管收縮素轉化酶（angiotensin converting enzyme II, ACE2）為RAS中的主要因子。Ang II會刺激發炎反應、纖維母細胞生長、心肌細胞凋零、細胞分化以及纖維化，進而導致組織修復與重塑，而ACE2會水解Ang II形成Ang 1-7，以抑制Ang II所造成之不良反應。多數的文獻顯示ACE2於RAS與心臟疾病中扮演重要角色，但其於心臟細胞中之表現調節功能仍尚未被釐清。 本研究主要目的為探討心臟細胞中ACE2與ace2基因之表現調節，以及ACE2的表現調節與第二型金屬基質蛋白酶（matrix metalloproteinases-2, MMP-2）的關聯性。我們以初代人類心纖維細胞（human cardiac fibroblasts, HCFs）來探討Ang II和Ang 1-7對於ACE2的表現調節，以冷光報導分析法來探討ace2啟動子上與ACE2表現調控有關的序列。此外，我們也用慢病毒（lentivirus）方法促使細胞高量表現ACE2或抑制ACE2生成，探討ACE2表現受到調控時，其對MMP-2表現之影響性。 本研究的重要結果歸納如下：(1) Ang II與Ang 1-7分別會經由AT1R及Mas活化ERK-MAPK訊息傳遞途徑以刺激ACE2表現; (2) ace2基因啟動子的-516/-481序列上之5’-ATTTGGA-3’為特定轉錄因子的結合位置並可藉此調控ACE2表現; (3) Ang II會經由AT1R與其下游ERK-MAPK訊息傳遞鏈刺激ace2基因啟動子的-516/+20序列藉此調控ACE2表現; (4) TGF-β1和TNF-α處理HCFs對於其ace2基因啟動子活性並無顯著影響; (5) ACE2高表現會造成MMP-2活性提昇，且Ang II-AT1R-ERK1/2傳遞途徑可降低MMP-2活性表現; (6) 相對於Ang II，Ang 1-7會經由Mas抑制ERK1/2的活化，但對於MMP-2的活性表現並無明顯之影響性; (7) Ang II-AT1R-ERK1/2及Ang 1-7-Mas-ERK1/2這兩條傳遞途徑也可調控腫瘤壞死因子-α轉化酶（tumor necrosis factor-α-converting enzyme; TACE or ADAM17）的表現以改變細胞膜上ACE2的脫落效應（shedding ACE2, shed ACE2）; (8) 我們重新建立了ACE2基因剔除鼠（ACE2 knockout (KO) mice）群，包括ACE2+/-、ACE2-/-及ACE2-/y基因型; (9) 相較於野生型小鼠，在ACE2 KO小鼠的心臟組織中，MMP-2活性表現量會顯著提昇。 本研究顯示ACE2會受到Ang II及Ang 1-7之表現調節，Ang II及Ang 1-7會提昇HCFs之ACE2表現，且Ang II會經由ace2啟動子中的-516/+20序列區段調控ace2基因表現。於HCFs，ACE2的高表現會提昇MMP-2之活性表現，而Ang II會降低HCFs/ACE2的ADAM17 mRNA表現，也使MMP-2與shed ACE2的活性表現下降。這些結果皆暗示ACE2於心臟重塑病程中擔任保護的角色，Ang II-AT1R-ERK1/2及Ang 1-7-Mas-ERK1/2這兩條傳遞途徑可調控ADAM17的mRNA表現，以及ACE2與shed ACE2之活性表現，且Ang II-AT1R-ERK1/2可調控MMP-2之活性表現。

Renin-angiotensin system (RAS) is an important regulation system in the human circulatory system. The abnormal RAS is associated with the pathogenesis of cardiovascular diseases. Angiotensin II (Ang II), angiotensin 1-7 (Ang 1-7), angiotensin converting enzyme (ACE) and angiotensin converting enzyme II (ACE2) are the mainly members in RAS. Ang II stimulates inflammation, fibroblasts growth, cardiac myocyte apoptosis, fibrogenesis, and differentiation to cause tissue repair/remodeling, and it has been hydrolyzed by ACE2 to obtain the Ang 1-7 to against the disadvantageous effect of Ang II. ACE2 plays a significant role in RAS and heart diseases, but the regulation mechanisms of ACE2 in the heart and cardiac cells are unclear. This study demonstrates the regulation mechanism of ACE2 and ace2 gene expression in cardiac cells, and the association between ACE2 and matrix metalloproteinases-2 (MMP-2) in the cells. The ACE2 expression in human cardiac fibroblasts (HCFs) treated with angiotensin peptides, Ang II and Ang 1-7, and analyzed the promoter activity of human ace2 using luciferase report assay to identify regulatory elements of ace2 gene. In addition, we also revealed the relationship with ACE2 and MMP-2 in HCFs utilized the technology of lentivirus to investigate the role of ACE2 regulation in cardiac cells. The major results of this study are: (1) Ang II-AT1R and Ang 1-7-Mas axes were via ERK-MAPK signal pathway to regulate ACE2 expression, respectively; (2) the sequences of 5’-ATTTGGA-3’ within -516/-481 domain of the ace2 promoter could regulate ACE2 expression and this sequences was the binding site of the transcription factor; (3) Ang II regulated ACE2 expression through AT1R to activate ERK-MAPK signal pathway to stimulate the -516/+20 domain within the ace2 promoter; (4) the ace2 promoter activity was no significant response when HCFs treated with TGF-β1 and TNF-α; (5) ACE2 overexpression enhanced MMP-2 activity and Ang II-AT1R-ERK1/2 axis decreased MMP-2 activity; (6) compared to Ang II, Ang 1-7 through Mas receptor inhibited the activation of ERK1/2, but no significant effect of MMP-2 activity; (7) the tumor necrosis factor-α-converting enzyme (TACE or ADAM17) expression could be regulated through Ang II-AT1R-ERK1/2 and Ang 1-7-Mas-ERK1/2 axes to alter ACE2 shedding; (8) the ACE2 knockout (KO) mice were re-established in our laboratory, including the mice with ACE2+/-, ACE2-/- and ACE2-/y genotypes; (9) ACE2 deficiency enhances MMP-2 activity in heart tissue of ACE2 KO mice compared to WT mice. This study reveals the ACE2 regulation by angiotensin peptides. Ang II and Ang 1-7 could enhance the ACE2 expression in HCFs and indicated that Ang II through the -516/+20 sequence domain within ace2 promoter to regulate ace2 gene expression. ACE2 overexpression enhances MMP-2 activity in HCFs, Ang II decrease the mRNA expression of ADAM17, MMP-2 activity and shed ACE2 activity in HCFs/ACE2. These results show ACE2 plays a protect role against the mechanism of heart remodeling by Ang II induction. Two mainly axes, Ang II-AT1R-ERK1/2 and Ang 1-7-Mas-ERK1/2, regulate the mRNA expression of ADAM17 and the activity of ACE2 and shed ACE2, and MMP-2 activity also could be regulated through Ang II-AT1R-ERK1/2 pathway.