Angiotensin-(1–7) promotes mitochondrial translocation of human telomerase reverse transcriptase in HUVECs through the TOM20 complex
Angiotensin-(1–7) promotes mitochondrial translocation of human telomerase reverse transcriptase in HUVECs through the TOM20 complex
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血管紧张素-(1-7)通过TOM20复合物促进HUVEC中人端粒酶逆转录酶的线粒体转位
DOI：
10.1016/j.abb.2022.109218
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发表时间：
2022
影响因子：
3.9
通讯作者：
Chenghong Yin
中科院分区：
文献类型：
--
作者：
Weikai Hu;Yinan Guo;Xueyan Wang;Tianyu Cui;Chunyun Li;Ruixia Liu;Chenghong Yin
血管紧张素(Ang)(1-7)是一种血管扩张多肽，可改善微循环功能障碍，增加细胞内端粒酶活性，发挥血管扩张、抗炎、抗氧化和抗细胞凋亡等作用。线粒体人类端粒酶逆转录酶(HTERT)在抗细胞凋亡、抗氧化应激、永生化等过程中发挥着重要作用。本研究旨在探讨血管紧张素转换酶(Ang(1-7))对端粒酶逆转录酶(HTERT)线粒体易位的影响。方法建立脂多糖(LPS)诱导的人脐静脉内皮细胞(HUVEC)炎症模型。在内毒素刺激前30min加入Ang(1-7)。在内毒素刺激前30min加入Ang(1-7)/mas受体拮抗剂A779加Ang(1-7)。将转位酶外膜(TOM)20高表达的HUVECs(HUVEC-TOM20OE)、TOM20基因敲除的HUVECs(HUVEC-TOM20KD)及相应的阴性对照细胞系通过慢病毒感染HUVECs的方法构建。单纯内毒素刺激、内毒素+血管紧张素转换酶(1-7)、内毒素+血管紧张素转换酶(1-7)和A779、赋形剂和不加任何处理的细胞分别称为内毒素组、内毒素+血管紧张素转换酶A组、内毒素+血管紧张素转换酶+血管紧张素转换酶A组和阴性对照组。免疫荧光染色检测hTERT在HUVECs细胞核和线粒体中的分布，并在线粒体中定位TOM20、Tom40和转位酶内膜(TIM)23。Western印迹法检测总hTERT、线粒体hTERT、TOM20、Tom40和TIM23的蛋白表达水平。用定量逆转录聚合酶链式反应(qRT-PCR)检测hTERT、TOM20、Tom40和TIM23的mRNA表达水平。内毒素+A组大鼠线粒体端粒酶逆转录酶蛋白水平显著高于内毒素组(P=0.001)，内毒素组线粒体端粒酶逆转录酶的表达明显高于对照组(P=0.001)。总hTERT水平在内毒素+A组和内毒素组之间无显著差异。内毒素+A+A+A779组线粒体端粒酶逆转录酶蛋白水平明显低于内毒素+A组(P=0.021)。HUVEC-TOM20KD与HUVEC-TOM20KD相比，HUVEC-TOM20KD-CON与HUVEC-CON相比，HUVEC-TOM20KD-LPSvsHUVEC-LPS，P=0.003；HUVEC-TOM20KD-LPS+AvS，ANG(1-7)不能恢复HUVEC-TOM20KD线粒体hTERT的下调。HUVEC-TOM20OE细胞线粒体端粒酶逆转录酶水平显著升高(HUVEC-TOM20OE-CON与HUVEC-CON相比，P=0.010)，而Ang(1-7)刺激(HUVEC-TOM20OE-LPS8+AvsHUVEC-TOM200OE-CON，P=0.011)。结论Ang(1-7)通过TOM20有效促进人脐静脉内皮细胞线粒体转位，提示端粒酶逆转录酶可能通过TOM20复合体转运至线粒体。此外，A779可阻断Ang(1-7)对人脐静脉内皮细胞的作用。
BackgroundAngiotensin (Ang) (1–7) is a vasodilator peptide that ameliorates microcirculation dysfunction, increases telomerase activity in cells, and exerts vasodilatory, anti-inflammatory, antioxidative stress, and antiapoptotic effects. Mitochondrial human telomerase reverse transcriptase (hTERT) plays an important role in the processes of antiapoptosis, antioxidative stress, and immortalization. This study aimed to investigate the effect of Ang(1–7) on the mitochondrial translocation of hTERT.MethodsAnin vitromodel of lipopolysaccharide (LPS)-induced inflammation was established in human umbilical vein endothelial cells (HUVECs). Ang(1–7) was added to cells 30 min before LPS stimulation. The Ang(1–7)/Mas receptor antagonist A779 plus Ang(1–7) were added to the cells 30 min before LPS stimulation. The translocase outer membrane (TOM)20-overexpression HUVECs (HUVEC-TOM20OE), TOM20-knockdown HUVECs (HUVEC-TOM20KD), and the corresponding negative control cell lines were constructed by lentiviral transfection of HUVECs. Cells subjected to LPS stimulation alone, LPS plus Ang(1–7), LPS plus Ang(1–7) and A779, vehicle and no treatment were termed the LPS group, LPS + A group, LPS + A + A779 group, Con group and Neg group, respectively. Immunofluorescence staining was used to detect the distribution of hTERT in the nuclei and mitochondria of HUVECs and to locate TOM20, TOM40, and translocase inner membrane (TIM)23 in the mitochondria. The protein expression levels of total hTERT, mitochondrial hTERT, TOM20, TOM40, and TIM23 were measured by Western blot. The mRNA expression levels of hTERT, TOM20, TOM40, and TIM23 were assessed by quantitative reverse transcription–polymerase chain reaction (qRT-PCR).ResultshTERT colocalized with TOM40, TOM20 and TIM23 in the mitochondria. The mitochondrial hTERT protein level of the LPS + A group was significantly greater than that of the LPS group (P= 0.001), and the LPS group showed significantly increased expression of mitochondrial hTERT compared with that of the control group (P= 0.001). No significant difference in the level of total hTERT was observed between the LPS + A and LPS groups. The mitochondrial hTERT protein level of the LPS + A + A779 group was significantly lower than that of the LPS + A group (P= 0.021). The protein level of mitochondrial hTERT in HUVEC-TOM20KDtreated with or without LPS alone or LPS + A was significantly decreased compared with the corresponding groups of control HUVECs (HUVEC-TOM20KD-Con vs. HUVEC-Con,P= 0.035; HUVEC-TOM20KD-LPS vs. HUVEC-LPS,P= 0.003; HUVEC-TOM20KD-LPS + A vs. HUVEC-LPS + A,P= 0.001), and treatment with Ang(1–7) did not restore the downregulation of mitochondrial hTERT in HUVEC-TOM20KD. HUVEC-TOM20OEshowed a significantly increased level of mitochondrial hTERT (HUVEC-TOM20OE-Con vs. HUVEC-Con,P= 0.010), which was further elevated by Ang(1–7) stimulation (HUVEC-TOM20OE-LPS + A vs. HUVEC-TOM20OE-Con,P= 0.011). Lastly, the protein expression levels of TOM40 (HUVEC-TOM20KD-Con vs. HUVEC-Con,P= 0.007) and TIM23 (HUVEC-TOM20KD-Con vs. HUVEC-Con,P= 0.001) were significantly increased in HUVEC-TOM20KDin comparison to HUVECs.ConclusionsAng(1–7) effectively promoted mitochondrial translocation of hTERT in HUVECs via TOM20, indicating that hTERT may be transported to the mitochondria through the TOM20 complex. In addition, A779 could block the effects of Ang(1–7) in HUVECs.