IRF-1 mediates the suppressive effects of mTOR inhibition on arterial endothelium
IRF-1 mediates the suppressive effects of mTOR inhibition on arterial endothelium
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IRF-1 介导 mTOR 抑制对动脉内皮的抑制作用
DOI：
10.1016/j.yjmcc.2020.02.006
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发表时间：
2020-03-01
影响因子：
5
通讯作者：
Sun, ChongXiu
中科院分区：
文献类型：
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作者：
Peng, Kai;Fan, Xing;Sun, ChongXiu
目的：已发现用于药物洗脱支架（DES）控制再狭窄的哺乳动物雷帕霉素靶点（mTOR）抑制剂可延迟内皮化，并通过尚未完全了解的机制增加迟发性血栓形成的发生率。我们发现，mTOR抑制（mTORi）上调细胞生长抑制因子IRF-1在原代人动脉内皮细胞（HAEC）的表达。本研究旨在研究如何mTOR调节IRF-1的表达有助于抑制mTORI对动脉内皮proliferation.Methods和结果：Western印迹，定量PCR，和双荧光素酶报告基因分析表明，mTOR抑制剂雷帕霉素和洛林1上调IRF-1的表达，并增加其转录活性。IRF-1进而通过介导HAEC凋亡和细胞周期阻滞而促进mTORi的抑制作用，部分通过上调半胱天冬酶1和下调细胞周期蛋白D3，如CCK-8测定、膜联蛋白V结合测定、活化半胱天冬酶3的测量、BrdU掺入测定和基质胶管形成测定所揭示的。在股动脉钢丝损伤的小鼠模型中，给予雷帕霉素抑制EC恢复，IRF-1的EC缺乏减轻了这种作用。HAEC染色质免疫沉淀试验和Irf 1（-/-）小鼠EC中野生型或显性阴性IRF-1的拯救表达证实了IRF-1对CASP 1和CCND 3表达的转录调控。此外，mTORi激活多个PKC成员，其中PKC zeta负责对HAEC的生长抑制作用。激活的PKC zeta通过JAK/STAT-1和NF-κ B信号通路增加IRF 1的转录。最后，过度表达野生型或突变猛禽不能结合mTOR表明，mTOR-无猛禽有助于PKC zeta激活mTOR抑制HAEC.Conclusions：这项研究揭示了IRF-1介导的机制，有助于抑制作用的mTORi对HAEC增殖。进一步的研究可能有助于制定有效的策略，以减少冠状动脉介入治疗中使用DES的副作用。
Aims: Mammalian target of rapamycin (mTOR) inhibitors used in drug-eluting stents (DES) to control restenosis have been found to delay endothelialization and increase incidence of late-scent thrombosis through mechanisms not completely understood. We revealed that mTOR inhibition (mTORi) upregulated the expression of cell growth suppressor IRF-1 in primary human arterial endothelial cells (HAEC). This study aimed to examine how mTOR-regulated IRF-1 expression contributes to the suppressive effect of mTORi on arterial endothelial proliferation.Methods and results: Western blotting, quantitative PCR, and a dual-luciferase reporter assay indicated that mTOR inhibitors rapamycin and Lorin 1 upregulated IRF-1 expression and increased its transcriptional activity. IRF-1 in turn contributed to the suppressive effect of mTORi by mediating HAEC apoptosis and cell cycle arrest in part through upregulation of caspase 1 and downregulation of cyclin D3, as revealed by CCK-8 assay, Annexin V binding assay, measurement of activated caspase 3, BrdU incorporation assay, and matrigel tube formation assay. In a mouse model of femoral artery wire injury, administration of rapamycin inhibited EC recovery, an effect alleviated by EC deficiency of IRF-1. Chromatin immunoprecipitation assay with HAEC and rescue expression of wild type or dominant-negative IRF-1 in EC isolated from Irf1(-/-) mice confirmed transcriptional regulation of IRF-1 on the expression of CASP1 and CCND3. Furthermore, mTORi activated multiple PKC members, among which PKC zeta was responsible for the growth-inhibitory effect on HAEC. Activated PKC zeta increased IRF1 transcription through JAK/STAT-1 and NF-kappa B signaling. Finally, overexpression of wild type or mutant raptor incapable of binding mTOR indicated that mTOR-free raptor contributed to PKC zeta activation in mTOR-inhibited HAEC.Conclusions: The study reveals an IRF-1-mediated mechanism that contributes to the suppressive effects of mTORi on HAEC proliferation. Further study may facilitate the development of effective strategies to reduce the side effects of DES used in coronary interventions.