Reventun Paula, Alique Matilde, Cuadrado Irene, Márquez Susana, Toro Rocío, Zaragoza Carlos, Saura Marta
From the Biology Systems Department, Physiology, School of Medicine and Health Sciences, Universidad Alcalá (IRYCIS), Madrid, Spain (P.R., M.A., S.M., M.S.); Cardiology Department, University Francisco de Vitoria/Hospital Ramón y Cajal Research Unit (IRYCIS), Madrid, Spain (C.Z.); and Cardiology Department, School of Medicine, Cádiz University, Spain (R.T.).
Arterioscler Thromb Vasc Biol. 2017 Jul;37(7):1272-1281. doi: 10.1161/ATVBAHA.117.309560. Epub 2017 May 25.
ILK (integrin-linked kinase) plays a key role in controlling vasomotor tone and is decreased in atherosclerosis. The objective of this study is to test whether nitric oxide (NO) regulates ILK in vascular remodeling.
We found a striking correlation between increased levels of inducible nitric oxide and decreased ILK levels in human atherosclerosis and in a mouse model of vascular remodeling (carotid artery ligation) comparing with iNOS (inducible NO synthase) knockout mice. iNOS induction produced the same result in mouse aortic endothelial cells, and these effects were mimicked by an NO donor in a time-dependent manner. We found that NO decreased ILK protein stability by promoting the dissociation of the complex ILK/Hsp90 (heat shock protein 90)/eNOS (endothelial NO synthase), leading to eNOS uncoupling. NO also destabilized ILK signaling platform and lead to decreased levels of paxillin and α-parvin. ILK phosphorylation of its downstream target GSK3-β (glycogen synthase kinase 3 beta) was decreased by NO. Mechanistically, NO increased ILK ubiquitination mediated by the E3 ubiquitin ligase CHIP (C terminus of HSC70-interacting protein), but ILK ubiquitination was not followed by proteasome degradation. Alternatively, NO drove ILK to degradation through the endocytic-lysosomal pathway. ILK colocalized with the lysosome marker LAMP-1 (lysosomal-associated membrane protein 1) in endothelial cells, and inhibition of lysosome activity with chloroquine reversed the effect of NO. Likewise, ILK colocalized with the early endosome marker EEA1 (early endosome antigen 1). ILK endocytosis proceeded via dynamin because a specific inhibitor of dynamin (Dyngo 4a) was able to reverse ILK endocytosis and its lysosome degradation.
Endocytosis regulates ILK signaling in vascular remodeling where there is an overload of inducible NO, and thus its inhibition may represent a novel target to fight atherosclerotic disease.
整合素连接激酶（ILK）在控制血管舒缩张力中起关键作用，且在动脉粥样硬化中表达降低。本研究的目的是检测一氧化氮（NO）是否在血管重塑过程中调节ILK。
我们发现，与诱导型一氧化氮合酶（iNOS）基因敲除小鼠相比，在人类动脉粥样硬化以及血管重塑小鼠模型（颈动脉结扎）中，诱导型一氧化氮水平升高与ILK水平降低之间存在显著相关性。iNOS诱导在小鼠主动脉内皮细胞中产生了相同的结果，并且这些效应可被NO供体以时间依赖性方式模拟。我们发现，NO通过促进ILK/Hsp90（热休克蛋白90）/eNOS（内皮型一氧化氮合酶）复合物的解离来降低ILK蛋白稳定性，导致eNOS解偶联。NO还使ILK信号平台不稳定，并导致桩蛋白和α-帕文水平降低。NO降低了其下游靶点糖原合酶激酶3β（GSK3-β）的ILK磷酸化水平。机制上，NO增加了由E3泛素连接酶CHIP（HSC70相互作用蛋白的C末端）介导的ILK泛素化，但ILK泛素化后并未发生蛋白酶体降解。另外，NO通过内吞-溶酶体途径促使ILK降解。在内皮细胞中，ILK与溶酶体标志物溶酶体相关膜蛋白1（LAMP-1）共定位，用氯喹抑制溶酶体活性可逆转NO的作用。同样，ILK与早期内体标志物早期内体抗原1（EEA1）共定位。ILK内吞通过发动蛋白进行，因为发动蛋白的特异性抑制剂（Dyngo 4a）能够逆转ILK内吞及其溶酶体降解。
内吞作用在诱导型NO过载的血管重塑过程中调节ILK信号，因此抑制内吞作用可能是对抗动脉粥样硬化疾病的新靶点。
