Store-operated calcium entry-activated autophagy protects EPC proliferation via the CAMKK2-MTOR pathway in ox-LDL exposure

Improving biological functions of endothelial progenitor cells (EPCs) is beneficial to maintaining endothelium homeostasis and promoting vascular re-endothelialization. Because macroautophagy/autophagy has been documented as a double-edged sword in cell functions, its effects on EPCs remain to be elucidated. This study was designed to explore the role and molecular mechanisms of store-operated calcium entry (SOCE)-activated autophagy in proliferation of EPCs under hypercholesterolemia. We employed oxidized low-density lipoprotein (ox-LDL) to mimic hypercholesterolemia in bone marrow-derived EPCs from rat. Ox-LDL dose-dependently activated autophagy flux, while inhibiting EPC proliferation. Importantly, inhibition of autophagy either by silencing Atg7 or by 3-methyladenine treatment, further aggravated proliferative inhibition by ox-LDL, suggesting the protective effects of autophagy against ox-LDL. Interestingly, ox-LDL increased STIM1 expression and intracellular Ca2+ concentration. Either Ca2+ chelators or deficiency in STIM1 attenuated ox-LDL-induced autophagy activation, confirming the involvement of SOCE in the process. Furthermore, CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2, β) activation and MTOR (mechanistic target of rapamycin [serine/threonine kinase]) deactivation were associated with autophagy modulation. Together, our results reveal a novel signaling pathway of SOCE-CAMKK2 in the regulation of autophagy and offer new insights into the important roles of autophagy in maintaining proliferation and promoting the survival capability of EPCs. This may be beneficial to improving EPC transplantation efficacy and enhancing vascular re-endothelialization in patients with hypercholesterolemia.改善内皮祖细胞（endothelial progenitor cells, EPCs）的生物学功能，有助于维持内皮稳态并促进血管再内皮化。由于巨自噬/自噬（macroautophagy/autophagy）在细胞功能中被证实是一把双刃剑，其对EPCs的作用仍有待阐明。本研究旨在探讨高胆固醇血症环境下，钙池操纵性钙内流（store-operated calcium entry, SOCE）激活的自噬在EPCs增殖中的作用及分子机制。我们采用氧化型低密度脂蛋白（oxidized low-density lipoprotein, ox-LDL）处理大鼠骨髓来源的EPCs，以模拟高胆固醇血症环境。ox-LDL呈剂量依赖性激活自噬流，同时抑制EPCs增殖。值得注意的是，通过沉默Atg7或使用3-甲基腺嘌呤抑制自噬，会进一步加重ox-LDL介导的增殖抑制效应，提示自噬对ox-LDL诱导的细胞损伤具有保护作用。有趣的是，ox-LDL可上调基质相互作用分子1（STIM1）的表达及细胞内钙离子浓度。钙离子螯合剂或STIM1基因敲除均可削弱ox-LDL诱导的自噬激活，证实了SOCE参与该调控过程。进一步研究发现，钙/钙调蛋白依赖性蛋白激酶激酶2（CAMKK2, calcium/calmodulin-dependent protein kinase kinase 2, β）的激活与哺乳动物雷帕霉素靶蛋白（MTOR, mechanistic target of rapamycin [丝氨酸/苏氨酸激酶]）的失活，与自噬的调控密切相关。综上，本研究揭示了SOCE-CAMKK2调控自噬的全新信号通路，为阐明自噬在维持EPCs增殖、提升其存活能力中的重要作用提供了新见解。该发现或有助于改善高胆固醇血症患者的EPC移植疗效，促进血管再内皮化进程。