Hou Meng, Cui Jinjin, Liu Jingjin, Liu Fang, Jiang Rui, Liu Kai, Wang Yongshun, Yin Li, Liu Wenhua, Yu Bo
Key Laboratories of Education Ministry for Myocardial Ischemia Mechanism and Treatment, Harbin, Province Heilongjiang, China ; Department of Cardiology, The Second Affiliated Hospital of Harbin Medical University, Harbin, Province Heilongjiang, China.
Key Laboratories of Education Ministry for Myocardial Ischemia Mechanism and Treatment, Harbin, Province Heilongjiang, China ; Intensive Care Unit (ICU) Department, The Second Affiliated Hospital of Harbin Medical University, Harbin, Province Heilongjiang, China.
PLoS One. 2014 Jan 21;9(1):e85808. doi: 10.1371/journal.pone.0085808. eCollection 2014.
Angiopoietin-like 4 (ANGPTL4) is a potential anti-apoptotic agent for various cells. We examined the protective effect of ANGPTL4 on hypoxia/serum deprivation (SD)-induced apoptosis of MSCs, as well as the possible mechanisms. MSCs were obtained from rat bone marrow and cultured in vitro. Apoptosis was induced by hypoxia/SD for up to 24 hr, and assessed by flow cytometry and TUNEL assay. Expression levels of Akt, ERK1/2, focal adhesion kinase (FAK), Src, Bcl-2, Bax, cytochrome C and cleaved caspase-3 were detected by Western blotting. Integrin β1 mRNA was detected by qRT-PCR. Mitochondrial membrane potential was assayed using a membrane-permeable dye. Hypoxia/SD-induced apoptosis was significantly attenuated by recombinant rat ANGPTL4 in a concentration dependent manner. Moreover, ANGPTL4 decreased the hypoxia/SD-induced caspase-3 cleavage and the cytochrome C release, but increased the Bcl-2/Bax ratio and the mitochondrial membrane potential. Decreased expression of integrin β1, the ANGPTL4 receptor was observed during hypoxia/SD conditions, however, such decrease was reversed by ANGPTL4. In addition, ANGPTL4 induced integrin β1-associated FAK and Src phosphorylation, which was blocked by anti-integrin β1 antibody. ANGPTL4 also reversed the hypoxia/SD-induced decrease of Akt and ERK 1/2 phosphorylation, and the effect of ANGPTL4 was abolished by inhibitors of either integrins, ERK1/2, or phosphatidylinositol 3-kinase (PI3K). Blocking integrinβ1, Akt or ERK largely attenuated anti-apoptotic effect of ANGPTL4. ANGPTL4 protects MSCs from hypoxia/SD-induced apoptosis by interacting with integrins to stimulate FAK complex, leading to downstream ERK1/2 and PI3K/Akt signaling pathways and mimicking the pathway in which MSCs contact with the extracellular matrix.
血管生成素样蛋白4（ANGPTL4）是一种对多种细胞具有潜在抗凋亡作用的因子。我们研究了ANGPTL4对缺氧/血清剥夺（SD）诱导的间充质干细胞（MSCs）凋亡的保护作用及其可能机制。从大鼠骨髓中获取MSCs并进行体外培养。通过缺氧/SD诱导凋亡长达24小时，然后用流式细胞术和TUNEL法进行评估。通过蛋白质免疫印迹法检测Akt、ERK1/2、粘着斑激酶（FAK）、Src、Bcl-2、Bax、细胞色素C和裂解的caspase-3的表达水平。通过qRT-PCR检测整合素β1 mRNA。使用膜通透性染料检测线粒体膜电位。重组大鼠ANGPTL4以浓度依赖性方式显著减轻缺氧/SD诱导的凋亡。此外，ANGPTL4减少了缺氧/SD诱导的caspase-3裂解和细胞色素C释放，但增加了Bcl-2/Bax比值和线粒体膜电位。在缺氧/SD条件下观察到ANGPTL4受体整合素β1的表达降低，然而，ANGPTL4可逆转这种降低。此外，ANGPTL4诱导整合素β1相关的FAK和Src磷酸化，而抗整合素β1抗体可阻断这种磷酸化。ANGPTL4还逆转了缺氧/SD诱导的Akt和ERK 1/2磷酸化降低，并且整合素、ERK1/2或磷脂酰肌醇3激酶（PI3K）抑制剂可消除ANGPTL4的作用。阻断整合素β1/Akt或ERK可大大减弱ANGPTL4的抗凋亡作用。ANGPTL4通过与整合素相互作用刺激FAK复合物，从而激活下游ERK1/2和PI3K/Akt信号通路，模拟MSCs与细胞外基质接触的途径，保护MSCs免受缺氧/SD诱导的凋亡。
