细胞外泛素抑制心肌细胞中β-AR 刺激的细胞凋亡：GSK-3β 和线粒体途径的作用。
Extracellular ubiquitin inhibits beta-AR-stimulated apoptosis in cardiac myocytes: role of GSK-3beta and mitochondrial pathways.
机构信息
Department of Physiology, James H Quillen College of Medicine, James H Quillen Veterans Affairs Medical Center, East Tennessee State University, PO Box 70576, Johnson City, TN 37614, USA.
出版信息
Cardiovasc Res. 2010 Apr 1;86(1):20-8. doi: 10.1093/cvr/cvp402. Epub 2009 Dec 16.
AIMS
Beta-adrenergic receptor (beta-AR) stimulation induces apoptosis in adult rat ventricular myocytes (ARVMs) via the activation of glycogen synthase kinase-3beta (GSK-3beta) and mitochondrial pathways. However, beta-AR stimulation induces apoptosis only in a fraction ( approximately 15-20%) of ARVMs. We hypothesized that ARVMs may secrete/release a survival factor(s) which protects 80-85% of cells from apoptosis.
METHODS AND RESULTS
Using two-dimensional gel electrophoresis followed by MALDI TOF and MS/MS, we identified ubiquitin (Ub) in the conditioned media of ARVMs treated with beta-AR agonist (isoproterenol). Western blot analysis confirmed increased Ub levels in the conditioned media 3 and 6 h after beta-AR stimulation. Inhibition of beta1-AR and beta2-AR subtypes inhibited beta-AR-stimulated increases in extracellular levels of Ub, whereas activation of adenylyl cyclase using forskolin mimicked the effects of beta-AR stimulation. Incubation of cells with exogenous biotinylated Ub followed by western blot analysis of the cell lysates showed uptake of extracellular Ub into cells, which was found to be higher after beta-AR stimulation (1.9 +/- 0.4-fold; P < 0.05 vs. control, n = 6). Pre-treatment with Ub inhibited beta-AR-stimulated increases in apoptosis. Inhibition of phosphoinositide 3-kinase using wortmannin and LY-294002 prevented anti-apoptotic effects of extracellular Ub. Ub pre-treatment inhibited beta-AR-stimulated activation of GSK-3beta and c-Jun N-terminal kinase (JNK) and increases in the levels of cytosolic cytochrome c. The use of methylated Ub suggested that the anti-apoptotic effects of extracellular Ub are mediated via monoubiquitination.
CONCLUSION
beta-AR stimulation increases levels of Ub in the conditioned media. Extracellular Ub plays a protective role in beta-AR-stimulated apoptosis, possibly via the inactivation of GSK-3beta/JNK and mitochondrial pathways.
目的
β-肾上腺素能受体（β-AR）刺激通过激活糖原合酶激酶-3β（GSK-3β）和线粒体途径诱导成年大鼠心室肌细胞（ARVMs）凋亡。然而，β-AR 刺激仅在一小部分（约 15-20%）ARVMs 中诱导凋亡。我们假设 ARVMs 可能会分泌/释放一种生存因子（s），保护 80-85%的细胞免受凋亡。
方法和结果
使用二维凝胶电泳，然后使用 MALDI TOF 和 MS/MS，我们在β-AR 激动剂（异丙肾上腺素）处理的 ARVMs 的条件培养基中鉴定出泛素（Ub）。Western blot 分析证实，β-AR 刺激后 3 和 6 小时，条件培养基中的 Ub 水平增加。β1-AR 和β2-AR 亚基的抑制抑制了β-AR 刺激引起的细胞外 Ub 水平升高，而使用 forskolin 激活腺苷酸环化酶则模拟了β-AR 刺激的作用。将细胞与外源性生物素化 Ub 孵育，然后对细胞裂解物进行 Western blot 分析，显示细胞外 Ub 被摄取到细胞内，在β-AR 刺激后发现摄取量更高（1.9 +/- 0.4 倍；P < 0.05 与对照组相比，n = 6）。Ub 预处理抑制了β-AR 刺激引起的细胞凋亡增加。使用wortmannin 和 LY-294002 抑制磷酯酰肌醇 3-激酶可防止细胞外 Ub 的抗凋亡作用。Ub 预处理抑制了β-AR 刺激引起的 GSK-3β 和 c-Jun N 末端激酶（JNK）的激活以及细胞质细胞色素 c 水平的升高。使用甲基化 Ub 表明，细胞外 Ub 的抗凋亡作用是通过单泛素化介导的。
结论
β-AR 刺激增加了条件培养基中 Ub 的水平。细胞外 Ub 在β-AR 刺激的凋亡中发挥保护作用，可能通过失活 GSK-3β/JNK 和线粒体途径。
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