Hanson C Jane, Bootman Martin D, Distelhorst Clark W, Wojcikiewicz Richard J H, Roderick H Llewelyn
Laboratory of Molecular Signalling, Babraham Institute, Babraham, Cambridge CB2 4AT, UK.
Cell Calcium. 2008 Sep;44(3):324-38. doi: 10.1016/j.ceca.2008.01.003. Epub 2008 Apr 14.
Cell survival is promoted by the oncoprotein Bcl-2. Previous studies have established that one of the pro-survival actions of Bcl-2 is to reduce cellular fluxes of Ca2+ within cells. In particular, Bcl-2 has been demonstrated to inhibit the release of Ca2+ from the endoplasmic reticulum. However, the mechanism by which Bcl-2 causes reduced Ca2+ release is unclear. In the accompanying paper [C.J. Hanson, M.D. Bootman, C.W. Distelhorst, T. Maraldi, H.L. Roderick, The cellular concentration of Bcl-2 determines its pro- or anti-apoptotic effect, Cell Calcium (2008)], we described that only stable expression of Bcl-2 allowed it to work in a pro-survival manner whereas transient expression did not. In this study, we have employed HEK-293 cells that stably express Bcl-2, and which are, therefore, protected from pro-apoptotic stimuli, to examine the effect of Bcl-2 on Ca2+ homeostasis and signalling. We observed that Bcl-2 expression decreased the Ca2+ responses of cells induced by application of submaximal agonist concentrations. Whereas, decreasing endogenous Bcl-2 concentration using siRNA potentiated Ca2+ responses. Furthermore, we found that Bcl-2 expression reduced mitochondrial Ca2+ uptake by raising the threshold cytosolic Ca2+ concentration required to activate sequestration. Using a number of different assays, we did not find any evidence for reduction of endoplasmic reticulum luminal Ca2+ in our Bcl-2-expressing cells. Indeed, we observed that Bcl-2 served to preserve the content of the agonist-sensitive Ca2+ pool. Endogenous Bcl-2 was found to interact with inositol 1,4,5-trisphosphate receptors (InsP3Rs) in our cells, and to modify the profile of InsP3R expression. Our data suggest that the presence of Bcl-2 in the proteome of cells has multiple effects on agonist-mediated Ca2+ signals, and can abrogate responses to submaximal levels of stimulation through direct control of InsP3Rs.
癌蛋白Bcl-2可促进细胞存活。以往研究表明，Bcl-2的促存活作用之一是减少细胞内Ca2+的流动。特别是，已证实Bcl-2可抑制内质网释放Ca2+。然而，Bcl-2导致Ca2+释放减少的机制尚不清楚。在随附论文[C.J. 汉森、M.D. 布特曼、C.W. 迪斯特尔霍斯特、T. 马拉尔迪、H.L. 罗德里克，Bcl-2的细胞浓度决定其促凋亡或抗凋亡作用，《细胞钙》(2008年)]中，我们描述了只有Bcl-2的稳定表达才能使其以促存活方式发挥作用，而瞬时表达则不能。在本研究中，我们使用稳定表达Bcl-2、因此免受促凋亡刺激的HEK-293细胞，来研究Bcl-2对Ca2+稳态和信号传导的影响。我们观察到，Bcl-2的表达降低了应用次最大激动剂浓度诱导的细胞Ca2+反应。而使用小干扰RNA降低内源性Bcl-2浓度则增强了Ca2+反应。此外，我们发现Bcl-2的表达通过提高激活螯合所需的胞质Ca2+阈值浓度来减少线粒体Ca2+摄取。使用多种不同的检测方法，我们在表达Bcl-2的细胞中未发现内质网腔Ca2+减少的任何证据。事实上，我们观察到Bcl-2有助于维持激动剂敏感Ca2+池的含量。我们发现内源性Bcl-2在我们的细胞中与肌醇1,4,5-三磷酸受体(InsP3Rs)相互作用，并改变InsP3R表达谱。我们的数据表明，细胞蛋白质组中Bcl-2的存在对激动剂介导的Ca2+信号有多种影响，并且可以通过直接控制InsP3Rs消除对次最大刺激水平的反应。
