Yip Kirk H M, Zheng Ming H, Steer James H, Giardina Tindaro M, Han Renzhi, Lo Susan Z, Bakker Anthony J, Cassady A Ian, Joyce David A, Xu Jiake
Molecular Orthopaedic Laboratory, School of Surgery and Pathology, and Western Australian Institute for Medical Research, Nedlands, WA 6009, Australia.
J Bone Miner Res. 2005 Aug;20(8):1462-71. doi: 10.1359/JBMR.050324. Epub 2005 Mar 28.
The mechanism by which TG modulates osteoclast formation and apoptosis is not clear. In this study, we showed a biphasic effect of TG on osteoclast formation and apoptosis through the regulation of ROS production, caspase-3 activity, cytosolic Ca2+, and RANKL-induced activation of NF-kappaB and AP-1 activities.
Apoptosis and differentiation are among the consequences of changes in intracellular Ca2+ levels. In this study, we investigated the effects of the endoplasmic reticular Ca2+-ATPase inhibitor, thapsigargin (TG), on osteoclast apoptosis and differentiation.
Both RAW264.7 cells and primary spleen cells were used to examine the effect of TG on RANKL-induced osteoclastogenesis. To determine the action of TG on signaling pathways, we used reporter gene assays for NF-kappaB and activator protein-1 (AP-1) activity, Western blotting for phospho-extracellular signal-related kinase (ERK), and fluorescent probes to measure changes in levels of intracellular calcium and reactive oxygen species (ROS). To assess rates of apoptosis, we measured changes in annexin staining, caspase-3 activity, and chromatin and F-actin microfilament structure.
At concentrations that caused a rapid rise in intracellular Ca2+, TG increased caspase-3 activity and promoted apoptosis in osteoclast-like cells (OLCs). Low concentrations of TG, which were insufficient to measurably alter intracellular Ca2+, unexpectedly suppressed caspase-3 activity and enhanced RANKL-induced osteoclastogenesis. At these lower concentrations, TG potentiated ROS production and RANKL-induced NF-kappaB activity, but suppressed RANKL-induced AP-1 activity and had little effect on ERK phosphorylation.
Our novel findings of a biphasic effect of TG are incompletely explained by our current understanding of TG action, but raise the possibility that low intensity or local changes in subcellular Ca2+ levels may regulate intracellular differentiation signaling. The extent of cross-talk between Ca2+ and RANKL-mediated intracellular signaling pathways might be important in determining whether cells undergo apoptosis or differentiate into OLCs.
三磷酸肌醇（TG）调节破骨细胞形成和凋亡的机制尚不清楚。在本研究中，我们发现TG通过调节活性氧（ROS）生成、半胱天冬酶-3活性、胞质钙离子（Ca2+）以及核因子κB（NF-κB）和激活蛋白-1（AP-1）活性的核因子κB受体活化因子配体（RANKL）诱导激活，对破骨细胞形成和凋亡具有双相作用。
凋亡和分化是细胞内Ca2+水平变化的结果之一。在本研究中，我们研究了内质网Ca2+ -ATP酶抑制剂毒胡萝卜素（TG）对破骨细胞凋亡和分化的影响。
使用RAW264.7细胞和原代脾细胞来检测TG对RANKL诱导的破骨细胞生成的影响。为了确定TG对信号通路的作用，我们使用报告基因检测法检测NF-κB和激活蛋白-1（AP-1）活性，使用蛋白质免疫印迹法检测磷酸化细胞外信号调节激酶（ERK），并使用荧光探针测量细胞内钙和活性氧（ROS）水平的变化。为了评估凋亡率，我们测量膜联蛋白染色、半胱天冬酶-3活性以及染色质和F-肌动蛋白微丝结构的变化。
在导致细胞内Ca2+迅速升高的浓度下，TG增加了半胱天冬酶-3活性并促进了破骨细胞样细胞（OLCs）的凋亡。低浓度的TG不足以显著改变细胞内Ca2+，却意外地抑制了半胱天冬酶-3活性并增强了RANKL诱导的破骨细胞生成。在这些较低浓度下，TG增强了ROS生成和RANKL诱导的NF-κB活性，但抑制了RANKL诱导的AP-1活性，并且对ERK磷酸化影响很小。
我们关于TG双相作用的新发现目前对TG作用的理解尚不能完全解释，但提出了细胞内亚细胞Ca2+水平的低强度或局部变化可能调节细胞内分化信号的可能性。Ca2+和RANKL介导的细胞内信号通路之间的相互作用程度在决定细胞是发生凋亡还是分化为OLCs方面可能很重要。
