Intracellular zinc fluxes associated with apoptosis in growth plate chondrocytes
Intracellular zinc fluxes associated with apoptosis in growth plate chondrocytes
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DOI：
10.1002/jcb.10446
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发表时间：
2003-04-01
影响因子：
4
通讯作者：
Wuthier, RE
中科院分区：
文献类型：
--
作者：
Sauer, GR;Smith, DM;Wuthier, RE
已知骨骺生长板软骨细胞释放的基质囊泡含有大量不稳定的Zn2+。鸡跖骨的分带分析显示，生长板的静止/增殖区含有高水平的Zn2+，而肥大软骨的含量明显较低，表明随着软骨细胞的成熟，细胞中Zn2+的损失。用荧光螯合剂甲苯磺酰氨基喹啉（TSQ）测定生长板软骨细胞原代培养物中细胞内不稳定锌（Zn2+）的含量，并用TSQ衍生物锌素（zinquin）进行多光子激光扫描显微镜（MPLSM）成像。短期暴露于Zn2+，无论是否存在吡硫酮，都会导致胞浆中Zn2+的显著增加。通过膜渗透的Zn2+螯合剂TPEN处理后，可迅速降低不稳定的Zn2+水平，并引发细胞凋亡。与已知的软骨细胞凋亡诱导剂孵育24小时后，细胞内Zn2+水平显著降低。细胞内Zn2+的损失伴随着胞质金属结合蛋白金属硫蛋白的显著减少。MPLSM检测Zn2+处理后的细胞，锌离子荧光均匀升高。用TPEN淬灭Zn2+荧光处理负载Zn2+的细胞，证实软骨细胞中观察到的荧光是由于细胞内Zn2+的存在。在一定浓度的Zn2+处理的细胞中，观察到锌荧光呈剂量依赖性增加。用诱导凋亡的化学物质短期处理培养的软骨细胞导致细胞内不稳定的Zn2+短暂增加。这些结果表明，在软骨细胞凋亡的早期阶段，Zn2+从细胞内结合位点被动员，随后从细胞中丢失。Zn2+的早期动员为其向基质囊泡和细胞外基质的移动提供了一种机制。(C) 2003 Wiley-Liss, Inc。
Matrix vesicles released by epiphyseal growth plate chondrocytes are known to contain a significant quantity of labile Zn2+. Zonal analysis of chicken metatarsal bones showed that the resting/proliferative region of the growth plate contained high levels of Zn2+ with significantly lower levels in the hypertrophic cartilage suggesting a loss of cellular Zn2+ as the chondrocytes mature. Intracellular labile Zn2+ was measured in primary cultures of growth plate chondrocytes by assay with the fluorescent Zn-chelator toluenesulfonamidoquinoline (TSQ) and imaged bymulti-photon laser scanning microscopy (MPLSM) with the TSQ derivative zinquin. Short-term exposure to Zn2+, both in the presence and absence of pyrithione resulted in significant increases in cytosolic Zn2+. Treatment with the membrane-permeant Zn2+ chelator TPEN rapidly reduced the levels of labile Zn2+ and triggered apoptosis. Cytosolic Zn2+ levels were significantly reduced following 24-h incubations with known inducers of chondrocyte apoptosis. The loss of intracellular Zn2+ was accompanied by a significant reduction in the cytosolic metal-binding protein metallothionein. Examination of Zn2+-treated cells with MPLSM showed uniformly higher zinquin fluorescence. Treatment of Zn2+ loaded cells with TPEN quenched zinquin fluorescence confirming that the observed fluorescence in chondrocytes is due to the presence of intracellular Zn2+. A dose-dependent increase inzinquin fluorescence was observed in cells treated with a range of Zn2+ concentrations. Short-term treatment of cultured chondrocytes with apoptosis-inducing chemicals resulted in transient increases in intracellular labile Zn2+. These results indicate that Zn2+ is mobilized from intracellular binding sites in the early stages of chondrocyte apoptosis and is subsequently lost from the cells. The early mobilization of Zn2+ provides a mechanism for its movementto matrix vesicles and the extracellular matrix. (C) 2003 Wiley-Liss, Inc.