75千道尔顿的沉默调节蛋白1可阻断肿瘤坏死因子α介导的人骨关节炎软骨细胞凋亡。
75-kd sirtuin 1 blocks tumor necrosis factor α-mediated apoptosis in human osteoarthritic chondrocytes.
作者信息
Oppenheimer Hanna, Gabay Odile, Meir Hadar, Haze Amir, Kandel Leonid, Liebergall Meir, Gagarina Viktoria, Lee Eun Jin, Dvir-Ginzberg Mona
机构信息
Laboratory of Cartilage Biology, Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University, Hadassah Ein Kerem, Jerusalem, Israel.
出版信息
Arthritis Rheum. 2012 Mar;64(3):718-28. doi: 10.1002/art.33407.
OBJECTIVE
Sirtuin 1 (SirT1) has been implicated in the regulation of human cartilage homeostasis and chondrocyte survival. Exposing human osteoarthritic (OA) chondrocytes to tumor necrosis factor α (TNFα) generates a stable and enzymatically inactive 75-kd form of SirT1 (75SirT1) via cathepsin B-mediated cleavage. Because 75SirT1 is resistant to further degradation, we hypothesized that it has a distinct role in OA, and the present study was undertaken to identify this role.
METHODS
The presence of cathepsin B and 75SirT in OA and normal human chondrocytes was analyzed. Confocal imaging of SirT1 was used to monitor its subcellular trafficking following TNFα stimulation. Coimmunofluorescence staining for cathepsin B, mitochondrial cytochrome oxidase subunit IV, and lysosome-associated membrane protein 1 together with SirT1 was performed. Human chondrocytes were tested for apoptosis by fluorescence-activated cell sorter analysis and immunoblotting for caspases 3 and 8. Human chondrocyte mitochondrial extracts were obtained and analyzed for 75SirT1-cytochrome c association.
RESULTS
Confocal imaging and immunoblot analyses following TNFα challenge of human chondrocytes demonstrated that 75SirT1 was exported to the cytoplasm and colocalized with the mitochondrial membrane. Consistent with this, immunoprecipitation and immunoblot analyses revealed that 75SirT1 is enriched in mitochondrial extracts and associates with cytochrome c following TNFα stimulation. Preventing nuclear export of 75SirT1 or reducing levels of full-length SirT1 and 75SirT1 augmented chondrocyte apoptosis in the presence of TNFα. Levels of cathepsin B and 75SirT1 were elevated in OA versus normal chondrocytes. Additional analyses showed that human chondrocytes exposed to OA-derived synovial fluid generated the 75SirT1 fragment.
CONCLUSION
These data suggest that 75SirT1 promotes chondrocyte survival following exposure to proinflammatory cytokines.
目的
沉默调节蛋白1（SirT1）与人类软骨内环境稳定及软骨细胞存活的调节有关。将人类骨关节炎（OA）软骨细胞暴露于肿瘤坏死因子α（TNFα）下，通过组织蛋白酶B介导的裂解可产生一种稳定的、无酶活性的75-kd形式的SirT1（75SirT1）。由于75SirT1对进一步降解具有抗性，我们推测它在OA中具有独特作用，因此开展本研究以确定该作用。
方法
分析OA和正常人软骨细胞中组织蛋白酶B和75SirT的存在情况。利用SirT1的共聚焦成像监测TNFα刺激后其亚细胞转运情况。对组织蛋白酶B、线粒体细胞色素氧化酶亚基IV、溶酶体相关膜蛋白1与SirT1进行共免疫荧光染色。通过荧光激活细胞分选分析和半胱天冬酶3和8的免疫印迹检测人类软骨细胞的凋亡情况。获取人类软骨细胞线粒体提取物并分析75SirT1与细胞色素c的结合情况。
结果
对人类软骨细胞进行TNFα刺激后的共聚焦成像和免疫印迹分析表明，75SirT1被转运至细胞质并与线粒体膜共定位。与此一致，免疫沉淀和免疫印迹分析显示，75SirT1在TNFα刺激后富集于线粒体提取物中并与细胞色素c结合。在TNFα存在的情况下，阻止75SirT1的核输出或降低全长SirT1和75SirT1的水平会增加软骨细胞凋亡。与正常软骨细胞相比，OA软骨细胞中组织蛋白酶B和75SirT1的水平升高。进一步分析表明，暴露于OA来源滑液中的人类软骨细胞会产生75SirT1片段。
结论
这些数据表明，75SirT1在暴露于促炎细胞因子后可促进软骨细胞存活。
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