Parkin clearance of dysfunctional mitochondria regulates ROS levels and increases survival of human chondrocytes.
Parkin clearance of dysfunctional mitochondria regulates ROS levels and increases survival of human chondrocytes.
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DOI：
10.1016/j.joca.2017.07.020
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发表时间：
2018-08
影响因子：
7
通讯作者：
Haqqi TM
中科院分区：
文献类型：
--
作者：
Ansari MY;Khan NM;Ahmad I;Haqqi TM
线粒体功能障碍、氧化应激和软骨细胞死亡是骨关节炎（OA）发生和发病的重要因素。在这项研究中，我们确定了帕金蛋白在病理条件下清除受损/功能障碍的线粒体、调节ROS水平和软骨细胞存活中的表达和作用。人软骨细胞来自膝关节OA软骨的未受影响区域（n=12），并且用IL-1β刺激以模拟病理条件。线粒体膜去极化和ROS水平测定使用特定的染料和流式细胞术。通过Western印迹法检测ATG 5、Beclin 1、免疫荧光染色和共聚焦显微镜观察自噬。通过qRT-PCR测定基因表达。使用Amaxa系统转染siRNA、野生型和突变型帕金质粒。PI染色和TUNEL法检测软骨细胞凋亡。IL-1β刺激OA软骨细胞后，细胞内ROS生成增多，线粒体膜损伤，线粒体损伤程度加重，细胞凋亡率增高。IL-1β对Parkin表达缺失的软骨细胞的刺激导致持续高水平的ROS、受损/功能障碍的线粒体的积累和增强的凋亡。在IL-1β刺激的OA软骨细胞中，需要Parkin易位到去极化/受损的线粒体和p62/SQSTM 1的募集来消除受损/功能障碍的线粒体。重要的是，我们证明了帕金消除去极化/受损的线粒体需要帕金泛素连接酶的活性，并导致在病理条件下OA软骨细胞中ROS水平降低和细胞凋亡抑制。我们的数据表明，帕金功能，以消除去极化/受损的线粒体在软骨细胞中，这是必要的线粒体质量控制，ROS水平的调节和软骨细胞在病理条件下的生存。
Mitochondrial dysfunction, oxidative stress and chondrocyte death are important contributors to the development and pathogenesis of osteoarthritis (OA). In this study, we determined the expression and role of Parkin in the clearance of damaged/dysfunctional mitochondria, regulation of ROS levels and chondrocyte survival under pathological conditions. Human chondrocytes were from the unaffected area of knee OA cartilage (n=12) and were stimulated with IL-1β to mimic pathological conditions. Mitochondrial membrane depolarization and ROS levels were determined using specific dyes and flow cytometry. Autophagy was determined by Western blotting for ATG5, Beclin1, immunofluorescence staining and confocal microscopy. Gene expression was determined by qRT-PCR. siRNA, wild-type and mutant Parkin plasmids were transfected using Amaxa system. Apoptosis was determined by PI staining of chondrocytes and TUNEL assay. IL-1β-stimulated OA chondrocytes showed high levels of ROS generation, mitochondrial membrane damage, accumulation of damaged mitochondria and higher incidence of apoptosis. IL-1β stimulation of chondrocytes with depleted Parkin expression resulted in sustained high levels of ROS, accumulation of damaged/dysfunctional mitochondria and enhanced apoptosis. Parkin translocation to depolarized/damaged mitochondria and recruitment of p62/SQSTM1 was required for the elimination of damaged/dysfunctional mitochondria in IL-1β-stimulated OA chondrocytes. Importantly we demonstrate that Parkin elimination of depolarized/damaged mitochondria required the Parkin ubiquitin ligase activity and resulted in reduced ROS levels and inhibition of apoptosis in OA chondrocytes under pathological conditions. Our data demonstrates that Parkin functions to eliminate depolarized/damaged mitochondria in chondrocytes which is necessary for mitochondrial quality control, regulation of ROS levels and chondrocyte survival under pathological conditions.
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影响因子：
2.3
作者：
Cillero-Pastor B;Rego-Pérez I;Oreiro N;Fernandez-Lopez C;Blanco FJ
通讯作者：
Blanco FJ
影响因子：
11.4
作者：
Jones, Dean P.
通讯作者：
Jones, Dean P.
DOI：
10.1083/jcb.201210111
发表时间：
2013-01-21
期刊：
The Journal of cell biology
影响因子：
--
作者：
Lazarou M;Narendra DP;Jin SM;Tekle E;Banerjee S;Youle RJ
通讯作者：
Youle RJ
影响因子：
4.4
作者：
Blanco, FJ;López-Armada, MJ;Maneiro, E
通讯作者：
Maneiro, E
DOI：
10.1083/jcb.201102095
发表时间：
2011-07-11
期刊：
The Journal of cell biology
影响因子：
--
作者：
Finkel T
通讯作者：
Finkel T