Articular chondrocyte network mediated by gap junctions: role in metabolic cartilage homeostasis.
Articular chondrocyte network mediated by gap junctions: role in metabolic cartilage homeostasis.
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DOI：
10.1136/annrheumdis-2013-204244
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发表时间：
2015-01
影响因子：
27.4
通讯作者：
Blanco FJ
中科院分区：
文献类型：
--
作者：
Mayan MD;Gago-Fuentes R;Carpintero-Fernandez P;Fernandez-Puente P;Filgueira-Fernandez P;Goyanes N;Valiunas V;Brink PR;Goldberg GS;Blanco FJ
这项研究调查了软骨基质中的软骨细胞是否具有通过电压门控缝隙连接(GJ)通道介导的细胞间连接进行通信的能力。冰冻软骨标本进行免疫荧光和免疫组织化学检测。样品在脱水前被包埋在硬脂酸盐缓冲液中进行扫描电子显微镜观察。免疫共沉淀实验和质谱分析鉴定与Cx43 C末端相互作用的蛋白质。通过原位电穿孔、电生理和染料注入实验研究了GJ通讯。利用Transwell分层培养系统和MS对转移氨基酸进行鉴定和定量。显微镜图像显示，基质内存在多个连接软骨细胞的细胞突起。这些突起的长度在5到150μm之间。MS数据分析表明，Cx43的C末端与几种与Cx转运和GJ组装有关的细胞骨架蛋白相互作用，包括α-微管蛋白和β-微管蛋白、肌动蛋白和纽蛋白。电生理实验表明，12聚体寡核苷酸可在注射后12分钟内在软骨细胞间转移。在22min和35min内，葡萄糖分布均匀。将葡萄糖电击入无GJS的A549细胞，未检测到转移。Transwell分层培养系统结合MS分析表明，连接蛋白可以介导L-赖氨酸和L-精氨酸在软骨细胞之间的转移。这项研究表明，软骨细胞之间的细胞间连接含有GJS，它通过交换包括葡萄糖和必需氨基酸在内的营养物质，在细胞与细胞之间的沟通和代谢功能中发挥关键作用。GJS介导的三维细胞网络可能通过调节代谢和生理动态平衡来维持软骨组织。
This study investigated whether chondrocytes within the cartilage matrix have the capacity to communicate through intercellular connections mediated by voltage-gated gap junction (GJ) channels. Frozen cartilage samples were used for immunofluorescence and immunohistochemistry assays. Samples were embedded in cacodylate buffer before dehydration for scanning electron microscopy. Co-immunoprecipitation experiments and mass spectrometry (MS) were performed to identify proteins that interact with the C-terminal end of Cx43. GJ communication was studied through in situ electroporation, electrophysiology and dye injection experiments. A transwell layered culture system and MS were used to identify and quantify transferred amino acids. Microscopic images revealed the presence of multiple cellular projections connecting chondrocytes within the matrix. These projections were between 5 and 150 μm in length. MS data analysis indicated that the C-terminus of Cx43 interacts with several cytoskeletal proteins implicated in Cx trafficking and GJ assembly, including α-tubulin and β-tubulin, actin, and vinculin. Electrophysiology experiments demonstrated that 12-mer oligonucleotides could be transferred between chondrocytes within 12 min after injection. Glucose was homogeneously distributed within 22 and 35 min. No transfer was detected when glucose was electroporated into A549 cells, which have no GJs. Transwell layered culture systems coupled with MS analysis revealed connexins can mediate the transfer of L-lysine and L-arginine between chondrocytes. This study reveals that intercellular connections between chondrocytes contain GJs that play a key role in cell-cell communication and a metabolic function by exchange of nutrients including glucose and essential amino acids. A three-dimensional cellular network mediated through GJs might mediate metabolic and physiological homeostasis to maintain cartilage tissue.
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DOI：
10.1083/jcb.129.3.805
发表时间：
1995-05
期刊：
The Journal of cell biology
影响因子：
--
作者：
Elfgang C;Eckert R;Lichtenberg-Fraté H;Butterweck A;Traub O;Klein RA;Hülser DF;Willecke K
通讯作者：
Willecke K
影响因子：
2.4
作者：
Chi, SS;Rattner, JB;Matyas, JR
通讯作者：
Matyas, JR
影响因子：
4.8
作者：
Goldberg, GS;Moreno, AP;Lampe, PD
通讯作者：
Lampe, PD
影响因子：
7
作者：
Holloway, I;Kayser, M;Knight, MM
通讯作者：
Knight, MM
影响因子：
2.5
作者：
DEALY, CN;BEYER, EC;KOSHER, RA
通讯作者：
KOSHER, RA