Oscillating fluid flow regulates gap junction communication in osteocytic MLO-Y4 cells by an ERK1/2 MAP kinase-dependent mechanism
Oscillating fluid flow regulates gap junction communication in osteocytic MLO-Y4 cells by an ERK1/2 MAP kinase-dependent mechanism
复制标题
DOI：
10.1016/s8756-3282(03)00167-4
复制
发表时间：
2003-07-01
期刊：
影响因子：
4.1
通讯作者：
Donahue, HJ
中科院分区：
文献类型：
--
作者：
Alford, AI;Jacobs, CR;Donahue, HJ
本研究旨在探讨振荡流对骨细胞样MLOY-4细胞间隙连接通讯（GJIC）和差距连接蛋白（Cx）43的影响。在平行板流动室中，将细胞暴露于剪切应力为+/-10达因/cm（2）和频率为1 Hz的振荡流体流1小时。将对照细胞在室中孵育，但不暴露于振荡流体流。GJIC的功能分析表明，MLOY-4细胞暴露于振荡流体流建立更多的间隙连接与一个独立的群体的染料标记的细胞比对照细胞。Cx43的磷酸化通过用抗Cx43抗体进行免疫沉淀，然后用抗磷酸丝氨酸抗体进行免疫印迹分析来定量。将每个样品中的磷酸丝氨酸归一化为Cx43。与对照细胞相比，在暴露于振荡流体流的细胞中，Cx43的磷酸丝氨酸含量增加约两倍。细胞外信号调节激酶（ERK 1/2）在流动诱导的GJIC上调的可能作用也进行了研究。ERK 1/2抑制剂PD-98059可显著减弱振荡液流对MLOY-4细胞GJIC的影响。这些结果表明，振荡流体流通过ERK 1/2 MAP激酶调节MLOY-4细胞中的GJIC。此外，Cx43的丝氨酸磷酸化增加与血流诱导的GJIC增加相关。(C)2003 Elsevier Science（美国）。All rights reserved.
The present work was designed to investigate the effects of oscillating fluid flow on gap junctional intercellular communication (GJIC) and the gap junction protein connexin (Cx) 43 in osteocyte-like MLOY-4 cells. Cells were exposed for 1 h to oscillating fluid flow at a shear stress of +/- 10 dyn/cm(2) and a frequency of 1 Hz in a parallel plate flow chamber. Control cells were incubated in the chamber but were not exposed to oscillating fluid flow. Functional analysis of GJIC indicated that MLOY-4 cells exposed to oscillating fluid flow established more gap junctions with an independent population of dye-labeled cells than did control cells. Phosphorylation of Cx43 was quantified by immunoprecipitation with an anti-Cx43 antibody followed by immunoblot analysis using an anti-phosphoserine antibody. Phosphoserine was normalized to Cx43 in each sample. Compared to control cells, phosphoserine content of Cx43 increased approximately twofold in cells exposed to oscillating fluid flow. The possible role of the extracellular signal regulated kinase (ERK1/2) in the flow-induced upregulation of GJIC was also investigated. The ERK1/2 inhibitor PD-98059 significantly attenuated the effects of oscillating fluid flow on MLOY-4 cells GJIC. These results indicate that oscillating fluid flow regulates GJIC in MLOY-4 cells via the ERK1/2 MAP kinase. In addition, increased serine phosphorylation of Cx43 correlates with the flow-induced increase in GJIC. (C) 2003 Elsevier Science (USA). All rights reserved.