Altered Actin Dynamics and Functions of Osteoblast-Like Cells in Parabolic Flight may Involve ERK1/2
Altered Actin Dynamics and Functions of Osteoblast-Like Cells in Parabolic Flight may Involve ERK1/2
复制标题
抛物线飞行中成骨细胞样细胞肌动蛋白动力学和功能的改变可能涉及 ERK1/2
DOI：
10.1007/s12217-010-9216-7
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发表时间：
2011-01-01
影响因子：
1.8
通讯作者：
Li, Yinghui
中科院分区：
文献类型：
--
作者：
Dai, Zhongquan;Tan, Yingjun;Li, Yinghui
成骨细胞对重力等机械压力敏感，并改变其细胞骨架和功能以适应;然而，重力对这种现象的贡献还不清楚。在这项研究中，我们研究了急性重力变化对抛物线飞行产生的成骨细胞ROS 17/2.8的结构和功能的影响。用Phalloidin标记的德克萨斯红和Alexa Fluor 488标记的DNase I分别对F-actin和G-actin进行免疫荧光染色，观察细胞微丝骨架的变化。分别采用改良Gomori法、放射免疫法和RT-PCR法检测成骨细胞碱性磷酸酶（ALP）活性、骨钙素分泌以及ALP、胶原α 1链（COL 1A 1）和骨钙素的表达。磷酸化p44/42和F-actin的双重荧光染色观察它们的共定位关系。利用建立的EGFP荧光强度半定量分析方法，检测MAPK抑制剂PD 98059或F-actin抑制剂细胞松弛素B对EGFP-ROS细胞中COL 1A 1启动子活性的影响。结果表明，重力改变诱导成骨细胞微丝骨架的重组。抛物线飞行3 h后，成骨细胞骨架中的F-actin变粗，方向性变强，而G-actin收缩，集中在细胞核边缘。骨钙素分泌减少，ALP活性降低，mRNA表达减少。共定位分析表明磷酸化的p44/42 MAPK与F-actin偶联。抑制剂PD 98059和细胞松弛素B可降低EGFP-ROS细胞的荧光强度。上述结果提示，短时间重力变化引起成骨细胞结构和功能的调整，ERK 1/2信号通路可能参与了这些反应。我们认为，结构改变抑制功能的发挥是成骨细胞对重力改变的一种适应方式。
Osteoblasts are sensitive to mechanical stressors such as gravity and alter their cytoskeletons and functions to adapt; however, the contribution of gravity to this phenomenon is not well understood. In this study, we investigated the effects of acute gravitational changes on the structure and function of osteoblast ROS17/2.8 as generated by parabolic flight. The changes in microfilament cytoskeleton was observed by immunofluorescence stain of Texas red conjugated Phalloidin and Alexa Fluor 488 conjugated DNase I for F-actin and G-actin, respectively. To examine osteoblast function, ALP (alkaline phosphatase) activity, osteocalcin secretions and the expression of ALP, COL1A1 (collagen type I alpha 1 chain) and osteocalcin were detected by modified Gomori methods, radioimmunity and RT-PCR, respectively. Double fluorescence staining of phosphorylated p44/42 and F-actin were performed to observe their colocalization relationship. The established semi-quantitative analysis method of fluorescence intensity of EGFP was used to detect the activity changes of COL1A1 promoter in EGFP-ROS cells with MAPK inhibitor PD98059 or F-actin inhibitor cytochalasin B. Results indicate that the altered gravity induced the reorganization of microfilament cytoskeletons of osteoblasts. After 3 h parabolic flight, F-actin of osteoblast cytoskeleton became thicker and directivity, whereas G-actin shrunk and became more concentrated at the edge of nucleus. The excretion of osteocalcin, the activity of ALP and the expression of mRNA decreased. Colocalization analysis indicated that phosphorylated p44/42 MAPK was coupled with F-actin. Inhibitor PD98059 and cytochalasin B decreased the fluorescence intensity of EGFP-ROS cells. Above results suggest that short time gravity variations induce the adjustment of osteoblast structure and functional and ERK1/2 signaling maybe involve these responses. We believe that it is an adaptive method of the osteoblasts to gravity alteration that structure alteration inhibits the function performing.