Actin microfilament mediates osteoblast Cbfa1 responsiveness to BMP2 under simulated microgravity.
Actin microfilament mediates osteoblast Cbfa1 responsiveness to BMP2 under simulated microgravity.
复制标题
DOI：
10.1371/journal.pone.0063661
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Li Y
中科院分区：
文献类型：
--
作者：
Dai Z;Wu F;Chen J;Xu H;Wang H;Guo F;Tan Y;Ding B;Wang J;Wan Y;Li Y
微重力降低成骨细胞的活性，诱导肌动蛋白微丝断裂，抑制成骨细胞对细胞因子的反应，但其机制仍是个谜。此前，F-肌动蛋白细胞骨架与微重力下观察到的细胞形态、功能和信号的多种变化有关。在这里，我们研究了微丝参与了微重力和BMP2诱导对Cbfa1活性的影响。为此，我们构建了Cbfa1活性的荧光报告细胞系(OSE-MG63)，通过稳定地将OSE2的6个串联拷贝和增强型绿色荧光蛋白(EGFP)上游的最小mOG2启动子组成的报告基因导入MG63细胞。OSE-MG63的荧光强度对骨相关细胞因子(IGF-I、维生素D3和BMP2)具有反应性，并与碱性磷酸酶(ALP)活性呈一致趋势。利用OSE-MG63报告荧光技术，对模拟微重力、微丝干扰剂(细胞松弛素B，CB)、微丝稳定剂(jasplakinolide，JAS)处理后的Cbfa1活性进行半定量分析。同时分析碱性磷酸酶(ALP)活性、Cbfa1与OSE2的DNA结合活性(ChIP)、F-肌动蛋白结构(免疫荧光)和绿色荧光蛋白(EGFP)的mRNA表达(RT-qPCR)。模拟微重力抑制Cbfa1活性，影响Cbfa1对细胞因子BMP2的反应性，并导致微丝变薄和分散分布。在正常重力条件下，CB显著减弱BMP2对Cbfa1活性的诱导以及Cbfa1与OSE2的DNA结合活性。JAS的加入逆转了微重力对Cbfa1对BMP2反应性的抑制作用。我们的研究表明，CB或模拟微重力破坏微丝组织会减弱Cbfa1对BMP2的反应性。JAS对微丝组织的稳定逆转了这种抑制。综上所述，这些结果提示，肌动蛋白微丝参与了骨形态发生蛋白2的S诱导的Cbfa1活性，它们的破坏可能是微重力抑制骨形态发生蛋白2的S成骨诱导的一个重要因素。
Microgravity decreases osteoblastic activity, induces actin microfilament disruption and inhibits the responsiveness of osteoblast to cytokines, but the mechanisms remains enigmatic. The F-actin cytoskeleton has previously been implicated in manifold changes of cell shape, function and signaling observed under microgravity. Here we investigate the involvement of microfilament in mediating the effects of microgravity and BMP2 induction on Cbfa1 activity. For this purpose we constructed a fluorescent reporter cell line (OSE-MG63) of Cbfa1 activity by stably transfecting MG63 cells with a reporter consisting of six tandem copies of OSE2 and a minimal mOG2 promoter upstream of enhanced green fluorescent protein (EGFP). The fluorescence intensity of OSE-MG63 showed responsiveness to bone-related cytokines (IGF-I, vitamin D3 and BMP2) and presented an accordant tendency with alkaline phosphatase (ALP) activity. Using OSE-MG63 reporter fluorescence, we performed a semi-quantitative analysis of Cbfa1 activity after treatment with simulated microgravity, microfilament-disrupting agent (cytochalasin B, CB), microfilament-stabilizing agent (Jasplakinolide, JAS) or any combination thereof. In parallel, ALP activity, DNA binding activity of Cbfa1 to OSE2 (ChIP), F-actin structure (immunofluorescence) and EGFP mRNA expression (RT-qPCR) were analyzed. Simulated microgravity inhibited Cbfa1 activity, affected the responsiveness of Cbfa1 to cytokine BMP2, and caused a thinning and dispersed distribution of microfilament. Under normal gravity, CB significantly attenuated BMP2 induction to Cbfa1 activity as well as DNA binding activity of Cbfa1 to OSE2. The addition of JAS reversed the inhibitory effects of microgravity on the responsiveness of Cbfa1 to BMP2. Our study demonstrates that disrupting the microfilament organization by CB or simulated microgravity attenuates the responsiveness of Cbfa1 to BMP2. A stabilization of the microfilament organization by JAS reverses this inhibition. Taken together, these results suggest that actin microfilament participates in BMP2’s induction to Cbfa1 activity and that their disruption might be an important contributor to microgravity’s inhibition on BMP2’s osteogenic induction.
登录
查看更多内容
DOI：
10.1159/000151719
发表时间：
2009
期刊：
Cells, tissues, organs
影响因子：
--
作者：
Javed A;Afzal F;Bae JS;Gutierrez S;Zaidi K;Pratap J;van Wijnen AJ;Stein JL;Stein GS;Lian JB
通讯作者：
Lian JB
DOI：
10.1083/jcb.200610046
发表时间：
2007-02-26
期刊：
The Journal of cell biology
影响因子：
--
作者：
Ge C;Xiao G;Jiang D;Franceschi RT
通讯作者：
Franceschi RT
影响因子：
2.7
作者：
Collins, Tony J.
通讯作者：
Collins, Tony J.
影响因子：
8.5
作者：
Dai, Z. Q.;Wang, R.;Li, Y. H.
通讯作者：
Li, Y. H.
影响因子：
--
作者：
Higashibata, Akira;Imamizo-Sato, Mari;Seki, Masaya;Yamazaki, Takashi;Ishioka, Noriaki
通讯作者：
Ishioka, Noriaki