Osteoblastic alkaline phosphatase mRNA is stabilized by binding to vimentin intermediary filaments
Osteoblastic alkaline phosphatase mRNA is stabilized by binding to vimentin intermediary filaments
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DOI：
10.1515/hsz-2014-0274
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发表时间：
2015-03
影响因子：
3.7
通讯作者：
Yvonne Schmidt;M. Biniossek;G. B. Stark;G. Finkenzeller;F. Simunovic
中科院分区：
文献类型：
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作者：
Yvonne Schmidt;M. Biniossek;G. B. Stark;G. Finkenzeller;F. Simunovic
血管化在骨组织工程中是必不可少的，近年来的研究主要集中在成骨细胞（hOBs）和内皮细胞（ECs）之间的相互作用。结果表明，共培养可提高成骨细胞碱性磷酸酶（ALP） mRNA的稳定性。我们研究了这一现象背后的机制，重点是mRNA结合蛋白。通过荧光素酶报告基因分析，我们发现ALP mRNA的3 ' -非翻译区（UTR）对于人脐静脉内皮细胞（HUVEC）介导的成骨细胞ALP mRNA的稳定是必需的。通过下拉实验和纳米流高效液相色谱质谱法，确定了vimentin与ALP mRNA的3′-UTR结合。Western blotting进行验证。用亚氨基二丙腈抑制中间纤维和用siRNA转染特异性抑制vimentin的功能实验显示ALP mRNA和蛋白水平降低。因此，ALP mRNA与vimentin结合并被vimentin稳定。这些数据增加了对ALP mRNA细胞内转运及其功能的理解，并可能在组织工程应用中产生影响。
Abstract Vascularization is essential in bone tissue engineering and recent research has focused on interactions between osteoblasts (hOBs) and endothelial cells (ECs). It was shown that cocultivation increases the stability of osteoblastic alkaline phosphatase (ALP) mRNA. We investigated the mechanisms behind this observation, focusing on mRNA binding proteins. Using a luciferase reporter assay, we found that the 3′-untranslated region (UTR) of ALP mRNA is necessary for human umbilical vein endothelial cells (HUVEC)-mediated stabilization of osteoblastic ALP mRNA. Using pulldown experiments and nanoflow-HPLC mass spectrometry, vimentin was identified to bind to the 3′-UTR of ALP mRNA. Validation was performed by Western blotting. Functional experiments inhibiting intermediate filaments with iminodipropionitrile and specific inhibition of vimentin by siRNA transfection showed reduced levels of ALP mRNA and protein. Therefore, ALP mRNA binds to and is stabilized by vimentin. This data add to the understanding of intracellular trafficking of ALP mRNA, its function, and have possible implications in tissue engineering applications.