Vimentin Inhibits ATF4-mediated Osteocalcin Transcription and Osteoblast Differentiation
Vimentin Inhibits ATF4-mediated Osteocalcin Transcription and Osteoblast Differentiation
复制标题
DOI：
10.1074/jbc.m109.052373
复制
发表时间：
2009-10-30
影响因子：
4.8
通讯作者：
Yang, Xiangli
中科院分区：
文献类型：
--
作者：
Lian, Na;Wang, Weiguang;Yang, Xiangli
转录激活因子4（ATF 4）是一种成骨细胞富集的转录因子，调节骨钙素转录和成骨细胞终末分化。为了鉴定ATF 4的功能伴侣，我们将ROS 17/2.8成骨细胞核提取物和纯化的重组His-ATF 4应用到Ni+亲和基质色谱柱上。采用液相色谱-质谱联用技术对波形蛋白进行鉴定。免疫共沉淀和pulldown实验表明，波形蛋白与ATF 4的第一个亮氨酸拉链结构域相互作用。DNA共转染和凝胶阻滞表明，波形蛋白通过阻止骨钙素启动子上的ATF 4结合位点OSE 1来抑制ATF 4对骨钙素的反式激活活性。北方杂交显示波形蛋白在未成熟成骨细胞中高水平表达，在完全分化成骨细胞中低水平表达。小干扰RNA下调波形蛋白诱导未成熟成骨细胞内源性骨钙素转录。相反，成骨细胞中波形蛋白的异位过表达抑制成骨细胞分化，表现为碱性磷酸酶活性降低、矿化延迟和成骨细胞标记基因（如骨唾液蛋白和骨钙素）表达降低。总之，我们的数据揭示了一种新的机制，即细胞骨架蛋白，波形蛋白，通过与ATF 4相互作用，在未成熟的成骨细胞中作为分化的突破。
Activating transcription factor 4 (ATF4) is an osteoblast-enriched transcription factor that regulates osteocalcin transcription and osteoblast terminal differentiation. To identify functional partners of ATF4, we applied ROS17/2.8 osteoblast nuclear extracts and purified recombinant His-ATF4 onto a Ni+ affinity matrix chromatography column. Vimentin was identified by liquid chromatography-mass spectrometry. Coimmunoprecipitation and pulldown assays revealed that vimentin interacted with ATF4 with its first leucine zipper domain. DNA cotransfection and gel retardation demonstrated that vimentin inhibited the transactivation activity of ATF4 on osteocalcin by preventing it to bind OSE1, the ATF4 binding site on the osteocalcin promoter. Northern hybridization revealed that vimentin was expressed at a high level in immature osteoblasts and a low level in fully differentiated osteoblasts. Down-regulation of vimentin by small interfering RNA induced endogenous osteocalcin transcription in immature osteoblasts. Conversely, ectopic overexpression of vimentin in osteoblasts inhibited osteoblast differentiation as shown by lower alkaline phosphatase activity, delayed mineralization, and decreased expression of osteoblast marker genes such as bone sialoprotein and osteocalcin. Together, our data uncover a novel mechanism whereby a cytoskeletal protein, vimentin, acts as a break on differentiation in immature osteoblasts by interacting with ATF4.