南方医科大学学报

南方医科大学学报 ›› 2013, Vol. 33 ›› Issue (03): 326-.

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CITED1 63-84 氨基酸片段是影响其细胞定位与成骨作用的关键区域

林振，袁亮，孟越，冯瑞强，付兆宗，杨德鸿   

  • 出版日期:2013-03-20 发布日期:2013-03-20

Serine residues at position 63-84 are important for CITED1 nuclear translocation and osteoblast differentiation

  • Online:2013-03-20 Published:2013-03-20

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摘要： 目的生物信息学分析发现CITED1 63-84氨基酸片段为其重要的功能片段，进一步研究CITED1 63-84氨基酸片段突变
(丝氨酸突变为丙氨酸）是否影响其进核及成骨分化，探讨其在成骨分化过程中的生物学调控功能。方法CITED1 63-84 突变
质粒（9S>A），CITED1质粒及空白质粒分别转染MC3T3-E1细胞，培养2 d，用100 nmol/L PTH（1-34）刺激细胞，行细胞免疫荧
光实验，共聚焦镜下观察细胞内CITED1位置变化。将CITED1 63-84突变质粒（9S>A），CITED1质粒及空白质粒按相应体系
转染MC3T3-E1 细胞，分为两组，一组成骨诱导4 周，另一组成骨诱导基础上进行10 nmol/L PTH（1-34）间歇刺激（每48 h 刺
激4 h）4周，测ALP酶活性及Ca离子浓度并进行ALP及茜素红染色。行RT-PCR实验分析成骨相关基因ALP2，RUNX2，OC的
表达量。结果PTH（1-34）可促进CITED1 进核，将CITED1 氨基酸63-84 片段突变后，可明显抑制该反应。4 周成骨诱导后，
CITED1过表达明显抑制成骨细胞分化，ALP及Ca离子浓度明显低于对照组，而CITED1突变质粒（9S>A）过表达ALP及Ca离
子浓度明显高于CITED1 组，与对照组基本相同。进一步研究表明，CITED1 过表达抑制PTH（1-34）诱导的成骨细胞分化，而
CITED1 突变质粒（9S>A）可逆转该反应。ALP染色及茜素红染色也验证了以上结论。RT-PCR结果提示：CITED1 突变质粒
（9S>A）过表达成骨相关基因ALP2，RUNX2，OC明显高于CITED1质粒过表达。结论CITED1 63-84片段丝氨酸为其重要的
功能位点，可影响其细胞进核及成骨分化。

Abstract: Objective To determine the role of serine residues at position 63-84 of CITED1 in the nuclear translocation of CITED1
and osteoblast differentiation. Methods We engineered all the 9 phosphorylated serine residues of CITED1 with a
serine-to-alanine mutation at position 63-84. MC3T3E1 cells transfected with pCDNA3-CFP-CITED1 63-84 (9S>A),
pCDNA3-CFP-CITED1, and vehicle plasmid were examined with confocal laser scanning microscopy before and after
treatment with 100 nmol/L parathyroid hormone [PTH(1-34)] to observe the changes in the intracellular localization of
CITED1. The transfected cells were induced for osteoblastic differentiation with mineralized solution in the absence or
presence of 10 nmol/L PTH(1-34), and the changes in ALP activity and Ca2+ concentration were measured; RT-PCR was used to
detect the changes in ALP2, RUNX2, and OC gene expressions after the treatments. Results PTH(1-34) promoted the nuclear
translocation of CITED1 in MC3T3-E1 cells. The (63-84) 9S>A mutation of CITED1 obviously suppressed its translocation and
increased ALP activity and Ca2 + levels in the cells, which led to enhanced mineralization in the cells with also increased
expressions of ALP2, RUNX2, and OC. Conclusion The serine residues at position 63-84 of CITED1 play a vital role in the
nuclear translocation of CITED1 and osteoblast differentiation.