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

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

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。

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 ceils 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 nmolFL parathyroid hormone [PTH（1-34）] to observe the changes in the intraceUular 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.