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 Ca(2+) concentration were measured; RT-PCR was used to detect the changes in ALP2, RUNX2, and OC gene expressions after the treatments.
Results: s 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 Ca(2+) 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.