Nuclear translocation of CBP/p300-interacting protein CITED1 induced by parathyroid hormone requires serine phosphorylation at position 79 in its 63-84 domain.

The transcriptional cofactor CITED1 inhibits osteoblastic differentiation and blunts the stimulation of osteoblastic differentiation by parathyroid hormone (PTH). In the MC3T3-E1 osteoblastic cell line, we found that CITED1 was located predominantly in the cytoplasm and that hPTH(1-34) increased translocation of CITED1 from the cytoplasm to the nucleus. This response to hPTH(1-34) was not observed when all 9 serine residues within the 63-84 domain of CITED1 were mutated to alanines (CITED1 9S>A) or when a single serine to alanine mutation was made at position 79 (CITED1 S(79)>A).

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

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.

[Effect of signal-selective parathyroid hormone analogue peptide on expressions of Wnt signaling factors].

Objective: To study the effect of signal-selective parathyroid hormone (PTH) analogue peptide on Wnt signaling factors in osteoblasts isolated from neonatal mouse, and provide theoretical basis for the mechanism of PTH's function in bone metabolism.

Methods: Osteoblasts were isolated from calvaria of 2-3-day-old C57BL neonatal mouse and identified by alkaline phosphatase (ALP) staining, and Alizarin red staining. The cells at passage 1 were divided into 4 groups: control group, PTH (1-34) group, G1R19 (1-34) group, and G1R19 (1-28) group.

[Fluorescence resonance energy transfer analysis for detecting protein kinase C activation].

Objective: To establish a sensitive and direct method for detecting the activation of protein kinase C (PKC) using fluorescence resonance energy transfer (FRET) technique.

Methods: HEK293 cells were transfected with C kinase activity reporter (CKAR) plasmid or/and parathyroid receptor 1 plasmid , and after incubation for 72 h, the fluorescence resonance energy transfer was measured with or without parathyroid or TPA stimulation.

Results: TPA reduced the efficiency of FRET and increased the emission ratio of CFP/YFP (C/Y) in HEK293 cells transfected with CKAR.