Objective: To characterize the interaction effects between 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and human transforming growth factor-beta1 (hTGF-beta1) on the proliferation and differentiation of human embryonic osteoblasts.
Methods: Human embryonic osteoblasts were cultured. 1,25(OH)(2)D(3), of the concentration of 50 nmol/L, hTGF-beta1 of different concentrations (1 x 10(-8) g/L, 1 x 10(-7) g/L, 1 x 10(-6) g/L, and 1 x 10(-5) g/L), and both hTGF-beta1 of different concentrations and 1,25(OH)(2)D(3) of the concentration of 50 nmol/L were added into the cultures with a stimulation time of 96 hours. 3-(4, 5-dimethylthiazol-2-yl) - 2, 5 - diphenyl - tetrazolium bromide (MTT) was added into the cultures. ELISA technique was used to measure the optical density values so as to observe cell's number and proliferation. Alkaline phosphatase (ALP) activity was tested by King's method. Osteocalcin (OC) was tested by radioimmunoassay. The mRNA expression level of SMAD proteins, the key proteins of the TGF-beta signal transduction passageway, was detected by reverse transcriptase polymerase chain reaction (RT-PCR).
Results: MTT staining showed an absorbance of 0.086 +/- 0.022 in the 1,25(OH)(2)D(3) group, significantly lower than that of the control group (0.124 +/- 0.031, P < 0.05). The ALP activity in the culture of 1,25(OH)(2)D(3) group, hTGF-beta1 groups, and combined 1,25 (OH)(2)D(3) and hTGF-beta1 groups were 1.3 - 2.0 times that of the control group (all P < 0.05). When the concentration of hTGF-beta1 is 1 x 10(-6) g/L, an interaction effect was found between 1,25(OH)(2)D(3) and hTGF-beta1 on the ALP activity of human embryonic osteoblasts. The OC value in cell medium was 5.3 micro g/L +/- 1.6 micro g/L and 5.4 micro g/L +/- 0.9 micro g/L respectively in the hTGF-beta1 1 x 10(-6) g/L and 1 x 10(-5) g/L combined with 1,25(OH)(2)D(3) (50 nmol/L) groups, significantly higher than that in the control group (P < 0.05), but no interaction effect was found between 1,25(OH)(2)D(3) and hTGF-beta1 on the OC production. hTGF-beta1 of different concentrations and hTGF-beta1 of different concentrations combined with 1,25(OH)(2)D(3) all increased the SMAD3 mRNA expression level in human embryonic osteoblasts (all P < 0.05). When the human embryonic osteoblasts were treated with 1,25(OH)(2)D(3) combined with 1 x 10(-6) g/L hTGF-beta1, its SMAD3 mRNA level reached the peak, about 6-fold that of the control group. 1,25(OH)(2)VD(3) and/or hTGF-beta1 did not influenced the SMAD4 mRNA expression.
Conclusion: 1,25(OH)(2)D(3) inhibits the proliferation of human embryonic osteoblasts and increases the expression of osteoblastic markers, such as ALP and OC, thus promoting its differentiation. An interaction effect exists between 1,25(OH)(2)D(3) and hTGF-beta1 on the differentiation of human embryonic osteoblasts, which may be partly induced by SMA D(3) protein in the TGF-beta signal transduction passageway.