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Abstract
Objecti
Several important roles of 1,25(OH)2D3 have been recognized in the immune system. The availability of 1,25(OH)2D3 at the cellular level is significantly influenced by the relative abundance of enzymes to synthesize (CYP27B1) and catabolize (CYP24) 1,25(OH)2D3. In this study, we examined the effect of 1,25(OH)2D3 on the expression of the CYP24 gene and the role of MAPK for the induction of CYP24 by 1,25(OH)2D3 in activated human macrophages.
Methods
For obtaining human activated macrophages, we treated U937 cells with PMA and we cultured these cells for 24 hours to adhere. After 24 hours treatment with PMA, the differentiated cells were washed with phosphate buffered saline (PBS), and then they were used for examining the effect of 1,25(OH)2D3 on the expression of the CYP24 gene. The mRNA expressions of the vitamin-D3 inducible genes were measured by real-time PCR, and the change of the protein expression by 1,25(OH)2D3 was measured by immunoblotting.
Results
1,25(OH)2D3 significantly induced the expression of CYP24 in the U937 cells and the 1,25(OH)2D3-induced expression of CYP24 was strongly augmented in the PMA-differentiated U937 cells. The 1,25(OH)2D3-induced expression of CYP24 was mediated by Erk1/2 and p38 MAPKs. Parallel to the induced expression of CYP24, 1,25(OH)2D3 induced the expression and phosphorylation of the CCAAT enhancer-binding protein (C/EBPβ).
Conclusion
In this study, we found that 1,25(OH)2D3 inducedthe expression of CYP24 via activation of MAPKs. These results suggest that MAPK inhibitors may be useful for the treatment of inflammatory conditions, in which active vitamin D3 can be used as the therapeutic molecule, by increasing the availability of 1,25(OH)2D3.
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Fig. 1.
1,25(OH)2D3 increases the production of CYP24 in the human monocyte cell line. (A) Fresh or PMAdifferentiated U937 cells were cultured for 24 hours in the presence or absence of 10 nM 1,25(OH)2D3 (B, C). U937 cells were cultured for 24 hours in the presence or absence of PMA. In (A-C), the mRNA was analyzedusing real time PCR. The data is shown as the mean±SD of triplicate determinants and the data is representative of more than three experiments.
Fig. 2.
The increase of the CYP24 expression by 1,25 (OH)2D3 is dose- and time-dependent. (A) The PMA-differentiated U937 cells were treated with the indicated dose of 1,25(OH)2D3 for 24 hours. (B) The PMA-differentiated U937 cells were treated with 10 nM 1,25(OH)2D3 for the indicated time. (C) The PMA-differentiated U937 cells were treated with 1,25(OH)2D3 for 24 hours in the presence or absence of 1 mg/mL cyclohexmide. In (A-C), the mRNA was analyzed using real time PCR. The data is shown as the mean±SD of triplicate determinants and the data is representative of more than three experiments.
Fig. 3.
The increase of the CYP24 expression by 1,25(OH)2D3 is mediated by ERK1/2 and p38 MAPKs. (A) The PMA-differentiated U937 cells were treated with 10 nM 1,25(OH)2D3 for the indicated time. The whole cell lyates were analyzed by immunoblotting. The data is representative of more than three experiments. (B) The PMA-differentiated U937 cells were treated with 1,25(OH)2D3 for 24 hours in the presence or absence of 20 M PD98059 and/or 20 M SB203580. The mRNA was isolated and then analyzed using real time PCR. The data is shown as the mean±SD of triplicate determinants and the data is representative of more than three experiments.
Fig. 4.
1,25(OH)2D3 increases the expression and phosphorylation of C/EBPa in the human macrophages derived from U937 cells. The PMA-differentiated U937 cells were treated with 1,25(OH)2D3 for 24 hours in the presence or absence of 20 M PD98059 and/or 20 M SB203580. (A) The whole cell lyates were analyzed by immunoblotting. The data is representative of more than three experiments. (B) The mRNA was isolated and then analyzed using real time PCR. The data is shown as the mean±SD of triplicate determinants and the data is representative of more than three experiments.
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