Figure 1.
Expression of CYP27A1 and CYP2R1 mRNA in hGF and hPDLC.
Expression of CYP27B1 mRNA was detected by real-time PCR in hGF and hPDLC from all five donors (donors are numbered 1–5). The expression levels of CYP27A1 and CYP2R1 mRNA were not significantly different in the two kinds of cells. The data are presented as the mean ± SD.
Figure 2.
Protein expression of CYP27A1 and CYP2R1 in hGF and hPDLC.
Protein expression of CYP27A1 was detected by Western blot in hGF and hPDLC from all five donors (donors are numbered 1–5). Protein expression of CYP2R1 was detected by Western blot in PC-3 cells, which were used as a positive control, but was not detected in hGF and hPDLC. β-actin was used as an internal control.
Figure 3.
Activity of 25-hydroxylases in hGF and hPDLC.
hGF and hPDLC from donors 2, 4 and 5 were incubated with 1000 nM vitamin D3 for the times indicated, and the production of 25OHD3 was determined in supernatants(A) and cell lysates (B). After incubation, the production of 25OHD3 was detected. The amount of 25OHD3 generated was not significantly different between hGF and hPDLC. The data are presented as the mean ± SE.
Figure 4.
1,25OH2D3 generation by hGF and hPDLC.
hGF and hPDLC from donors 2, 4 and 5 were incubated with 1000 nM vitamin D3 for 48 h, and the production of 1,25OH2D3 was determined in supernatants and cell lysates. The amount of 1,25OH2D3 generated was not significantly different between hGF and hPDLC. The data are presented as the mean ± SE.
Figure 5.
The efficiency of RNA interference against CYP27A1 and CYP2R1.
hGF and hPDLC from donors 2, 4 and 5 were transfected with a siRNA oligonucleotide for CYP27B1, a siRNA oligonucleotide for CYP2R1, or a non-silencing control. Using real-time PCR as a measure, the efficiency of RNA interference against CYP27A1 and CYP2R1 was over 70% in hGF and hPDLC. The data are presented as the mean ± SD. * denotes difference from negative controls (p<0.05).
Figure 6.
Effect of knockdown of 25-hydroxylases on 25OHD3 generation.
hGF and hPDLC from donors 2, 4 and 5 were treated with vitamin D3 at various concentrations indicated in the figure for 12 h after transfection with a siRNA oligonucleotide for CYP27A1, a siRNA oligonucleotide for CYP2R1, or a non-silencing control. 25OHD3 production was measured in supernatants of hGF (A), supernatants of hPDLC (B), cell lysates of hGF (C), and cell lysates of hPDLC (D). When CYP27A1 or CYP2R1 was not knocked down, the production of 25OHD3 increased with an increasing concentration of 25OHD3. When CYP27A1 was knocked down in hGF and hPDLC, the generation of 25OHD3 decreased significantly compared to when CYP27A1 was not knocked down. When CYP2R1 was knocked down in hGF (A, C), the generation of 25OHD3 was not significantly different from that when CYP2R1 was not knocked down. When CYP2R1 was knocked down in hPDLC (B, D), the generation of 25OHD3 was only slightly different at some time points from that when CYP2R1 was not knocked down. The data are presented as the mean ± SE. * hGF or hPDLC generated significantly less 25OHD3 with the same amount of added vitamin D3 when CYP27A1 or CYP2R1 was knocked down (p<0.05). # hGF or hPDLC generated significantly more 25OHD3 with the same amount of added vitamin D3 when CYP27A1 or CYP2R1 was knocked down (p<0.05).
Figure 7.
The effect of 25-hydroxylase knockdown on 1,25OH2D3 generation.
hGF and hPDLC from donors 2, 4 and 5 were treated with 1000 nM vitamin D3 for 48 h after transfection with a siRNA oligonucleotide for CYP27A1 or a non-silencing control, and 1,25OH2D3 production was measured in supernatants(A) and cell lysates (B). When CYP27A1 was knocked down, the generation of 1,25OH2D3 decreased significantly compared to when CYP27A1 was not knocked down. The data are presented as the mean ± SE. * hGF or hPDLC generated significantly less 1,25OH2D3 with 1000 nM vitamin D3 when CYP27A1 was knocked down (p<0.05).
Figure 8.
Preliminary investigation of CYP27A1 regulation by inflammatory stimuli in hGF and hPDLC.
hGF and hPDLC from donors 2, 3, 4 and 5 were stimulated with different treatments indicated in the figure for 24 h, and CYP27A1 mRNA expression was determined by real-time PCR. IL-1β and Pg-LPS significantly up-regulated CYP27A1 mRNA expression and the higher dose of IL-1β or Pg-LPS raised higher CYP27A1 mRNA up-regulation in both hGF and hPDLC. Sodium butyrate did not significantly influence CYP27A1 mRNA expression. Additionally, the characteristics of CYP27A1 regulation in hGF and hPDLC were not significantly different. The data are presented as the mean ± SE. * CYP27A1 mRNA expression was significantly different from that of the vehicle group (p<0.05). # CYP27A1 mRNA expression was significantly different from that of the 1 ng/mL IL-1β group (p<0.05). ∧ CYP27A1 mRNA expression was significantly different from that of the 1 µg/mL Pg-LPS group (p<0.05). IL-1ß : interleukin-1β. Pg-LPS: Porphyromonas gingivalis lipopolysaccharide.
Table 1.
Primer sequences used for PCR or real-time PCR.