Abstract
BackgroundWe previously demonstrated that 25-hydroxyvitamin D3 concentrations in gingival crevicular fluid are 300 times higher than those in the plasma of patients with aggressive periodontitis. Here we explored whether 25-hydroxyvitamin D3 can be synthesized by periodontal soft tissue cells. We also investigated which of the two main kinds of hydroxylases, CYP27A1 and CYP2R1, is the key 25-hydroxylase in periodontal soft tissue cells.Methodology/Principal FindingsPrimary cultures of human gingival fibroblasts and periodontal ligament cells from 5 individual donors were established. CYP27A1 mRNA, CYP2R1 mRNA and CYP27A1 protein were detected in human gingival fibroblasts and periodontal ligament cells, whereas CYP2R1 protein was not. After incubation with the 25-hydroxylase substrate vitamin D3, human gingival fibroblasts and periodontal ligament cells generated detectable 25-hydroxyvitamin D3 that resulted in the production of 1α,25-dihydroxyvitamin D3. Specific knockdown of CYP27A1 in human gingival fibroblasts and periodontal ligament cells using siRNA resulted in a significant reduction in both 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 production. Knockdown of CYP2R1 did not significantly influence 25-hydroxyvitamin D3 synthesis. Sodium butyrate did not influence significantly CYP27A1 mRNA expression; however, interleukin-1β and Porphyromonas gingivalis lipopolysaccharide strongly induced CYP27A1 mRNA expression in human gingival fibroblasts and periodontal ligament cells.ConclusionsThe activity of 25-hydroxylase was verified in human gingival fibroblasts and periodontal ligament cells, and CYP27A1 was identified as the key 25-hydroxylase in these cells.
Highlights
Vitamin D plays an important role in the regulation of bone metabolism and immunological reactions [1,2]
CYP27A1 and CYP2R1 mRNA were detected in all the cells of the five donors, and no significant difference was found between the mRNA levels in Human gingival fibroblasts (hGF) and human periodontal ligament cells (hPDLC) (Fig. 1)
CYP27A1 protein was detected in all cells of the five donors, whereas CYP2R1 was not detected, with the premise that anti-CYP2R1 antibody was able to recognize the protein in PC-3 cells, which were used as a positive control (Fig. 2)
Summary
Vitamin D plays an important role in the regulation of bone metabolism and immunological reactions [1,2]. In the early years of biochemical research, a mitochondrial cytochrome P450 (CYP27A1), an important enzyme in the bile acid synthesis pathway [8,9], was demonstrated to be 25hydroxylase. Afterwards, Cheng et al identified a microsomal cytochrome P450 (CYP2R1) with vitamin D 25-hydroxylase activity [10,11]. Other cytochrome P450 enzymes, such as CYP2C11, CYP2D25, CYP3A4 and CYP2J2, were all identified as vitamin D 25-hydroxylases [12,13,14], and the two most active 25-hydroxylases were found to be CYP27A1 and CYP2R1 [10]. Mutations in human and mouse genes encoding CYP27A1 protein influenced bile acid synthesis, but had no consequence on vitamin D metabolism [15,16,17,18]. We investigated which of the two main kinds of hydroxylases, CYP27A1 and CYP2R1, is the key 25-hydroxylase in periodontal soft tissue cells
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