Activity of 25-Hydroxylase in Human Gingival Fibroblasts and Periodontal Ligament Cells

Background We 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 Findings Primary 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. Conclusions The 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.


Introduction
Vitamin D plays an important role in the regulation of bone metabolism and immunological reactions [1,2]. In humans, vitamin D, in the form of vitamin D 3 , is derived from dietary sources or made from 7-dehydrocholesterol in the skin by exposure to ultraviolet rays [3,4,5,6,7]. Then, vitamin D 3 is metabolized by two-step hydroxylations: first 25-hydroxylation in the liver to form 25-hydroxyvitamin D 3 (25OHD 3 ), the major circulating metabolite of vitamin D 3 , followed by 1,a-hydroxylation in the kidney to form 1a,25-dihydroxyvitamin D 3 (1,25OH 2 D 3 ), the biologically active metabolite of vitamin D 3 [6,7].
Human gingival fibroblasts (hGF) and human periodontal ligament cells (hPDLC) are two kinds of periodontal fibroblasts and are important components of periodontal soft tissues. Our previous study demonstrated that local 25OHD 3 levels in gingival crevicular fluid were about 300 times higher than that in the plasma of patients with aggressive periodontitis [27,28]. Since there is abundant 25OHD 3 around periodontal soft tissues, it was hypothesized that hGF and hPDLC have 25-hydroxylase activity, and can synthesize 25OHD 3 . The objective of this study was to test this hypothesis.

Results
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 hGF and hPDLC (Fig. 1). CYP27A1 protein was also 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). This indicated that CYP27A1 might be the key 25-hydroxylase in hGF and hPDLC.
After confirming the expression of 25-hydroxylase in hGF and hPDLC, the function of 25-hydroxylase was investigated. Whereas 1000 nM vitamin D 3 did not have a significant cytotoxic effect on any of the cells within 48 h, hGF and hPDLC generated 25OHD 3 in response to vitamin D 3 (Figs. 3A, B). The fact that extra-and intracellular 25OHD 3 was generated in the presence of vitamin D 3 provides direct and convincing evidence of the existence of 25hydroxylase in hGF and hPDLC. At all time points, there was no significant difference in the levels of intracellular and extracellular 25OHD 3 between the two cell types.
Additionally, exposure to vitamin D 3 also resulted in the synthesis of 1,25OH 2 D 3 in hGF and hPDLC (Fig. 4). The observation that hGF and hPDLC could synthesize 1,25OH 2 D 3 when exposed to 25OHD 3 [29] is further evidence of 25hydroxylase activity in hGF and hPDLC.
Based on the above direct evidence for 25-hydroxylase activity in hGF and hPDLC, we examined the effect of 25-hydroxylase knockdown. The efficiency of RNA interference against both CYP27A1 and CYP2R1 was both over 70% (Fig. 5). The generation of 25OHD 3 increased with increasing vitamin D 3 concentrations, but dropped significantly when CYP27A1 was knocked down using specific siRNA (Figs. 6A-D). However, knockdown of CYP2R1 did not significantly influence 25OHD 3 generation by hGF (Figs. 6A, C), and only slightly influenced 25OHD 3 generation by hPDLC (Figs. 6B, D). These results suggest that CYP27A1 might be the key 25-hydroxylase in hGF and hPDLC. In addition, knockdown of CYP27A1 resulted in a significant reduction of 1,25OH 2 D 3 generation (Figs. 7A-B). This is additional evidence for the activity of CYP27A1 as the 25hydroxylase in hGF and hPDLC.
After the comprehensive confirmation of 25-hydroxylase activity in hGF and hPDLC, and the verification of CYP27A1 as the key 25-hydroxylase, the regulation of CYP27A1 in hGF and hPDLC was investigated. Interleukin-1b (IL-1b) and Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) strongly induced CYP27A1 expression (Fig. 8). Additionally, dose-dependent increases in expression of CYP27A1 mRNA in hGF and hPDLC following incubation with IL-1b or Pg-LPS were demonstrated (Fig. 8). By contrast, sodium butyrate did not influence significantly CYP27A1 mRNA expression in hGF and hPDLC (Fig. 8). In addition, no significant differences between hGF and hPDLC were observed in the regulation of CYP27A1.

Discussion
In the present study, our hypothesis that hGF and hPDLC have 25-hydroxylase activity, and that they can synthesize 25OHD 3 was verified. Therefore, the origin of high 25OHD 3 concentrations in gingival crevicular fluid [27,28] might be hGF and hPDLC. Having demonstrated 1a-hydroxylase activity in hGF and hPDLC [29], we could consider that the conversion of vitamin D 3 to 1,25OH 2 D 3 in hGF and hPDLC consisted of two steps: s from vitamin D 3 to 25OHD 3 , under the action of 25-hydroxylase CYP27A1; t from 25OHD 3 to 1,25OH 2 D 3 , under the action of 1a-hydroxylase CYP27B1. This two-step conversion is similar to that observed in human keratinocytes [7,19,30,31,32]. In addition, Slominski et al. reported an alternate pathway of vitamin D 3 metabolism by cytochrome P450scc (CYP11A1) [33,34,35,36]. P450scc activity in hGF and hPDLC is worth further investigation in our future study.
We can then calculate and compare the amount of 1,25OH 2 D 3 synthesized from 1000 nM vitamin D 3 and from 1000 nM 25OHD 3 . According to the present study, the amount of 1,25OH 2 D 3 generated would be: s In hGF exposed to 1000 nM vitamin D 3 for 48 h, 9 fmol/10000 cells in supernatants +14 fmol/10000 cells in cell lysates = 23 fmol/10000 cells (Fig. 4). t In hPDLC exposed to 1000 nM vitamin D 3 for 48 h, 13 fmol/ 10000 cells in supernatants +16 fmol/10000 cells in cell lysates = 29 fmol/10000 cells (Fig. 4). According to our previous study [29], the amount of 1,25OH 2 D 3 generated would be the following: u In hGF exposed to 1000 nM 25OHD 3 for 48 h, 5 fmol/10000 cells in supernatants +13 fmol/10000 cells in cell lysates = 18 fmol/10000 cells. v In hPDLC exposed to 1000 nM 25OHD 3 for 48 h, 13 fmol/10000 cells in supernatants +14 fmol/ 10000 cells in cell lysates = 27 fmol/10000 cells. It is interesting that 1000 nM vitamin D 3 could induce hGF and hPDLC to generate even more 1,25OH 2 D 3 than 1000 nM 25OHD 3 . Particular attention should be paid to the observation that after 1000 nM vitamin D 3 treatment for 48 h, the 25OHD 3 concentration in the cell supernatants of hGF and hPDLC were only about 45 nM-64 nM and 30 nM-50 nM respectively, much lower than the added 1000 nM vitamin D 3 . So, why was less 25OHD 3 converted to more 1,25OH 2 D 3 ? One reason might be that after vitamin D 3 treatment, 25OHD 3 is found not only in the supernatant, but also in the cell lysates, allowing intracellular 25OHD 3 to act directly as substrate of 1a-hydroxylase. On the other hand, exogenous 25OHD 3 should enter the cells before eliciting a response. Thus, the direct availability at the site of action might be of great importance.
After comprehensive verification of 25-hydroxylase activity and the demonstration of CYP27A1 as the key 25-hydroxylase in hGF and hPDLC, the regulation of CYP27A1 in these cells was preliminarily investigated. IL-1b in gingival crevicular fluids of patients with periodontitis decreases significantly after initial periodontal therapy, indicating that IL-1b is associated with periodontitis [28]. Porphyromonas gingivalis is an important pathogen of periodontitis and butyrate is one of its metabolites [37]. It was demonstrated that the butyrate concentrations in gingival crevicular fluids of patients with periodontitis are significantly higher than those of healthy controls, and that butyrate concentrations in gingival crevicular fluids are significantly correlated with periodontal inflammation [38,39]. To investigate the regulation of CYP27A1 in hGF and hPDLC, IL-1b, Pg-LPS and sodium butyrate were chosen for the present study. It should be considered, however, that although stimuli with periodontal characteristics were used to simulate a periodontitis-like condition, this does not properly model the chronic disease situation in vivo, and can only help to investigate the regulation of CYP27A1 in hGF and hPDLC. The NF-kB activator, IL-1b, was demonstrated to be a potent up-regulator of CYP27A1 mRNA in hGF and hPDLC (Fig. 8). Pg-LPS could also up-regulate significantly the   expression of CYP27A1 mRNA, whereas sodium butyrate could not. It was reported that Pg-LPS is the ligand of Toll-like receptor 2 (TLR2) and TLR4 [40,41] and that both hGF and hPDLC expressed TLR2 and TLR4 [42]. Upon ligand binding, TLR2 or TLR4-mediated signaling could activate signal transduction, leading to NF-kB activation [43,44]. Thus, NF-kB might be involved in the regulation of CYP27A1 expression, an observation that warrants further investigation.
Each donor supplied both hGF and hPDLC in the present study. Although hGF and hPDLC are two different kinds of cells, they shared many features in 25-hydroxylase expression, activity and regulation, and only subtle differences were detected. As shown in Fig. 6, when CYP2R1 was knocked down, 25OHD 3 generation by hGF was not changed significantly, whereas 25OHD 3 generation by hPDLC was affected slightly. However, the difference did not affect our conclusion that CYP27A1 might be the key 25-hydroxylase in hGF and hPDLC.
Since 1,25OH 2 D 3 may enhance the antibacterial defense of human gingival epithelial cells [45] and hGF and hPDLC could synthesize 1,25OH 2 D 3 with 25OHD 3 [29], the confirmation of 25-hydroxylase activity in hGF and hPDLC implies that these cells could generate 25OHD 3 as a substrate for 1,25OH 2 D 3 . From this perspective, 25-hydroxylase activity in hGF and hPDLC may be involved in the innate immune defense of the oral cavity. Recently, it was reported that oral calcium and vitamin D supplementation have a positive effect on periodontal health [46,47]. However, topical application of vitamin D has not been reported. Since hGF and hPDLC have the ability to synthesize 25OHD 3 and then to synthesize 1,25OH 2 D 3 , the topical application of vitamin D 3 might fulfill the function of 1,25OH 2 D 3 . Thus, our data suggest a potential benefit of topical application of vitamin D 3 in periodontal therapy.
In conclusion, hGF and hPDLC were identified as new extrahepatic sites of 25OHD 3 synthesis for the first time, and CYP27A1 might be the key 25-hydroxylase in these cells.

Ethics Statement
The study protocol was approved by the institutional review board of Peking University School and Hospital of Stomatology (PKUSSIRB-2011007) and written informed consent was obtained from each participant in accordance with the Declaration of Helsinki.

Cell Culture
Primary culture of hGF and hPDLC was carried out according to our previous methods [29]. In brief, hPDLC were obtained from extracted third molars of 5 young healthy volunteers, and hGF was isolated from the gingiva of the same 5 donors. The periodontal ligament tissues attached to the middle third of the roots were curetted gently by a surgical scalpel, minced and placed in 24-well plates. Gingivae were also minced and transferred into 24-well plates. Tissue explants were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; PAA, Coelbe, Germany), 100 U/mL penicillin G and 100 mg/mL streptomycin. Cultures were maintained in a humidified atmosphere of 5% (v/v) CO 2 at 37uC. After reaching 80% confluence, hGF and hPDLC were digested with a mixture of 0.25% (w/v) trypsin and 0.02% (w/v) EDTA, and subcultured at a 1:3 ratio. The prostate cancer cell line, PC-3 (American Type Culture Collection, Rockville, MD, USA), was cultured in RPMI 1640 (Gibco, Gaithersburg, MD, USA) supplemented with 10% (v/v) FBS (FBS; PAA, Coelbe, Germany) in a humidified atmosphere of 5% CO 2 at 37uC and was used when the cells were in the logarithmic phase and reached 80% confluence.

Cytotoxicity Test of Vitamin D 3
hGF and hPDLC of three donors were used in the cytotoxicity test. hGF and hPDLC in their logarithmic growth phase were plated into 96-well plates at a density of 3000 cells/well in DMEM with 10% DCC-FBS, and the medium was replaced by DMEM without DCC-FBS after 24 h. After another 24 h, the medium 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 6 SD. * denotes difference from negative controls (p,0.05).

Detection of 25-hydroxylase Expression
hGF and hPDLC from all five donors were seeded into six-well plates at a density of 5000 cm 22 in DMEM supplemented with 10% DCC-FBS. Four days later, a portion of the cells were harvested using Trizol agent (Dongsheng Biotech, Guangzhou, China). RNA was extracted using Trizol according to the manufacturer's instructions, and was reverse transcribed to cDNA using a reverse transcription kit (Bio-Rad, Hercules, CA, USA). Real-time PCR reactions were accomplished using SYBRH Premix Ex Taq TM II (TaKaRa Biotechnology, Dalian, China) in an ABI 7500 real-time Thermocycler (Applied Biosystems, Foster City, CA, USA). The data were analyzed using the SDS software, according to the manufacturer's instructions.
Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as an internal control. Data were presented as relative mRNA levels calculated by the equation 2 2DCt (DCt = Ct of target gene minus Ct of GAPDH) [48]. The primers used are listed in Table 1. PC-3 cells and the remaining hGF and hPDLC were harvested using lysis buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerol phosphate and 2 mM Na 3 VO 4 supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany)] [29] for Western blotting. The protein Figure 6. Effect of knockdown of 25-hydroxylases on 25OHD 3 generation. hGF and hPDLC from donors 2, 4 and 5 were treated with vitamin D 3 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. 25OHD 3 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 25OHD 3 increased with an increasing concentration of 25OHD 3 . When CYP27A1 was knocked down in hGF and hPDLC, the generation of 25OHD 3 decreased significantly compared to when CYP27A1 was not knocked down. When CYP2R1 was knocked down in hGF (A, C), the generation of 25OHD 3 was not significantly different from that when CYP2R1 was not knocked down. When CYP2R1 was knocked down in hPDLC (B, D), the generation of 25OHD 3 was only slightly different at some time points from that when CYP2R1 was not knocked down. The data are presented as the mean 6 SE. * hGF or hPDLC generated significantly less 25OHD 3 with the same amount of added vitamin D 3 when CYP27A1 or CYP2R1 was knocked down (p,0.05). # hGF or hPDLC generated significantly more 25OHD 3 with the same amount of added vitamin D 3 when CYP27A1 or CYP2R1 was knocked down (p,0.05). doi:10.1371/journal.pone.0052053.g006 concentration was determined using the Bicinchoninic Acid Protein Assay Kit (Applygen, Beijing, China). Twenty micrograms of total protein from each sample were loaded onto a gel comprising a 5% (w/v) stacking gel and a 10% (w/v) running gel. At the end of the electrophoresis, samples were transferred onto nitrocellulose blotting membranes (Hybond TM ; Amersham Pharmacia, Little Chalfont, UK). Blots were probed with a goat polyclonal antibody to CYP27A1 (  were treated with 1000 nM vitamin D 3 for 48 h after transfection with a siRNA oligonucleotide for CYP27A1 or a non-silencing control, and 1,25OH 2 D 3 production was measured in supernatants(A) and cell lysates (B). When CYP27A1 was knocked down, the generation of 1,25OH 2 D 3 decreased significantly compared to when CYP27A1 was not knocked down. The data are presented as the mean 6 SE. * hGF or hPDLC generated significantly less 1,25OH 2 D 3 with 1000 nM vitamin D 3 when CYP27A1 was knocked down (p,0.05). doi:10.1371/journal.pone.0052053.g007 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-1b and Pg-LPS significantly up-regulated CYP27A1 mRNA expression and the higher dose of IL-1b 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 6 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-1b group (p,0.05). ' CYP27A1 mRNA expression was significantly different from that of the 1 mg/mL Pg-LPS group (p,0.05). IL-1ß : interleukin-1b. Pg-LPS: Porphyromonas gingivalis lipopolysaccharide. doi:10.1371/journal.pone.0052053.g008

Detection of 25OHD 3 Production
Cells from 3 donors were treated with 1000 nM vitamin D 3 (Sigma, St. Louis, MO, USA) for 1, 4, 12, 24 or 48 h, after which supernatants were collected, and the cells were scraped in PBS containing 0.2% Triton X-100 and stored at 280uC. Prior to use, cell lysates were sonicated on ice in a sonifier cell disrupter for 2615 s. The levels of 25OHD 3 in cell supernatants and cell lysates were detected using a 25OHD 3 radioimmunoassay kit (DiaSorin, Stillwater, MN, USA) with a sensitivity of 1.5 ng/mL.

Detection of 1,25OH 2 D 3 Production
Cells from 3 donors were treated with 1000 nM vitamin D 3 (Sigma, St. Louis, MO, USA) for 48 h and then supernatants were collected and cells were scraped in PBS containing 0.2% Triton X-100 and stored at 280uC. Prior to use, cell lysates were sonicated on ice in a sonifier cell disrupter for 2615 s. The levels of 1,25OH 2 D 3 in cell supernatants and cell lysates were determined using a 1,25OH 2 D 3 radioimmunoassay kit (DiaSorin, Stillwater, MN, USA). The sensitivity of the assay was 2.0 pg/mL.

RNA Interference of 25-hydroxylase
To confirm the dependence of vitamin D 3 conversion to 25OHD 3 on 25-hydroxylase, the highly specific technique of RNA interference was utilized. Cells were seeded at a density of 15000 cm 22 in six-well plates. Eight hours later, the cells were transfected with either CYP27A1 siRNA (10 nM) or CYP2R1 siRNA (10 nM), or a non-silencing control siRNA using Hiperfect TM transfection reagent (Qiagen, Duesseldorf, Germany), according to the manufacturer's instructions. The target sequence of CYP27A1 siRNA was 59-CACGCTGACATGGGCCCTGTA -39, the target sequence of CYP2R1 siRNA was 59-TGGGTTGATCACAGACGATTA -39, and the non-silencing control was a non-homologous, scrambled sequence equivalent.
Sixty hours after transfection, cells were harvested, RNA and cDNA were obtained, and real-time PCR was performed as described earlier to test the effect of RNAi.
After confirming the effect of RNAi, 25OHD 3 production after RNAi was determined. Cells were first transfected with CYP27A1 siRNA (10 nM) or CYP2R1 siRNA (10 nM), or non-silencing control siRNA. Twelve hours after transfection, these cells were treated with 100 nM, 200 nM, 400 nM, 600 nM or 1000 nM vitamin D 3 (Sigma, St. Louis, MO, USA) for another 12 h. Then, the 25OHD 3 concentrations in the cell supernatants and cell lysates were determined as described earlier.
Some other cells were first transfected with CYP27A1 siRNA (10 nM), or non-silencing control siRNA, and 12 h after transfection, these cells were treated with 1000 nM vitamin D 3 (Sigma, St. Louis, MO, USA) for another 48 h. Then, the 1,25OH 2 D 3 concentrations in the cell supernatants and cell lysates were detected as described earlier.

Regulation of CYP27A1 in hGF and hPDLC
Cells from four donors were seeded into six-well plates at a density of 5000 cm 22 in DMEM supplemented with 10% DCC-FBS. Four days later, cells were incubated with IL-1b (PeproTech, London, UK; 1 ng/mL and 10 ng/mL), Pg-LPS (Invivogen, San Diego, CA, USA; 1 mg/mL and 10 mg/mL) or sodium butyrate (SCRC, Shanghai, China; 4 mM) for 24 h. Then mRNA expression was detected by real-time PCR as described previously.

Statistical Methods
The Shapiro-Wilk test was used to determinate the distribution of the variants. The paired samples t-test was used to compare differences of the mRNA expression levels of CYP27A1 and CYP2R1 between hGF and hPDLC, differences of 25OHD 3 generation by hGF and hPDLC, and the effect of RNA interference. Comparison of 25OHD 3 generation with and without knockdown of 25-hydroxylase, and 1,25OH 2 D 3 generation with and without knockdown of CYP27A1 were also performed using a paired samples t-test. The impact of stimulation on CYP27A1 mRNA expression was analyzed using a pairedsamples t-test, and the difference between CYP27A1 regulation in hGF and hPDLC was analyzed using a Wilcoxon test.
Statistical analyses were accomplished using the SPSS 11.5 software package (SPSS Inc., Chicago, IL, USA). A p value ,0.05 was considered statistically significant.