OBJECTIVE: There is asingle nucleotide polymorphism (SNP) in the exon 2 of the vitamin D receptor (VDR) gene that can be distinguished using the restriction endonuclease FokI, and accordingly divided into three genotypes: FF, Ff and ff. VDR-FokI polymorphism was the only known SNP that could alter the protein structure of VDR. CYP24A1 is the gene encoding vitamin D 24 hydroxylase and is a vitamin D responsive gene. The influence of rs2228570 on transcriptional activation by VDR in human gingival fibroblasts (hGF) and periodontal ligament cells (hPDLC) was investigated in this study. METHODS: hGF and hPDLC of 12 donors' were primarily cultured and genomic DNA was extracted. A part of genomic DNA with the length of 267 bp was obtained using PCR, which contained the SNP. VDR-Fok I genotypes were determined according to the results of restriction fragment length polymorphism. hGF and hPDLC were stimulated with 10 nmol/L 1α,25 dihydroxy vitamin D₃ (1,25OH₂D₃) or 1 000 nmol/L 25 hydroxy vitamin D₃ (25OHD₃) for 48 h before RNA was extracted. Then VDR antagonist ZK159222 was used or not used during 1,25OH₂D₃ or 25OHD₃ stimulation with hGF and hPDLC. After 1,25OH₂D₃ stimulation for 48 h, the proteins in hGF and hPDLC were also collected. The protein expressions of CYP24A1 and VDR were detected using Western blot. RESULTS: Among the 12 donors' cell cultures, the number of FF, ff and Ff genotypes was 4, 3 and 5, respectively.After stimulation with 1,25OH₂D₃ or 25OHD₃ for 48 h,CYP24A1 mRNA levels in FF-hGF were significantly higher than those in other hGF genotypes(1,25OH₂D₃: F=31.147, P<0.01; 25OHD₃: F= 32.061,P <0.01), as was in FF-hPDLC (1,25OH₂D₃: F=23.347, P<0.01; 25OHD₃: F=32.569,P<0.01). When ZK159222 was used before 1,25OH₂D₃ stimulation, this statistically significant difference disappeared (hGF: F=0.246, P=0.787; hPDLC: F=0.574, P=0.583). When ZK159222 was used before 25OHD₃ stimulation, the trend was similar (hGF: F=1.636, P=0.248; hPDLC: F=0.582, P=0.578).After stimulation with 1,25OH₂D₃ for 48 h, CYP24A1 protein levels in FF-hGF were significantly higher than those in the other hGF genotypes (F=12.368, P <0.01), as was in FF-hPDLC (F=15.749, P <0.01). In hGF and hPDLC, the mRNA or protein expression of VDR of different genotypes was not significantly different under different stimulation conditions.The paired comparison showed that there was no statistically significant difference between the expression of CYP24A1 in hGF and that in hPDLC under all the stimulation conditions, as was the expression of VDR. CONCLUSION In hGF and hPDLC, the FF-VDR genotype is associated with the more remarkable up-regulation of CYP24A1than the other genotypes, indicating that transcriptional activation of FF-VDR might be higher than those of other vitamin D receptors.
Fibroblasts/metabolism* ; Genotype ; Humans ; Periodontal Ligament/metabolism* ; Polymorphism, Genetic ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; Receptors, Calcitriol/genetics* ; Vitamin D3 24-Hydroxylase/metabolism*

AIMTo investigate the effects of 1 alpha hydroxylase and serum calcium on the expression of 24-hydroxylase gene in mice kidney.

METHODSMice with targeted deletion of the 25-hydroxyvitamin D 1 alpha hydroxylase gene(1alpha (OH)ase-/-), and the vitamin D receptor gene (VDR-/-) were used. The study of each mutant had two groups which were (1) mutant with high calcium diet, which maintained fertility but left mice hypocalcaemia; (2) mutant with high lactose diet, which normalized calcium in two mutant. Mice in same litter were as control. There were six groups in total and each group had five mice. All mice were killed at 10-week-old. Serum calcium was determined by an autoanalyzer. RNA was isolated from mouse kidney and the express of 1 alpha hydroxylase gene and 24-Hydroxylase gene were studied by RT-PCR.

RESULTSOn the high calcium intake, all mutant animals were hypocalcaemia (1alpha (OH)ase-/- (78 +/- 10.4) mg/L, P < 0.05; VDR-/- (68 +/- 9.8) mg/L, P < 0.05. WT (111 +/- 16.5 mg/L), but when the high lactose diet was administered, serum calcium levels in two mutant mice rose to wild-type levels. The 1 alpha hydroxylase gene was expressed at very higher levels in the vitamin D receptor mutant mice than in wild-type mice when animals received a high calcium intake; This was reduced by eliminating hypocalcaemia with the high lactose diet. Expression of the 24(OH)ase gene was extremely down-regulated in two mutant mice on the high calcium diet but was restored to wild-type levels on the high lactose diet.

CONCLUSIONThe express of 24-hydroxylase gene was directly regulated by serum calcium rather than 1 alpha-hydroxylase. These studies indicate that both the serum calcium and 1 alpha-hydroxylase exert effects on the expression of 24-hydroxylase gene, but 1 alpha-hydroxylase take the effects by elevated the concentration of serum calcium. There are no direct interaction between 1 alpha-hydroxylase gene and 24-hydroxylase gene.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase ; genetics ; Animals ; Calcium ; blood ; Gene Expression ; Mice ; Mice, Knockout ; Serum ; chemistry ; Steroid Hydroxylases ; genetics ; metabolism ; Vitamin D3 24-Hydroxylase