Fibroblast Growth Factor 23 Regulation by Systemic and Local Osteoblast-Synthesized 1,25-Dihydroxyvitamin D

Abstract

Circulating levels of fibroblast growth factor 23 (FGF23) increase during the early stages of kidney disease, but the underlying mechanism remains incompletely characterized. We investigated the role of vitamin D metabolites in regulating intact FGF23 production in genetically modified mice without and with adenine-induced uremia. Exogenous calcitriol (1,25-dihydroxyvitamin D) and high circulating levels of calcidiol (25-hydroxyvitamin D) each increased serum FGF23 levels in wild-type mice and in mice with global deficiency of the Cyp27b1 gene encoding 25-hydroxyvitamin D 1-α-hydroxylase, which produces 1,25-hydroxyvitamin D. Compared with wild-type mice, normal, or uremic mice lacking Cyp27b1 had lower levels of serum FGF23, despite having high concentrations of parathyroid hormone, but administration of exogenous 1,25-dihydroxyvitamin D increased FGF23 levels. Furthermore, raising serum calcium levels in Cyp27b1-depleted mice directly increased FGF23 levels and indirectly enhanced the action of ambient vitamin D metabolites via the vitamin D receptor. In chromatin immunoprecipitation assays, 25-hydroxyvitamin D promoted binding of the vitamin D receptor and retinoid X receptor to the promoters of osteoblastic target genes. Conditional osteoblastic deletion of Cyp27b1 caused lower serum FGF23 levels, despite normal circulating levels of vitamin D metabolites. In adenine-induced uremia, only a modest increase in serum FGF23 levels occurred in mice with osteoblastic deletion of Cyp27b1 (12-fold) compared with a large increase (58-fold) in wild-type mice. Therefore, in addition to the direct effect of high circulating concentrations of 25-hydroxyvitamin D, local osteoblastic conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D appears to be an important positive regulator of FGF23 production, particularly in uremia.

Keywords: Activated Vitamin D; FGF23; Vitamin D; calcium; parathyroid hormone.

Figure 1.

Circulating FGF23 is influenced by serum calcium and VDR levels. Basal serum biochemical parameters of WT, VDR^−/−, and 1(OH)ase^−/− mice. Serum Ca, P, PTH, and FGF23 are shown in panels (A–D), respectively. WT mice were maintained on normal chow. 1(OH)ase^−/− and VDR^−/− mice were maintained either on a high-Ca diet or a rescue diet, denoted as 1(OH)ase^−/−C and VDR^−/−C, and 1(OH)ase^−/−R and VDR^−/−R, respectively. Bars represent the mean±SEM of measured data from six to eight animals of each genotype. ^♦♦♦P < 0.001 compared with age-matched WT; ***P<0.001 and **P≤0.01 compared with mice of the same genotype on a high-Ca diet.

Figure 2.

FGF23 increases induced by vitamin D metabolites are enhanced by increasing the serum calcium. Comparison of the FGF23 responses to exogenous 1,25(OH)₂D and 25(OH)D treatment of WT mice (A) and of 1(OH)ase^−/−C mice (on a high-Ca diet) and 1(OH)ase^−/−R mice (on a rescue diet) (B). Serum Ca levels are shown in WT mice (C) and in 1(OH)ase^−/−C and 1(OH)ase^−/−R mice (D) after treatment with 1,25(OH)₂D and 25(OH)D. Intraperitoneal injections of 1,25(OH)₂D (6 ng/g) or 25(OH)D (100 ng/g) were given every 2 days for 1 week. Alternatively, exogenous 1,25(OH)₂D (50 pg/g) was administered for 2 months (2M) to 1(OH)ase^−/−C mice (on a high-Ca diet). Bars represent the mean±SEM. Significant differences between groups were determined by one-way ANOVA followed by Bonferroni test. *P≤0.05; **P≤0.01; ***P≤0.001; and ****P≤0.0001 compared with vehicle-treated mice of the same genotype on the same diet; ^‡P≤0.05 compared with 1(OH)ase^−/−C mice.

Figure 3.

Increased VDR expression in osteoblasts is induced by 1,25(OH)₂D and further amplified by increasing serum calcium. Representative sections of VDR expression in bone analyzed by immunohistochemistry of femur from WT, VDR^−/−, and 1(OH)ase^−/− mice. The decalcified bone tissue was paraffin-embedded and stained for antibodies to VDR. Positive labeling for VDR in WT mice on normal chow is clearly evident in brown and denoted by black arrows (A). VDR expression in VDR^−/− mice on a rescue diet was a negative control and showed no staining (D). VDR expression in femurs of 1(OH)ase^−/− mice on a high-Ca diet contained undetectable to low levels of VDR (B), and no change was observed after treatment with exogenous 1,25(OH)₂D (C). VDR expression in 1(OH)ase^−/− mice was upregulated by a rescue diet (E) and increased further after exogenous treatment with 1,25(OH)₂D (F). Original magnification, ×300 in A and D; ×200 in B, C, E, and F.

Figure 4.

1,25(OH)₂D and 25(OH)D stimulate VDR and RXR recruitment to the VDRE site on the promoters of osteoblastic proteins. Chromatin was extracted from intact MC3T3 or UMR106 osteoblastic cells that had been treated with vehicle or 10^–7 M 1,25(OH)₂D or 10 ^–6 M 25(OH)D for 2 hours, in the presence of 25 mM ketoconazole to inhibit 1-hydroxylation of 25(OH)D. Extracts were then crosslinked and subjected to immunoprecipitation with VDR or RXR antibody. Nonimmunoprecipitated (Input) and Immunoprecipitated DNA from MC3T3-E3 cells were subjected to PCR using specific primers designed according to the VDRE site located in the promoter region of the target genes osteopontin, osteocalcin, and Cyp24a1 (A). PCR products were analyzed using 2% agarose gels, and representative agarose gels of 2–3 independent experiments are shown. Control was DNA immunoprecipitated with IgG. For assessment of VDR and RXR recruitment to the promoter of the FGF23 gene chromatin extracted from UMR106 cells and DNA immunoprecipitated by ChIP assay as above were analyzed by qPCR using a SsoFast-EvaGreen real-time PCR kit. Expression was normalized to the expression of input. Values represent results of three independent experiments (B).

Figure 5.

Circulating FGF23 levels, are reduced in non-uremic mice deficient in osteoblastic 1(OH)ase and are increased by exogenous 1,25(OH)₂D but not 25(OH)D. Serum biochemistry of OB-1(OH)ase^−/− mice. WT and OB-1(OH)ase^−/− mice were maintained on normal chow and were euthanized at 3 months of age. Blood was collected for biochemical assays as described in Concise Methods. Serum 1,25(OH)₂D (A), Ca (B), P (C), PTH (D), FGF23 (E), 25(OH)D (F), and 24,25(OH)₂D (G) are shown in WT, 1(OH)ase^−/−C, and OB-1(OH)ase^−/− mice after treatment with vehicle (V) or with exogenous 1,25(OH)₂D (6 ng/g every 2 days for 1 week) or with 25(OH)D (100 ng/g every 2 days for 1 week). Bars represent the mean±SEM of 6–8 animals per group; significant differences between groups were determined by one-way ANOVA followed by Bonferroni test. *P≤0.05; ***P<0.001; and ****P≤0.001 relative to mice of the same genotype on the same diet; ^‡P≤0.05; ^‡‡P≤0.01; and ^‡‡‡‡P≤0.001 compared with WT mice.

Figure 6.

The FGF23 increase in uremic mice is absent after global deletion of the 1(OH)ase and markedly reduced after specific deletion of the osteoblastic 1(OH)ase. Comparison of biochemical abnormalities in adenine-induced CKD in WT, 1(OH)ase^−/−C, and OB-1(OH)ase^−/− mice. Serum urea nitrogen (A), Ca (B), P (C), 1,25(OH)₂D (D), PTH (E), and FGF23 (F) levels are shown. Levels of mRNA encoding FGF23 extracted from long bone of WT and OB-1(OH)ase^−/− mice and determined by qPCR are shown in (G) and serum 24,25(OH)₂D levels in (H). Bars represent the mean±SEM of measured data from six to eight animals per group. Significant differences between adenine-treated mice compared with age-matched control mice on a regular diet were determined by one-way ANOVA followed by a Bonferroni test. *P≤0.05; **P≤0.01; ***P<0.001; and ****P≤0.0001 relative to mice of the same genotype on a regular diet; ^♦P<0.05 and ^♦♦P<0.01 compared with age-matched WT mice on the same diet.

Figure 7.

Model of the regulation of FGF23 by vitamin D metabolites and minerals showing the important role of renal-derived and osteoblast- derived 1,25(OH)₂D. (A) In the presence of normal renal function. Circulating 25(OH)D can be converted in the kidney to 1,25(OH)₂D via the 1(OH)ase (Cyp27b1). 1,25(OH)₂D can stimulate 24(OH)ase and 25(OH)D can be degraded by the 24(OH)ase (Cyp24a1) to 24,25(OH)₂D (24,25D). 1,25(OH)₂D can also be converted to 1,24,25(OH)₃D (1,24,25D) by the 24(OH)ase. 1,25(OH)₂D can itself stimulate 24(OH)ase activity. Renal-derived circulating 1,25(OH)₂D can then enter the mature osteoblast/osteocyte and bind the VDR which then complexes with the RXR. In the nucleus, this complex may bind to an FGF23 VDRE and increase FGF23 transcription, resulting in increased FGF23 production and release. Circulating 25(OH)D may also enter the mature osteoblast/osteocyte and be converted by the osteoblastic 1(OH)ase to 1,25(OH)₂D or be degraded by the osteoblastic 24(OH)ase to 24,25(OH)₂D. The intracellular 1,25(OH)₂D produced from 25(OH)D by the osteoblast may act as an intracrine factor and bind to VDR and increase FGF23 production. At high circulating concentrations of 25(OH)D, this vitamin D metabolite may also act by directly binding to VDR and stimulating FGF23 production. Both Ca, by unknown mechanisms, and 1,25(OH)₂D, by stimulation of transcription, can increase VDR concentrations and facilitate the actions of 1,25(OH)₂D and 25(OH)D. Ca and P can increase FGF23 production by unknown mechanisms. (B) In the absence of renal function. Renal conversion of 25(OH)D to 1,25(OH)₂D is impaired, and renal degradation of 25(OH)D is also impaired. Osteoblastic production of 1,25(OH)₂D becomes the major source of 1,25(OH)₂D in the mature osteoblast/osteocyte and a major regulator of FGF23 production. Circulating 25(OH)D levels may be influenced by nutritional intake of vitamin D as well as ultraviolet light–induced production of vitamin D.