BASIC RESEARCH www.jasn.org Fibroblast Growth Factor 23 Regulation by Systemic and Local Osteoblast-Synthesized 1,25-Dihydroxyvitamin D † ‡ | Loan Nguyen-Yamamoto,* Andrew C. Karaplis, Rene St–Arnaud,* § and David Goltzman* Departments of *Medicine, ‡Surgery, and §Human Genetics, and †Department of Medicine, Sir Mortimer B. Davis Jewish General Hospital, McGill University, Montreal, Canada; and |Research Centre, Shriners Hospital for Children, Montreal, Canada 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-a-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 Dmetabolitesvia the vitamin D receptor. In chromatin immunoprecipitation assays, 25-hydroxyvitamin D pro- moted 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. J Am Soc Nephrol 28: 586–597, 2017. doi: 10.1681/ASN.2016010066 Fibroblast growth factor 23 (FGF23) is a 32 kD and parathyroid hormone (PTH).10–13 Vitamin D re- (251 aa) hormone secreted mainly by skeletal sponse elements (VDREs) in the FGF23 promoter osteocytes and osteoblasts1 and was originally dis- have been reported14–16 and several in vitro studies 14–16 covered as a cause of autosomal dominant hypo- have shown that both 1,25(OH)2D and phosphatemic rickets.2 Subsequent studies have PTH10–13 can directly increase FGF23 gene transcription. confirmed its role in other congenital and acquired hypophosphatemic/phosphaturic conditions in humans. Additionally, spontaneous and genetically Received January 19, 2016. Accepted July 10, 2016. engineered mouse models have demonstrated the Published online ahead of print. Publication date available at potent effect of circulating FGF23 on inhibiting re- www.jasn.org. 3–8 nal phosphate reabsorption. Correspondence: Dr. David Goltzman, McGill University Health The production of FGF23 has been reported to Centre, Glen Site, 1001 Decarie Blvd, Room EM1.3220, Mon- be stimulated by systemic factorssuchascirculating treal, QC, Canada, H4A 3J1. Email: [email protected] 9 calcitriol (1,25-dihydroxyvitamin D [1,25(OH)2D]) Copyright © 2017 by the American Society of Nephrology 586 ISSN : 1046-6673/2802-586 J Am Soc Nephrol 28: 586–597, 2017 www.jasn.org BASIC RESEARCH 34,35 However, 1,25(OH)2D/vitamin D receptor (VDR) induction of described, its exact function is unclear. Alternatively, 25 an intermediate factor appears to be a requirement for a full (OH)D, and also 1,25(OH)2D, can be 24-hydroxylated by a increase in FGF23 production.15 25-hydroxyvitamin D 24-hydroxylase (24[OH]ase) enzyme, Although dietary phosphate (P),17–19 serum P,20 and cal- CYP24A1, as a first step in the catabolism of these vitamin 20 cium (serum Ca) are able to increase circulating levels of D metabolites. In a negative feedback loop, 1,25(OH)2Dcan FGF23, the precise molecular mechanism whereby these inhibit renal CYP27B1, but 1,25(OH)2D can also stimulate ions regulate FGF23 is unclear. Serum Ca–mediated increases renal CYP24A1, thus reducing further 1,25(OH)2Dsynthesis in serum FGF23 required a threshold level of serum phospho- and enhancing its own elimination in order to maintain 36 rus and, likewise, P-elicited increases in FGF23 were markedly appropriate circulating levels of 1,25(OH)2D. FGF23 also blunted if serum Ca was below a threshold level.20 Conse- acts at the proximal renal tubule to downregulate the quently, the best correlation between Ca and P and serum expression of CYP27B1 and stimulate the expression FGF23 was found between FGF23 and the Ca3P product.20 of CYP24A1.37,38 Decreases in CYP27B1 activity due Nevertheless, the use of a Ca3P product as a determinant of a to FGF23 may also result from a defect in translational or physiologic or pathologic event has been questioned, particu- post-translational modification of the enzyme.39 The 21 larly as it relates to ectopic calcification. Iron deficiency may decreased circulating 1,25(OH)2D that occurs as a conse- also cause high levels of both intact and carboxy-terminal quence of these actions of FGF23 can then decrease intestinal FGF23.22 In addition, circulating FGF23 levels increase very Ca absorption. 23 soon after kidney disease occurs ; however, the mechanism The active form of vitamin D, 1,25(OH)2D, can bind to the underlying this early rise is poorly understood. Finally, several VDR and form a heterodimer with the retinoid X receptor local bone-derived factors, such as PHEX, DMP1, and MEPE, (RXR). This complex can then bind to VDREs to regulate may act in an autocrine/paracrine mode to regulate FGF23 gene transcription.40,41 It has also been reported that, at expression in bone.24 Nevertheless, their mechanism of regu- high concentrations, 25(OH)D may bind as an agonist to lating FGF23 production still remains to be determined. VDR, and may be transcriptionally active.42–47 Thus, a complete lack of DMP1 in the context of normal renal Although local production of 1,25(OH)2Dbybonecells function results in increased circulating levels and bone ex- has been hypothesized to contribute to regulation of FGF23 pression of FGF23.25,26 However, overexpression of DMP1 production,48 direct evidence for an autocrine function of does not cause the inverse phenotype, that is, DMP1 excess 1,25(OH)2D in this regulation has not yet been reported. In does not suppress FGF23 expression.27,28 Furthermore, a si- this study, we used genetically engineered mouse models to multaneous increase in both DMP1 and FGF23 expression was assess the role of vitamin D metabolites in FGF23 regulation, 2 2 reported in osteocytes of patients with CKD, which also ap- and probed the contribution of skeletal 1(OH)ase / in mod- pears contrary to the concept that DMP1 acts to suppress ulating FGF23 production in the presence and absence of renal 29 FGF23 expression. 1,25(OH)2D has been reported to regu- dysfunction. late FGF23 expression by repressing DMP1 via the VDR path- way;30 however, in a study in dialysis patients treated with active vitamin D, bone-intact FGF23 increased but DMP1 RESULTS fragments were altered with therapy.31 Consequently, further understanding of the effects of DMP1 fragments on FGF23 Studies in Mice with Normal Renal Function production and the role of vitamin D in mediating this effect Serum Ca and P levels were significantly reduced and PTH 2 2 2 2 appears to be needed. levels elevated in the 1(OH)ase / mice and the VDR / mice FGF23 acts by binding to FGF receptors (FGFRs), which are on a high-Ca diet compared with wild-type (WT) mice (Fig- transmembrane phosphotyrosine kinases, and complexing ure 1, A–C). Serum Ca, P, and PTH were normalized on the 32,33 with a Klotho, an essential coreceptor for FGF23. Thus rescue diet compared with the high-Ca diet. 1,25(OH)2D lev- 2 2 coexpression of FGFR and Klotho appears to define the target els were undetectable in the 1(OH)ase / mice on either diet 2 2 tissue specificity of FGF23 action. FGF23 exerts its phospha- andwereelevatedintheVDR / mice on both diets (not turic action by reducing the sodium-P cotransporters, Npt2a shown), as previously described.49 FGF23 levels were low in 2 2 and Npt2c, in the renal proximal tubule and thus decreasing P the 1(OH)ase / mice on the high-Ca diet (Figure 1D) in 3–7 reabsorption. association with the undetectable circulating 1,25(OH)2D, Vitamin D is derived either via ultraviolet irradiation of a even though PTH levels were elevated (Figure 1C). When 2 2 2 2 skin precursor, or via intestinal absorption from the diet, and the 1(OH)ase / mice were fed a rescue diet (1(OH)ase / R) can then be enzymatically converted in the liver to calcidiol and serum Ca and P were normalized, FGF23 levels rose (25-hydroxyvitamin D [25(OH)D]), the most abundant despite a fall in PTH levels and an undetectable level of circulating vitamin D metabolite. Subsequently, CYP27b1, en- 1,25(OH)2D (Figure 1D). Consequently, PTH did not seem coding 25-hydroxyvitamin D 1a-hydroxylase (1[OH]ase), to be a critical determinant of FGF23 levels in this setting. In the 2/2 converts 25(OH)D to its active form, 1,25(OH)2D, in the kid- VDR mice, FGF23 levels were low on the high-Ca diet even in ney. Although extrarenal skeletal 1(OH)ase has been the presence of high circulating 1,25(OH)2D, indicating that the J Am Soc Nephrol 28: 586–597, 2017 FGF23 Regulation by Vitamin D 587 BASIC RESEARCH www.jasn.org 1,25(OH)2D, a stable and sustained increase in FGF23 levels was seen (Figure 2B).