Fibroblast Growth Factor 23 Is a Counter-Regulatory Phosphaturic Hormone for Vitamin D
Shiguang Liu,
Wen Tang,
Jianping Zhou,
Jason R. Stubbs,
Qiang Luo,
Min Pi and
L. Darryl Quarles
Department of Internal Medicine and the Kidney Institute, University of Kansas Medical Center, Kansas City, Kansas
Address correspondence to: Dr. Shiguang Liu, Department of Internal Medicine and the Kidney Institute, University of Kansas Medical Center, 3901 Rainbow Boulevard, Room 6020 WHE, MS 3018, Kansas City, KS 66160. Phone: 913-588-0705; Fax: 913-588-9251; E-mail: sliu{at}kumc.edu
Received for publication November 14, 2005.
Accepted for publication March 4, 2006.
The regulation of the phosphaturic factor fibroblast growthfactor 23 (FGF23) is not well understood. It was found thatadministration of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) tomice rapidly increased serum FGF23 concentrations from a basallevel of 90.6 ± 8.1 to 213.8 ± 14.6 pg/ml at 8h (mean ± SEM; P < 0.01) and resulted in a four-foldincrease in FGF23 transcripts in bone, the predominate siteof FGF23 expression. In the Hyp-mouse homologue of X-linkedhypophosphatemic rickets, administration of 1,25(OH)2D3 furtherincreased circulating FGF23 levels. In Gcm2 null mice, low 1,25(OH)2D3levels were associated with a three-fold reduction in FGF23levels that were increased by administration of 1,25(OH)2D3.In osteoblast cell cultures, 1,25(OH)2D3 but not calcium, phosphate,or parathyroid hormone stimulated FGF23 mRNA levels and resultedin a dose-dependent increase in FGF23 promoter activity. Overexpressionof a dominant negative vitamin D receptor inhibited 1,25(OH)2D3stimulation of FGF23 promoter activity, and mutagenesis of theFGF23 promoter identified a vitamin D-responsive element (–1180GGAACTcagTAACCT –1156) that is responsible for the vitaminD effects. These data suggest that 1,25(OH)2D3 is an importantregulator of FGF23 production by osteoblasts in bone. The physiologicrole of FGF23 may be to act as a counterregulatory phosphaturichormone to maintain phosphate homeostasis in response to vitaminD.
Fibroblast growth factor 23 (FGF23) is a recently discoveredphosphaturic hormone (1–5). Impaired degradation of FGF23,as a result of mutations in a proprotein convertase cleavagesite, accounts for the hypophosphatemia and impaired 1,25-dihydroxyvitaminD3 (1,25[OH]2D3) production in autosomal dominant hypophosphatemicrickets (1). Increased production of FGF23 is the cause of phosphaturiaand disordered vitamin D metabolism in tumor-induced osteomalacia(6,7), X-linked hypophosphatemic rickets (XLH) (7,8), and McCune-Albrightsyndrome (9). In addition, transgenic mice that overexpressa cleavage-resistant mutant form of FGF23 have hypophosphatemia(10). Conversely, decrements in FGF23 function, as a resultof either loss-of-function mutations of FGF23 in hereditaryforms of tumoral calcinosis (11,12) or FGF23 deficiency producedby the deletion of FGF23 in mice (4,13), cause hyperphosphatemia.
FGF23 also suppresses 1 hydroxylase activity in the proximalrenal tubule, leading to reduced circulating levels of 1,25(OH)2D3(2,10,14,15). The significance of FGF23 regulation of 1,25(OH)2D3production is not clear, but the findings that FGF23 is producedpredominantly by osteoblasts in bone and that FGF23 regulatesphosphate reabsorption and 1,25(OH)2D3 production by the kidneyraise the possibility that FGF23 may be involved in a bone-kidneyaxis that controls phosphate and vitamin D homeostasis (16,17).How FGF23 is integrated with the vitamin D-parathyroid hormone(PTH) axis, which plays a central role in calcium homeostasis,skeletal development, and mineralization (18), however, is notclear. Understanding the effects of 1,25(OH)2D3 on FGF23 productionis important, because vitamin D therapy often is used to treatFGF23-mediated hypophosphatemic disorders, such as XLH (19).
In an effort to understand more fully the regulation of FGF23expression in osteoblasts and bone, we assessed the effect of1,25(OH)2D3 administration on circulating levels of FGF23 inwild-type Gcm2 null (20) and Hyp mice (21), as well as the effectsof 1,25(OH)2D3 on the FGF23 transcripts in bone. In addition,we investigated the ability of 1,25(OH)2D3 to regulate endogenousFGF23 transcripts and the activity of a transfected murine FGF23promoter luciferase reporter in osteoblasts. Our findings demonstratethe importance of bone as a target for vitamin D-mediated incrementsin FGF23 production and suggest that FGF23 production servesas a counterregulatory hormone to enhance renal phosphate clearancein response to vitamin D-mediated increments in gastrointestinalphosphate absorption and decrements in the phosphaturic hormonePTH.
1,25(OH)2D3 and PTH Administration
Both Hyp mice (21) and C57BL/6J mice were purchased from JacksonLaboratory (Bar Harbor, ME). Male and female Gcm2+/– micewere mated to generate homozygous Gcm2 null mice that lackedparathyroid glands (22). All mice were maintained and used inaccordance with recommendations in the Guide for the Care andUse of Laboratory Animals, prepared by the Institute on LaboratoryAnimal Resources, National Research Council (Department of Health& Human Services Publication NIH 86-23, National AcademyPress, 1996) and by guidelines established by the InstitutionalAnimal Care and Use Committee of the University of Kansas MedicalCenter. Mice were fed with Harlan Teklad Rodent Diet (W) 8604(Harlan Teklad, Madison, WI). Calcitriol (American PharmaceuticalPartners, Inc., Schaumburg, IL) was diluted in 0.9% sodium chloridefor intraperitoneal injection. The same volume of 0.9% sodiumchloride was injected in the control group. Serum and tissuesamples were collected from the mice before injection for baselinemeasurements and at various time points as indicated.
Serum Bioassays
Serum FGF23 levels were measured using an FGF23 ELISA kit (KainosLaboratories, Inc., Tokyo, Japan). Serum calcium and phosphatewere measured, respectively, using Calcium (CPC) Liquicolor(Stanbio Laboratory, Boerne, TX) and the phosphomolybdate-ascorbicacid method as described previously (23). Serum PTH was determinedusing the Mouse Intact PTH ELISA Kit (Immunotopics, San Clemente,CA).
RNA Isolation and Quantitative Reverse Transcription-PCR
Total RNA was extracted from snap-frozen tissues and from culturedcells with Trizol (Invitrogen, Carlsbad, CA) and then treatedwith RNase-Free DNAse using an RNeasy column (Qiagen, Valencia,CA). First-strand cDNA was synthesized using iScript cDNA SynthesisKit (Bio-Rad Laboratories, Hercules, CA). One microgram of totalRNA was used in each 20 µl of reverse transcription reaction.A total of 400 ng and 20 ng of input RNA were used to amplifyFGF23 and cyclophilin A, respectively. The forward primer 5'-TTGGATCGTATCACTTCAGC-3'and reverse primer 5'-TGCTTCGGTGACAGGTAG-3' were used to amplifyrat FGF23 from ROS17/2.8 cells. Forward primer 5'-TTTCCCAGGTTCGTCTAGG-3'and reverse primer 5'-CTCGCAGGTGACTCTCAG-3' were used to amplifyFGF23 from mouse samples. Forward primer 5'-GAAGGCATGAACATTGTGGAAG-3'and reverse primer 5'-ACAGAAGGAATGGTTTGATGGG-3' were used toamplify mouse and rat cyclophilin A. The iCycler iQ Real-TimePCR Detection System and iQ SYBR Green Supermix (Bio-Rad Laboratories)were used for real-time PCR analysis (24).
Isolation of the Murine FGF23 5'-Flanking Region
A mouse BAC clone that contained the mouse FGF23 gene was purchasedfrom Children’s Hospital Oakland Research Institute. WithRP23-195E18 as a template, we amplified a 3550-bp fragment from–1 to –3550, relative to the translation start codon,by PCR with BIO-X-ACT DNA polymerase (Bioline USA, Inc., Randolph,MA), using forward primer 5'-GAGGTACCTCATCTATGGAGTAGACTC-3'and reverse primer 5'-GCAAGCTTTGCACAGCACTGAGTGGCTAATGC-3'. ThePCR product was cloned into a pCR II-TOPO vector. The nucleotidesequence was confirmed by DNA sequence analysis.
The putative promoter and transcriptional start site were predictedby Neural Network Promoter Prediction (http://www.fruitfly.org/seq_tools/promoter.html),and transcriptional factor binding sites were analyzed by Matinspectorand DiAlign TF from Genomatix (http://www.genomatix.de/company/index.html).The nuclear hormone receptor response elements were screenedby nuclear hormone receptor-scan (25).
FGF23 Promoter/Reporter and Other Constructs
Luciferase constructs were numbered according to the translationstart site of the FGF23 gene. A 3550-bp FGF23 5'-flanking regionfrom –3550 to –1 relative to the translation startsite ATG was subcloned into a pGL3-Basic vector (Promega, Madison,WI) between KpnI and Hind III restriction sites (p3550Fgf23-luc)to create FGF23 promoter/firefly luciferase reporter construct.For generation of luciferase constructs that contained different5' deletions of the FGF23 promoter, forward primers startingat –2000, –1300, –1000, –600, and thesame reverse primer ending at –1 were used for PCR amplificationof constructs (Table 1). To confirm the putative vitamin D responsiveelement (VDRE), we deleted the putative VDRE located in –1180to –1166 of the FGF23 5' flanking region using a PCR mutagenesismethod. An RL-TK construct (Promega) was used as an internalcontrol for transfection efficiency. pBK-CMV-PTHr expressingthe rat PTH receptor driven by a cytomegalovirus promoter wasgenerated as described previously (26). A dominant mutant formof vitamin D receptor (VDR)-expressing construct pSG5E420A wasprovided by Dr. Mark R. Haussler (University of Arizona [27]).
Table 1. Primers used in PCR for generation of FGF23 promoter constructsa
Mammalian Cell Culture, Transient Transfection, and Promoter/Reporter Assays
ROS17/2.8 and UMR-106 osteoblasts were grown as described previously(28,29) in a humidified incubator with 5% CO2 at a temperatureof 37°C. Using the FuGENE 6 Transfection Reagent (RocheApplied Science, Indianapolis, IN), we transfected ROS17/2.8osteoblasts with 1 µg of p3550Fgf23-luc and 0.01 µgof pRL-TK per well in six-well plates. To assess PTH effects,we used 0.6 µg of p3550Fgf23-luc, 0.4 µg of pBK-PTHr,and 0.01 µg of pRL-TK. Different concentrations of 1,25(OH)2D3 (Sigma-Aldrich, St. Louis, MO), PTH (1-34 fragment; Sigma-Aldrich),phosphate (potassium phosphate monobasic/sodium phosphate dibasic[pH 7.2]), and calcium chloride were added with fresh culturemedium 24 h after transfection, and cells were harvested afteranother 24-h culture period using Passive Lysis Buffer (Promega).For the FGF23 promoter deletion study, a similar transfectionmethod and 10–8 M 1,25(OH)2D3 concentration were used.The Firefly and renilla luciferase activities were measuredwith a Dual-Luciferase Reporter Assay System (Promega). To investigatethe regulation of the endogenous promoter of the FGF23 gene,we also cultured ROS17/2.8 cells with the same concentrationof 1,25(OH)2D3, PTH (fragment 1-34), phosphate, and calciumchloride for 24 h as in the transfection study, and then totalRNA was isolated for real-time reverse transcription-PCR (RT-PCR).
Statistical Analyses
We evaluated the differences between the two groups with a ttest or Wilcoxon test when the distribution was not normal.We used one-way ANOVA for multiple sample comparisons. All valuesare expressed as mean ± SEM. All computations were performedusing the Statgraphic statistical graphics system (STSC, Inc.,Rockville, MD).
Effects of 1,25(OH)2D3 on Serum FGF23 Concentrations
To determine whether 1,25(OH)2D3 increases circulating FGF23levels, we measured serum FGF23 concentrations 4, 8, and 24h after intraperitoneal injections of 1,25(OH)2D3 (100 ng/kgbody wt) into wild-type mice (Figure 1A). We observed that serumFGF23 levels were significantly increased at 4 h from a baselinevalue of 90.6 ± 8.1 to 141.0 ± 6.9 pg/ml (mean± SEM; P < 0.01). The increment reached a peak of213.8 ± 14.6 pg/ml at 8 h and declined to 142.7 ±7.0 pg/ml at 24 h (Figure 1A), remaining significantly elevatedabove baseline (P < 0.01). Next, we examined the dose-dependenteffects of 1,25(OH)2D3 to stimulate serum FGF23 levels (Figure 1B).Intraperitoneal administration of 1,25(OH)2D3 at a dose of 10ng/kg body wt had no effect on circulating FGF23 concentrations,whereas incremental increases in serum FGF23 levels were observed8 h after the administration of 50, 100, and 1000 ng/kg bodywt, achieving a three-fold increase at the highest dose (Figure 1B).Table 2 shows the mean serum FGF23, PTH, calcium, and phosphateconcentrations at 8 h after vehicle or 1,25(OH)2D3 treatment.The increase in FGF23 levels after 1,25(OH)2D3 treatment wasassociated with a decrease in serum PTH levels from 37.6 ±7.6 to 19.1 ± 1.8 pg/ml (mean ± SEM; P < 0.05)at 8 h but no changes in calcium or phosphate.
Figure 1. Time- and dose-dependent effects of calcitriol on serum fibroblast growth factor 23 (FGF23) levels in mice. (A) Female C57BL/6J mice received intraperitoneal injections of equal volumes of vehicle or calcitriol (100 ng/kg body wt). Serum samples were collected at 4, 8, or 24 h after injection for FGF23 measurements. We also collected serum from untreated age-matched mice as a baseline. Serum FGF23 concentrations after calcitriol administration are significantly different from vehicle at all time points (P < 0.05). (B) Serum FGF23 was measured in C57BL/6J mice 8 h after intraperitoneal administration of calcitriol at doses from 10 to 1000 ng/kg body wt. Values from different doses that share the same superscript letters are not significantly different at P < 0.05. Serum FGF23 was measured using an ELISA kit as described in Materials and Methods. FGF23 levels are expressed as mean ± SEM (n = 6 in each group).
Table 2. Serum markers at 8 h after injection of calcitriol in wild-type and Gcm2 null micea
To investigate whether changes in PTH might contribute to theregulation of FGF23 and the potential interrelationships amongphosphate, PTH, and 1,25(OH)2D3, we examined Gcm2 null mice,which are characterized by hyperphosphatemia and low serum PTHand 1,25(OH)2D3 levels (Table 3) as a result of the failureof parathyroid gland development (20). Despite hyperphosphatemiaand low serum PTH levels, we observed a three-fold decreasein serum FGF23 levels associated with a four-fold decrease of1,25(OH)2D3 in Gcm2 null mice compared with age- and gender-matchedwild-type controls (Table 3). To determine whether low levelsof 1,25(OH)2D3 contributed to the decreased FGF23 levels inGcm2 null mice, we measured serum FGF23 concentrations 8 h afterintraperitoneal injections of 1,25(OH)2D3. We found that administrationof 1,25(OH)2D3 to Gcm2 null mice resulted in a significant increasein circulating FGF23 levels, attaining maximal response similarto that attained in wild-type mice (Table 2).
Table 3. Serum data from Gcm2 null and wild-type mice at 12 wk of agea
We confirmed that Hyp mice, the murine model of XLH, have hypophosphatemiaand increased FGF23 as a result of inactivating mutations ofthe Phex gene (Table 4). To investigate whether calcitriol treatmentof Hyp mice further increases FGF23 levels, we administered1,25(OH)2D3 to Hyp mice for up to 16 d. Chronic administrationof 1,25(OH)2D3 resulted in a two-fold increase in FGF23 associatedwith significant reductions in PTH and nonsignificant increasesin serum calcium and phosphate concentrations (Table 4).
Table 4. Effects of calcitriol on serum markers in Hyp micea
Effects of 1,25(OH)2D3 on FGF23 Transcripts in Bone and Osteoblast Cultures
To evaluate whether vitamin D stimulation of circulating FGF23levels was associated with increased levels of FGF23 transcriptin bone, we measured FGF23 message levels by real-time quantitativeRT-PCR in the calvaria isolated from mice 8 h after treatmentwith 1,25(OH)2D3 (Figure 2A). 1,25(OH)2D3 treatment resultedin a 3.5-fold increase in mRNA levels in calvaria (P < 0.05),consistent with the increased production of FGF23 from bone.To confirm that increased production of FGF23 from bone representsa direct effect on osteoblasts, we evaluated the effects of1,25(OH)2D3 to stimulate endogenous FGF23 transcripts in culturedROS17/2.8 osteoblasts by real-time PCR (Figure 2B). Treatmentof ROS17/2.8 osteoblasts with 1,25(OH)2D3 (10–8 M) for8 and 24 h increased FGF23 message levels approximately eight-and 100-fold, respectively. DNA sequence analysis confirmedthat the PCR product was 100% homologous to rat FGF23 mRNA (datanot shown).
Figure 2. Analysis of FGF23 mRNA expression in bone and ROS17/2.8 cells after treatment with 1,25-dihydroxyvitamin D3 (1,25[OH]2D3). (A) Female C57BL/6J mice were treated with vehicle or calcitriol at 100 ng/kg body wt administered by intraperitoneal injection, and expression in calvaria was assessed by real-time PCR, as described in Materials and Methods. Calcitriol administration increased FGF23 transcripts by approximately 3.5-fold in calvaria after 8 h. (B) ROS17/2.8 cells were stimulated with 1,25(OH) 2D3 at concentrations of 10–8 M, and FGF23 transcripts were quantified by real-time PCR at 8 and 24 h. The FGF23 transcript was increased eight-fold at 8 h and 100-fold at 24 h in ROS17/2.8 cells stimulated with 1,25(OH) 2D3. (C) ROS17/2.8 cells were stimulated with phosphate (5 mM), calcium (5 mM), or parathyroid hormone (PTH; 10 nM). The FGF23 expressions were measured by real-time PCR 24 h after the addition of phosphate, calcium, or PTH. Phosphate, calcium, and PTH did not stimulate FGF23 transcript in ROS17/2.8 osteoblasts.
Cloning of the Mouse FGF23 Promoter and its 5' Untranslated Region
To investigate further the mechanism whereby 1,25(OH)2D3 increasesthe FGF23 message, we cloned and sequenced the 3550-bp 5' flankingregion of the FGF23 gene (Figure 3A). Using a promoter predictionprogram (Neural Network Promoter Prediction, The Berkeley DrosophilaGenome Project, Berkeley, CA), we located two potential transcriptionstart sites at –98 and –108 bp, respectively, upstreamof the ATG. The upstream promoter contains a TATA-box. In addition,we found that the FGF23 promoter region is highly conservedbetween mouse, rat, and human. The mouse FGF23 promoter is 79%homologous to the rat FGF23 promoter over the proximate 2300bp and is 67% homologous to the human promoter over the proximate800 bp (data not shown). Predicted transcriptional factor bindingsites that are conserved between mouse, rat, and human overthe initial 2000 bp of their respective promoter regions areshown in Figure 3A.
Figure 3. Isolation of the FGF23 promoter and its cell-type specificity of the FGF23 promoter activities response to 1,25(OH)2D3. (A) Nucleotide sequence of the 5' flanking region of the FGF23 gene. We cloned and sequenced the 3550-bp 5' flanking region of the FGF23 gene. Shown is the sequence of the 3550-bp 5' flanking region and partial exon 1. The predicted transcription start sites are shown by . We identified putative binding sites for GATA-binding factor (GATA), positive regulatory domain I binding factor (PRDF), RAR-related orphan receptor 1 (RORA), ETS1 factors (ETSF), Brn POU domain factor (BRNF), hepatic nuclear factor 4 (HNF4), myelin transcription factor 1 (MYT1) zinc finger protein, GC-Box factors SP1/GC (SP1F), cAMP-responsive element binding protein (CREB), CCAAT/enhancer binding protein (CEBP), ecotropic viral integration site 1-myeloid transforming protein (EVI1), CAS interacting zinc finger protein (CIZF), nuclear factor of activated T cells (NFAT), glioma-associated oncogene homolog (GLI)-Krueppel-related transcription factor (E4FF), gut-enriched Krueppel-like binding factor (GKLF), TATA-binding protein factor (TBPF), enhancer CCAAT binding factors (ECAT), and promoter CCAAT binding factors (PCAT). VDRE, vitamin D responsive element. The primers used to generate promoter/reporter constructs are underlined, and the putative transcriptional factor binding sites conserved between mouse, rat, and human are double underlined. (B) Promoter activity of the 3550-bp 5' flanking region of FGF23. The promoter/reporter construct p3550Fgf23-luc consists of the sequence from –3550 to –1 relative to translation starting site ATG, which was subcloned into pGL3-Basic vector. The relative luciferase activity of p3550Fgf23-luc is approximately six-fold greater than the empty vector in ROS17/2.8 cells, indicating that the 5' flanking region from –3550 to –1 has promoter activity in ROS17/2.8 in vitro. *Values that are significantly different from empty vector (P < 0.05).
We subcloned the region from –3550 to –1 relativeto translation starting site ATG into pGL3-Basic to create apromoter/reporter construct (p3550Fgf23-luc) and examined theactivity of this promoter/reporter construct in ROS17/2.8 osteoblasts.The relative luciferase activity of p3550Fgf23-luc was approximatelysix-fold greater than the empty vector in ROS17/2.8 cells (Figure 3B),indicating that the 5' flanking region from –3550 to –1has promoter activity.
Effects of 1,25(OH)2D3, PTH, Calcium, and Phosphate on FGF23 Promoter Activity
Next, we evaluated the effects of 1,25(OH)2D3 (10–10 to10–8 M), PTH (1-34; 1 to 100 nM), phosphate (1 to 4 mM),and calcium (1 to 5 mM) on p3550Fgf23-luc activity in ROS17/28osteoblasts (Figure 4). Compared with vehicle treatments, 1,25(OH)2D3resulted in a dose-dependent stimulation of luciferase activityin ROS17/2.8 cells transfected with p3550Fgf23-luc. The maximalincrease was approximately two-fold at 10–8 M 1,25(OH)2D3(Figure 4A). In contrast, we observed a small (35%) but significantinhibition of FGF23 promoter activity by PTH (Figure 4B). Theaddition of neither phosphate (1 to 4 mM) nor calcium (1 to5 mM) to the media affected FGF23 promoter activity in ROS17/2.8osteoblasts (Figure 4, C and D). To exclude the possibilitythat the 3500-bp FGF23 promoter lacks necessary cis-acting elements,we examined the effects of PTH (10 nM), phosphate (5 mM), orcalcium (5 mM) on endogenous FGF23 expression in ROS17/2.8 osteoblastsby quantitative RT-PCR. In contrast to the stimulation of endogenousFGF23 expression by 1,25(OH)2D3 (Figure 2B), we did not observeany stimulation of FGF23 message levels by PTH, phosphate, orcalcium (Figure 2C). To determine whether this lack of calciumand phosphate stimulation was a unique feature of ROS17/2.8osteoblasts, we examined the effects of 1,25(OH)2D3 (10–8M), phosphate (5 mM), and calcium (5 mM) to stimulate FGF23expression in UMR-106 osteoblasts. 1,25(OH)2D3 but not phosphateor calcium increased FGF23 transcripts by real-time PCR in UMR-106osteoblasts (data not shown).
Figure 4. Effect of 1,25(OH)2D3, PTH, phosphate, and calcium on FGF23 promoter activities. ROS17/2.8 osteoblasts were transiently transfected with p3550Fgf23-luc and the pRL-TK construct, which expresses renilla luciferase as an internal control for transfection efficiency. Twenty-four hours after transfection, cells were treated with 10–10 to 10–8 M 1,25(OH) 2D3 (A), 1 to 100 mM PTH (1-34; B), 1 to 4 mM phosphate (C), 1 to 5 mM calcium chloride (D), or vehicle as control for 24 h. Data represent relative luciferase activity expressed as the mean ± SEM of at least three independent transfection experiments. *Values that are significantly different from vehicle (P < 0.05).
Mapping the 1,25(OH)2D3, Response Region
Finally, to identify the vitamin D responsive region of theFGF23 promoter, we compared the function of the full-lengthpromoter (p3550Fgf23-luc) with successive 5' deletion mutants(p2000Fgf23-luc, p1300Fgf23-luc, p1000Fgf23-luc, and p600Fgf23-luc)transfected into ROS17/2.8 osteoblasts. Promoter activity waslower in the p3550Fgf23-luc and p2000Fgf23-luc constructs comparedwith the more truncated constructs, consistent with the presenceof a suppressor region in the distal FGF23 promoter (Figure 5A).1,25(OH)2D3 stimulated the p3550FGF23-luc, p2000Fgf23-luc, andp1300Fgf23-luc but did not stimulate the promoter activity ofthe p1000Fgf23-luc or p600Fgf23-luc constructs transfected intoROS17/2.8 cells, indicating localization of the VDRE in theregion between –1300 and –1000 bp (Figure 5A). Toconfirm the vitamin D responsive region, we created serial deletionconstructs using p2000Fgf23-luc as a template to generate constructswith different lengths of deletions between –1393 and–1000 using a restriction digestion and PCR methods. Analysisof these constructs further localized the vitamin D responsiveregion to between –1240 and –1161 (data not shown).Alignment of the mouse and rat sequences that corresponded tothis region revealed 83% identity and the presence of a conservedVDRE (–1180 GGAACTcagTAACCT –1156). Next, we deletedthis 15-bp region from the p2000Fgf23-luc construct using PCRmutagenesis methods to create pDelFgf23-luc. 1,25(OH)2D3 failedto stimulate luciferase activity in ROS17/2.8 cells transfectedwith pDelFgf23-luc (Figure 5B). Finally, to establish a rolefor the VDR, we assessed the response to 1,25(OH)2D3 in ROS17/2.8cells co-transfected p3500Fgf23-luc and pGS5A420E, a dominantnegative human VDR expression construct. Overexpression of thehuman VDR A420E mutant significantly inhibited 1,25(OH)2D3-stimulatedFGF23 promoter activity in ROS17/2.8 (Figure 5C).
Figure 5. Deletion analysis of the FGF23 promoter activities response to 1,25(OH)2D3 and the response is abolished by dominant mutant vitamin D receptor (VDR). (A) ROS17/2.8 osteoblasts were transiently transfected with a promoterless construct pGL3-Basic or with the indicated FGF23 promoter-luciferase constructs that contained successive 5' deletions along with the pRL-TK construct, which expresses renilla luciferase as an internal control for transfection efficiency. Twenty-four hours after transfection, the transfected cells were treated with 10–8 M 1,25(OH)2D3 or the same amount of ethanol (vehicle) for 24 h. Promoter activities were measured by the Firefly luciferase activities normalized by Renilla luciferase activities and expressed as luciferase activities relative to the activities from pGL3-Basic. All values are mean ± SEM; n = at least 4 in each group. *P < 0.05 versus vehicle in each group. (B) Deletion analysis of VDRE in p2000Fgf23-luc. ROS17/2.8 cells were transfected with wild-type and deletion construct. Promoter activities and their response to 1,25(OH)2D3 were assayed as described above. (C) Co-transfection of p3550Fgf23-luc with pSG5 vector or pSG5E420A in ROS17/2.8 osteoblasts. E420A dominant mutant VDR expression abolished 1,25(OH)2D3 response in p3550Fgf23-luc promoter/reporter construct. All values are mean ± SEM; n = 3 in each group. *P < 0.05 versus vehicle in each group.
This study supports a physiologic role for 1,25(OH)2D3 in theregulation of FGF23 production by osteoblasts in bone. We demonstrateby in vivo administration of calcitriol (Figure 1) and by assessingthe effects of direct exposure of osteoblast cultures to 1,25(OH)2D3(Figure 2B) that 1,25(OH)2D3 stimulates the production of FGF23in bone and osteoblasts. Additional studies indicate that 1,25(OH)2D3results in an increase in FGF23 transcripts in calvaria andcultured osteoblasts (Figure 2), as well as activation of a3500-bp FGF23 promoter-reporter construct transfected into ROS17/2.8osteoblasts (Figures 4 and 5A).
We identified a VDRE located between –1180 and –1161that is required for vitamin D effects on the FGF23 promoter/reporterconstruct in ROS17/2.8 cells (Figure 5B). In addition, expressionof a dominant negative VDR inhibited 1,25(OH)2D3 stimulationof FGF23 promoter activity (Figure 5C), indicating that FGF23is regulated directly by 1,25(OH)2D3 in osteoblasts. It is interestingthat 1,25(OH)2D3 also is reported to suppress PHEX mRNA levelsin osteoblasts and bone (30,31) and reductions in PHEX can leadto increased FGF23 expression in osteoblasts (32,33). Therefore,there seem to be both direct and indirect mechanisms for 1,25(OH)2D3to regulate FGF23 production by osteoblasts.
1,25(OH)2D3 regulation of FGF23 production has been reportedpreviously (34–37). In this regard, VDR null mice havelow circulating FGF23 levels (35), and 1,25(OH)2D3 administrationstimulates FGF23 levels in mice independent of PTH (35,38).Also, 1,25(OH)2D3 has been shown to upregulate FGF23 messageexpression in UMR-106 osteoblasts (39) and FGF23 promoter activityin K562 erythroleukemia cells (36). These studies more completelycharacterize the mechanism of vitamin D-stimulated FGF23 productionand identified osteoblasts and bone as the principle targetfor vitamin D effects on this phosphaturic hormone. Moreover,our preliminary findings lead us first to propose that the physiologicrole of FGF23 is to act as a counterregulatory hormone for 1,25(OH)2D3(40).
1,25(OH)2D3 stimulation of FGF23 production by bone and osteoblastsmay be important for several reasons. First, FGF23 providesa mechanism to maintain systemic phosphate homeostasis in thesetting of 1,25(OH)2D3 inhibition of osteoblast-mediated mineralizationof bone (31). The overall physiologic effect would be to increaserenal phosphate clearance under circumstances in which phosphatewas not needed for mineralization of extracellular matrix. Second,1,25(OH)2D3 stimulation of FGF23 production by osteoblasts inbone provides a mechanism to maintain phosphate homeostasisin the setting of suppressed PTH secretion (41). In the presenceof 1,25(OH)2D3 stimulation of calcium and phosphate absorptionby the gastrointestinal tract (42), increments in calcium (aswell as 1,25[OH]2D3 itself) act on the parathyroid gland tosuppress PTH, thereby limiting the ability of the kidney toexcrete the increased phosphate absorbed from the gastrointestinaltract. 1,25(OH)2D3 stimulation of the production of FGF23 bybone and the resulting increase in renal phosphate excretionmaintain phosphate homeostasis in the setting of suppressedPTH (Figure 6).
Figure 6. Proposed model showing interrelationship between FGF23 and vitamin D, PTH, calcium, and phosphorus. 1,25(OH)2D3 causes concomitant suppression of the calcemic and phosphaturic hormone PTH as a result of increases in gastrointestinal calcium absorption and directly suppresses PTH production by the parathyroid glands. Consequent to the loss of PTH-mediated phosphaturia, hyperphosphatemia might occur in response to 1,25(OH)2D3-mediated increases in gastrointestinal phosphate absorption if not for the presence of a counterregulatory phosphaturic factor, such as FGF23. Dietary phosphate regulation of FGF23 production by bone likely occurs through unknown intermediate steps, because hyperphosphatemia per se does not directly stimulate FGF23 production by osteoblasts.
Gcm2 null mice, which have low circulating PTH along with reduced1,25(OH)2D3 levels, hypocalcemia, and hyperphosphatemia (Table 3)as a result of the failure of the parathyroid gland development,also had low serum FGF23 levels (Table 3). That the administrationof calcitriol raised serum FGF23 levels to values identicalto those in wild-type mice stimulated with calcitriol, and theinability to demonstrate direct stimulation of the FGF23 promoter(Figure 4) or endogenous FGF23 transcription (Figure 2C) inROS1/7/2.8 cells by PTH, suggest that the low levels of FGF23are due to decreased 1,25(OH)2D3 in Gcm2-deficient mice.
Surprisingly, hyperphosphatemia in Gcm2 null mice did not resultin increased circulating FGF23 concentrations (Tables 2 and3), and the addition of phosphate to osteoblast cultures failedto increase FGF23 transcripts (Figure 4). These observationsdo not mean that phosphate is not an important regulator ofFGF23, because several studies indicate that phosphate administrationand restriction, respectively, increase and decrease circulatingFGF23 levels in both mice and humans (34,43–45). Also,high phosphate in medium that contains 1,25(OH)2D3 has beenreported to stimulate FGF23 promoter activity in erythroleukemiacells (36). That hyperphosphatemia per se is not associatedwith increased FGF23 levels, however, suggests possible complexmechanisms whereby serum phosphate regulates FGF23 productionor vice versa. Indeed, other factors in Gcm2 null mice, suchas decreased PTH, calcium, and 1,25(OH)2D3 levels (46), mayhave prevented phosphate-mediated regulation of FGF23 production,or, conversely, the low FGF23 and PTH levels in Gcm2 null micemay be responsible for the high serum phosphate.
Finally, our findings that calcitriol further increases FGF23production in Hyp mice raises possible concerns about the useof active vitamin D analogues to treat hypophosphatemia in patientswith XLH (Table 4). By further increasing FGF23 levels, suchtreatment would offset the effects of vitamin D to increaseserum phosphate levels. On the basis of these observations,it would be interesting to determine whether treatment withactive vitamin D analogues causes similar increments in FGF23levels in patients with XLH and explains the variation in FGF23levels that is observed in these patients (7).
1,25(OH)2D3 is an important regulator of FGF23 production byosteoblasts through a VDRE in the FGF23 promoter. We proposethat FGF23 may provide a means to maintain renal phosphate excretionin the setting of 1,25(OH)2D3-mediated stimulation of gastrointestinalabsorption of phosphate and suppression of the phosphaturichormone PTH.
Acknowledgments
This work was supported by National Institutes of Health grantsRO1-AR45955 from National Institute of Arthritis an Musculoskeletaland Skin Diseases and P20 RR-17708 from the National Centerfor Research Resources.
Portions of this work were published previously in abstractform (J Am Soc Nephrol 15: 282A, 2004) at the American Societyof Nephrology Meeting; October 27 to November 1, 2004; St. Louis,MO.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
hydroxylase activity in the proximal renal tubule, leading to reduced circulating levels of 1,25(OH)2D3 (2,10,14,15). The significance of FGF23 regulation of 1,25(OH)2D3 production is not clear, but the findings that FGF23 is produced predominantly by osteoblasts in bone and that FGF23 regulates phosphate reabsorption and 1,25(OH)2D3 production by the kidney raise the possibility that FGF23 may be involved in a bone-kidney axis that controls phosphate and vitamin D homeostasis (16,17). How FGF23 is integrated with the vitamin D-parathyroid hormone (PTH) axis, which plays a central role in calcium homeostasis, skeletal development, and mineralization (18), however, is not clear. Understanding the effects of 1,25(OH)2D3 on FGF23 production is important, because vitamin D therapy often is used to treat FGF23-mediated hypophosphatemic disorders, such as XLH (19). 
. We identified putative binding sites for GATA-binding factor (GATA), positive regulatory domain I binding factor (PRDF), RAR-related orphan receptor 1 (RORA), ETS1 factors (ETSF), Brn POU domain factor (BRNF), hepatic nuclear factor 4 (HNF4), myelin transcription factor 1 (MYT1) zinc finger protein, GC-Box factors SP1/GC (SP1F), cAMP-responsive element binding protein (CREB), CCAAT/enhancer binding protein (CEBP), ecotropic viral integration site 1-myeloid transforming protein (EVI1), CAS interacting zinc finger protein (CIZF), nuclear factor of activated T cells (NFAT), glioma-associated oncogene homolog (GLI)-Krueppel-related transcription factor (E4FF), gut-enriched Krueppel-like binding factor (GKLF), TATA-binding protein factor (TBPF), enhancer CCAAT binding factors (ECAT), and promoter CCAAT binding factors (PCAT). VDRE, vitamin D responsive element. The primers used to generate promoter/reporter constructs are underlined, and the putative transcriptional factor binding sites conserved between mouse, rat, and human are double underlined. (B) Promoter activity of the 3550-bp 5' flanking region of FGF23. The promoter/reporter construct p3550Fgf23-luc consists of the sequence from –3550 to –1 relative to translation starting site ATG, which was subcloned into pGL3-Basic vector. The relative luciferase activity of p3550Fgf23-luc is approximately six-fold greater than the empty vector in ROS17/2.8 cells, indicating that the 5' flanking region from –3550 to –1 has promoter activity in ROS17/2.8 in vitro. *Values that are significantly different from empty vector (P < 0.05).
