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. 2011 Nov 15;108(46):E1146-55.
doi: 10.1073/pnas.1110905108. Epub 2011 Oct 17.

Iron deficiency drives an autosomal dominant hypophosphatemic rickets (ADHR) phenotype in fibroblast growth factor-23 (Fgf23) knock-in mice

Emily G Farrow  1 , Xijie Yu, Lelia J Summers, Siobhan I Davis, James C Fleet, Matthew R Allen, Alexander G Robling, Keith R Stayrook, Victoria Jideonwo, Martin J Magers, Holly J Garringer, Ruben Vidal, Rebecca J Chan, Charles B Goodwin, Siu L Hui, Munro Peacock, Kenneth E White
Affiliations

Iron deficiency drives an autosomal dominant hypophosphatemic rickets (ADHR) phenotype in fibroblast growth factor-23 (Fgf23) knock-in mice

Emily G Farrow et al. Proc Natl Acad Sci U S A. .

Abstract

Autosomal dominant hypophosphatemic rickets (ADHR) is unique among the disorders involving Fibroblast growth factor 23 (FGF23) because individuals with R176Q/W and R179Q/W mutations in the FGF23 (176)RXXR(179)/S(180) proteolytic cleavage motif can cycle from unaffected status to delayed onset of disease. This onset may occur in physiological states associated with iron deficiency, including puberty and pregnancy. To test the role of iron status in development of the ADHR phenotype, WT and R176Q-Fgf23 knock-in (ADHR) mice were placed on control or low-iron diets. Both the WT and ADHR mice receiving low-iron diet had significantly elevated bone Fgf23 mRNA. WT mice on a low-iron diet maintained normal serum intact Fgf23 and phosphate metabolism, with elevated serum C-terminal Fgf23 fragments. In contrast, the ADHR mice on the low-iron diet had elevated intact and C-terminal Fgf23 with hypophosphatemic osteomalacia. We used in vitro iron chelation to isolate the effects of iron deficiency on Fgf23 expression VSports手机版. We found that iron chelation in vitro resulted in a significant increase in Fgf23 mRNA that was dependent upon Mapk. Thus, unlike other syndromes of elevated FGF23, our findings support the concept that late-onset ADHR is the product of gene-environment interactions whereby the combined presence of an Fgf23-stabilizing mutation and iron deficiency can lead to ADHR. .

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Conflict of interest statement

Conflict of interest statement: K. E V体育安卓版. W. receives royalties for licensing the FGF23 gene to Kyowa Hakko Kirin Co. Ltd. , and M. P. is involved in a clinical trial with Kyowa Hakko Kirin Co. Ltd.

"V体育平台登录" Figures

Fig. 1.
Fig. 1.
Mineral homeostasis in WT and ADHR mice. (A) Male (M) and female (F) WT and ADHR mice receiving control (Ctl; iron 45 mg/kg) and low-iron (Low; iron 0 mg/kg) diets gained weight continuously over the time course (n = 5–10 mice per group). (B) The low-iron diet regimen resulted in significantly reduced total serum iron in the WT and ADHR low-iron groups (*P < 0.01; n = at least 4 mice per group). (C) qPCR of liver RNA showed reduced Hamp mRNA concentrations at 8 and 12 wk in WT and ADHR mice provided low-iron diets (*P < 0.01; n = 5–10 mice per group). (D) Serum phosphate was reduced significantly at 8 and 12 wk of treatment in ADHR mice provided the low-iron diet compared with WT mice receiving control or low-iron diets and with ADHR mice provided the control diet. Serum calcium was unchanged. Serum alkaline phosphatase was elevated in ADHR mice on the low-iron diet. PTH was not different among groups (***P < 0.0001, **P < 0.001, *P < 0.01, ‡P < 0.05 vs. all groups; n = at least 12 mice per group).
Fig. 2.
Fig. 2.
Fgf23 following dietary intervention. (A) Intact Fgf23: WT mice receiving control or low-iron diets showed no differences between groups in intact Fgf23 at all time points. Fgf23 was elevated at 6 wk and reduced at 12 wk (*P < 0.01) in the ADHR mice on the low-iron diet. At 6 wk, the percentage of mice with intact Fgf23 concentrations >2.5 SD the WT control diet group mean is listed above the intact Fgf23 concentration for each cohort (‡P < 0.05 for ADHR mice on the low-iron diet v. all groups). (B) C-terminal Fgf23: C-terminal Fgf23 ELISA demonstrated significantly elevated Fgf23 in all low-iron groups (***P < 0.0001). (C) Immunoprecipitation and Western blot analysis of Fgf23 from bone lysates at 12 wk revealed that, compared with the largely intact Fgf23 protein produced by mice receiving the control diet, the low-iron diet resulted in increased immunoreactive Fgf23 proteolytic fragments in both WT and ADHR mice (arrows and bracket). Actin in the starting lysates was used as the loading control, and the negative control with no primary antibody (Neg. Ab) resulted in no immunoreactive protein. Molecular mass markers (M) are shown in kDa. (D) (Left) The intact/C-terminal Fgf23 concentrations showed a positive association in WT and ADHR mice receiving the control diet (P < 0.0001). (Right) For the low-iron diet intact did not change with C-terminal Fgf23 in WT mice, but ADHR mice had a positive correlation (P < 0.001 for difference in slopes). (E) Fgf23 transcripts were measured in total RNA from femur/tibia by qPCR. Transcript levels were not different between WT and ADHR mice receiving the control diet for 8 or 12 wk. In mice provided the low-iron diet, Fgf23 mRNA was significantly increased in WT and ADHR mice at 8 and 12 wk [**P < 0.001; ‡P < 0.05 vs. WT mice on the low-iron diet; n = 6–14 mice (mixed sexes) per group].
Fig. 3.
Fig. 3.
ADHR skeletal phenotype. (A) (Top) Goldner's staining of femur distal metaphysis revealed that WT mice receiving the control and low-iron diets and ADHR mice given the control diet had normal mineralization at 8 wk of treatment. ADHR mice receiving the low-iron diet had marked osteomalacia (3× magnification). (Middle) Enlarged images (20× magnification) of boxed areas in the top row demonstrated areas of intense osteomalacia in the ADHR low-iron group (arrows). (Bottom) Analysis of cortical bone demonstrated areas of osteoid surrounding osteocytes (arrows) that were adjacent to normal-appearing cells in the ADHR mice provided the low-iron diet (20× magnification). (B) Quantitative histomorphometric analyses demonstrated significantly increased osteoid surface/bone surface percent (OS/BS%) as well as increased osteoid thickness (‡P < 0.05 vs. all groups; n = 3–6 mice per group).
Fig. 4.
Fig. 4.
Molecular manifestations of ADHR in the kidney. (A) Immunofluorescent analyses on fixed kidney sections revealed that at 8 wk of dietary treatment the ADHR mice on the low-iron diet (Lower Right; n = 4–6 mice per group) had detectable but reduced Npt2a (green) protein compared with WT mice receiving the low-iron diet (Lower Left; n = 4–6 mice per group). The apical membrane control Nherf1 (red; Insets) was similar in sections from the same animals. (B) WT mice provided control or low-iron diets and ADHR mice receiving the control diet showed no kidney p-Erk1/2 but were positive for Klotho (KL) (green), whereas ADHR mice on the low-iron diet showed increased p-Erk1/2 activity (red) that localized with Klotho. (C) qPCR analyses demonstrated suppression of Cyp27b1 (Upper) and increased expression of Cyp24 (Lower) mRNAs in the ADHR low-iron cohort compared with the other groups. For Cyp27b1, the horizontal line indicates the normalized mean of WT mice on the control diet (*P < 0.01; n = at least 10 mice per group). (Lower Inset) Serum 1,25D concentrations at 8 wk of treatment (**P < 0.001 vs. mice on the control diet; ‡P < 0.05 vs. WT mice on the low-iron diet; n = 6–9 mice per group).
Fig. 5.
Fig. 5.
Regulation of Fgf23 in vitro. (A) (Upper) UMR-106 cells were treated with 25 and 50 μM DFO, resulting at 48 h in dose-dependent increases in Fgf23 mRNA of 12- and 20-fold, respectively (*P < 0.01 vs. vehicle). The Mek inhibitor U0126 (10 μM) blunted the increase in Fgf23 mRNA by more than 70% (#P < 0.05 vs. DFO treatment). (Inset) As a positive control for iron reduction in the cultures by chelation, TfRc1 mRNA was increased over the same treatment course (#P < 0.05 vs. vehicle). (Lower) Western blot analyses of UMR-106 cell lysates demonstrated that p-Erk1/2 was increased at 48 h following 25- or 50-μM DFO treatment. p-Erk1/2 activity was inhibited by U0126 (10 μM); the control total-Erk1/2 was similar across treatments. (B) The EGLN inhibitor l-MIM (50 μM) significantly increased Fgf23 mRNA (*P < 0.01; **P < 0.001). (C) DFO and l-MIM (50 or 100 μM) were associated with stabilized HIF1α but not HIF2α protein. (D) Transcriptional activation of Fgf23 was examined with actinomycin D (0.1 μg/mL) before DFO or l-MIM treatment (24 h). Actinomycin D treatment abolished the Fgf23 mRNA increases (#P < 0.05 vs. both vehicle control and actinomycin D treatment).
Fig. P1.
Fig. P1.
Iron deficiency and the regulation of FGF23 in late-onset ADHR. Iron deficiency causes increased bone expression of Fgf23 mRNA and protein in vivo. Moreover, iron chelation and activation of HIF1α are associated with increased Fgf23 mRNA in an osteoblastic cell line. Further, we show that excess wild-type FGF23 hormone is proteolytically cleaved to maintain normal serum concentrations of intact FGF23 and appropriate phosphate metabolism (blue pathways). In contrast, the proteolysis of R176Q-FGF23 is compromised, resulting in increased serum concentration of intact FGF23, hypophosphatemia, and osteomalacia (red pathways).
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References

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