FGF23 is dependent on α-Kl for regulation of vitamin D synthesis in kidney (A–E). (A) Serum concentrations of FGF23 in WT and α-kl ^−/− were measured by ELISA. WT (open bars) and α-kl ^−/− (filled bars) mice (n = 4/group) were injected with recombinant hFGF23 (0.2 mg/kg) or PBS control. Mice were killed 4 h after injection, and serum concentrations of 1,25(OH)₂D (B) were measured. Cyp27b1 (C), Cyp24 (D), and α-kl (E) mRNA levels in kidney were analyzed by RT–quantitative PCR. In this and all other figures, error bars represent mean ± SD and are plotted as fold change. Data were derived from 8- to 10-week-old male mice on vitamin D–deficient diets. *P < 0.05; **P < 0.01. FGFR1 binds to α-Kl and is phosphorylated by FGF23 in the kidney (F–I). (F) Kidney lysates were precipitated with anti-FGFR1 antibody or with control IgG. Input is 0.01% of the kidney whole extract used for the immunoprecipitation. (G) Kidney lysates were precipitated with anti-FGFR4 antibody or with control IgG. Input is 0.2% of the kidney whole extract used for the immunoprecipitation. (H) The kidney lysates of WT and α-kl ^−/− mice were immunoprecipitated with the anti-phospho-FGFRs or the anti-FGFR1 antibody. The immunoprecipitates were separated by SDS/PAGE and blotted with anti-FGFR1 antibody. (I) Kidney lysates of WT mice were immunoprecipitated with the anti–phospho-FGFRs or the anti-FGFR4 antibody and then blotted with anti-FGFR4 antibody.

Schematic representation of α-Kl/FGF23 and β-Kl/FGF15 systems. (A) Regulatory network of mineral homeostasis illustrated by the mutual positive/negative feedback actions of α-Kl, FGF23, and 1,25(OH)₂D. (B) Regulatory network of bile acid/cholesterol metabolism represented by the mutual positive/negative feedback actions of β-Kl, FGF15, and bile acids.

FGF19 is dependent on β-Kl for regulation of bile acid synthesis in liver (A–D). (A) mRNA levels of Fgf15 in terminal ileum in WT and β-kl ^−/− were measured by RTQ-PCR. WT mice (open bars) and β-kl ^−/− mice (filled bars) (n = 5/group) were injected with recombinant hFGF19 (1 mg/kg) or control medium. Mice were killed 6 h after injection and Cyp7a1 (B) and Cyp8b1 (C) mRNA levels in liver were measured by RT–quantitative PCR. Data were derived from 14- to 16-week-old male mice on standard diet. Egr-1 induction mediated by FGF19 in liver (D). hFGF19 (1 mg/kg) or control medium were injected into WT and β-kl^−/− male mice (12–14 weeks old) on standard diet. Thirty minutes after injection, tissues in WT (Upper) and β-kl ^−/− (Lower) mice (n = 4/group) were excised. Egr-1 mRNA levels were measured by RT-quantitative PCR. The expression levels of FGF19-injected mice (filled bars) and vehicle injected mice (open bars) are plotted as fold change. *P < 0.05; **P < 0.01.

FGFR4 binds to β-Kl and is phosphorylated by FGF19 in liver. (A) Liver lysates were precipitated with anti-FGFR4 antibody or with control IgG. Input is 1% of the liver whole extract used for the immunoprecipitation. The immunoprecipitates were separated by SDS/PAGE and blotted with anti-FGFR4 antibody. (B, D, and E) Ten minutes after injection of hFGF19 or control medium, livers were excised. Liver lysates from WT mice (B) and β-kl ^−/− mice (D) were immunoprecipitated with the anti–phospho-FGFRs or the polyclonal anti-FGFR4 antibody. Immunoprecipitates were blotted with anti-FGFR4 antibody. The arrowhead indicates a nonspecific band (Fig. S2). (C) Whole liver extracts of WT and β-kl ^−/− mice were blotted with anti-FGFR4 antibody or anti–β-actin antibody for a loading control. (E) Liver lysates from WT and β-kl ^−/− were immunoblotted with anti-phospho-ERK1/2 or anti-ERK1/2 antibodies (n = 2 in each case).

β-Kl is not essential for FGF21-mediated signaling (A–I). Thirty minutes after injection, tissues in WT (n = 5/group) (A) and β-kl ^−/− mice (n = 4/group) (B) were excised. Egr-1 mRNA levels were measured by RT-quantitative PCR. The expression levels of hFGF21 injected mice (filled bars) and vehicle injected mice (open bars) mice are plotted as fold change. Data were derived from 7- to 10-week-old male mice on standard diet. (C) Serum FGF21 concentrations of WT and β-kl ^−/− mice (n = 5–6/group) were measured by RIA. (D) Fgf21 mRNA levels in livers of WT and β-kl ^−/− mice (n = 5–6/group) were measured by RT-quantitative PCR and are plotted as fold change. Data were derived from 15- to 20-week-old male mice on standard diet. hFGF21 (0.4 mg/kg) or control medium were injected into WT and β-kl^−/− mice. (E) WT mice were injected with recombinant hFGF21 (0.4 mg/kg) or control medium (n = 5/group). Mice were killed 4 h after injection and β-kl mRNA levels in WAT were analyzed by RT-quantitative PCR. Data were derived from 10-week-old male mice on standard diet. (F) A 15-ng quantity of each FGF was pulled down (PD) by α-/β-Kl in the presence (R1, R3, or R4) or absence (–) of FGFRs. Input was 8% of samples used for the pull-down assay. Samples of pulled down by α-/β-Kl were analyzed by SDS/PAGE and blotted with antibodies (anti-His for FGFs, anti-Human Fc for FGFRs, and anti-GFP for α-/β-Kl). Arrowhead indicates a nonspecific band. (G) A 50-ng quantity of FGF19, 150 ng of FGF23, or 500 ng of FGF21 was precipitated by β-Kl in the presence or absence of FGFR4. Input was 1% of samples used for the assay. (H and I) hsl and atgl mRNA levels in WAT and BAT were analyzed by RT-quantitative PCR. Data were derived from 9- to 14-week-old female β-kl ^+/+ and β-kl ^−/− mice (n = 5/group). *P < 0.05; **P < 0.01.