FGF23 expression is stimulated in transgenic α-Klotho longevity mouse model

Abstract

Observations in transgenic α-Klotho (Kl) mice (KlTg) defined the antiaging role of soluble Klotho (sKL130). A genetic translocation that elevates sKL levels in humans is paradoxically associated with increased circulating fibroblast growth factor 23 (FGF23) levels and the potential of both membrane KL (mKL135) and sKL130 to act as coreceptors for FGF23 activation of fibroblast growth factor receptors (FGFRs). Neither FGF23 expression nor the contributions of FGF23, mKL135, and sKL130 codependent and independent functions have been investigated in KlTg mice. In the current study, we examined the effects of Kl overexpression on FGF23 levels and functions in KlTg mice. We found that mKL135 but not sKL130 stimulated FGF23 expression in osteoblasts, leading to elevated Fgf23 bone expression and circulating levels in KlTg mice. Elevated FGF23 suppressed 1,25(OH)2D and parathyroid hormone levels but did not cause hypophosphatemic rickets in KlTg mice. KlTg mice developed low aldosterone-associated hypertension but not left ventricular hypertrophy. Mechanistically, we found that mKL135 and sKL130 are essential cofactors for FGF23-mediated ERK activation but that they inhibited FGF23 stimulation of PLC-γ and PI3K/AKT signaling. Thus, increased longevity in KlTg mice occurs in the presence of excess FGF23 that interacts with mKL and sKL to bias FGFR pathways.

Keywords: Bone Biology; Cardiovascular disease.

Figure 1. α-Klotho isoforms and FGF23 expression in α-Klotho–transgenic mice.

(A and B) Multiple-tissue real-time RT-PCR analyses for membrane (mKL¹³⁵, A) and alternative spliced (sKl ⁷⁰, B) Klotho transcripts. (C) Kidney Western blot analysis for mKL¹³⁵/sKL¹³⁰ and sKL⁷⁰/sKL1⁷⁰ protein levels. (D) Serum immunoprecipitation of serum soluble Klotho proteins (sKL¹³⁰ or sKL⁷⁰/sKL1⁷⁰) with anti-Klotho KL1 rat mAb (KL-234). (E) Multiple-tissue real-time RT-PCR analyses for Fgf23 transcripts. (F) Regulation of Fgf23 promoter-luciferase reporter activities by transient overexpression of α-Klotho isoforms. Data are expressed as the mean ± SD from 8–10 mice or 3 independent experiments in triplicate. The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range. *P < 0.05, **P < 0.01, and ***P < 0.001 versus wild-type or vector alone control group.

Figure 2. Heart and blood pressure phenotypes in Kl^Tg mice.

(A) Cross sections of the heart in H&E staining. (B and C) Heart weight/body weight (HW/BW) and heart weight/femur length (HW/FL) ratios. (D–G) Echocardiography for cardiac function. LVPW; s, left ventricular posterior wall thickness at end-systole; LVPW;d, left ventricular posterior wall thickness at end-diastole; LVEF, left ventricular ejection fraction. (H) Blood pressure (BP) measurement. SBP, systolic BP; DBP, diastolic BP; MBP, mean BP. Data are expressed as the mean ± SD from 8–10 mice. The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range. **P < 0.01 versus wild-type control mice.

Figure 3. Bone phenotypes in Kl^Tg mice.

Effects of mKL¹³⁵ transgene on bone mineral density (BMD) (A), structure of femurs (B), and periosteal mineral apposition rate (MAR) (C). Original magnification, ×20. Data are expressed as the mean ± SD from 8–10 mice. The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range. Ct.Th, cortical thickness.

Figure 4. Gross appearance and survival curve in compound Fgf23 knockout and Kl^Tg mice.

(A) Gross appearance and body weight. (B) Kaplan-Meier survival curve of Fgf23^–/–, Kl^Tg Fgf23^–/–, wild-type, and Kl^Tg mice. Data are expressed as the mean ± SD from 8–10 mice or 3 independent experiments in triplicate. ***P < 0.001 versus wild-type.

Figure 5. Effects of different α-Klotho isoforms on FGF23-mediated activation of ERK activities.

(A) Schematic illustration of α-Klotho isoform constructs. (B) Cotransfection of different α-Klotho isoform constructs with ERK reporter. (C) Cotransfection of mKL¹³⁵ with TACE (ADAM17) cDNA. (D) Addition of recombinant α-Klotho (rKL) into HEK293T cells transfected with ERK reporter in response to FGF23 plus heparan sulfate (HS) stimulation. (E and F) NF-κB reporter activities. Data are expressed as the mean ± SD from 3 independent experiments in triplicate. The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range. ***P < 0.001 versus vehicle control group. ^###P < 0.001 versus vector plus mKL¹³⁵ or FGF23 plus HS or vector plus TNF-α or vector plus FGF23 and HS group.

Figure 6. The effects of the FGF23 on FGF23-induced signaling in HEK293T cells transfected with either empty expression vector or human α-Klotho isoforms.

(A) Western blot analysis of FGF23/ERK, FGF23/PLC-γ, and FGF23/AKT signaling. Ab, antibody; HA, hemagglutinin tag antibody. (B) Quantification of the pERK/tERK ratio, pPLC-γ/tPLC-γ ratio, and pAKT/tAKT ratio. Data are expressed as the mean ± SD from 3 independent experiments. The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range. ***P < 0.001 versus vector plus vehicle or FGF23 group. ^###P < 0.001 versus mKL135 plus FGF23 group. t, total.