Research ArticleDISEASE PREVENTION

PAI-1 is a critical regulator of FGF23 homeostasis

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Science Advances  13 Sep 2017:
Vol. 3, no. 9, e1603259
DOI: 10.1126/sciadv.1603259

Figures

  • Fig. 1 Effect of PAI-1 on circulating levels of cFGF23 and iFGF23 in Pai-1stab transgenic and Klotho mice.

    Plasma levels of cFGF23 (black filled bars) and iFGF23 (gray filled bars) were measured in WT, untreated, and TM5441-treated Pai-1stab mice (A) and in WT, kl/kl, TM5441-treated kl/kl, and kl/klpai-1−/− mice (B). As seen in (A), changes in cFGF23 levels due to PAI-1 overexpression and TM5441 treatment were both significant in Pai-1stab mice. Similarly, TM5441 treatment and PAI-1 deficiency significantly reduced cFGF23 and iFGF23 levels in kl/kl mice. #P = 0.0003, $P = 0.0001 versus kl/kl cFGF23; *P = 0.03, **P = 0.001 versus kl/kl iFGF23. Analysis of the measurements by one-way analysis of variance (ANOVA) yielded significant differences (P < 0.0001) for both Pai-1stab and kl/kl mice groups.

  • Fig. 2 Effect of pharmacological inhibition or genetic deficiency of PAI-1 on the circulating levels of endogenous iFGF23 and clearance of rhFGF23 injected via the tail vein in mice with FA-induced AKI.

    (A) AKI induces plasma PAI-1 levels 10-fold higher in FA-treated WT mice (n = 13) compared to vehicle-treated WT mice (n = 5). (B) Mouse iFGF23 (miFGF23) levels in WT (n = 7), WT with AKI (n = 21), TM5441-treated WT with AKI (n = 15), PAI-1−/− with AKI (n = 9), and pai-1−/− untreated (n = 6). *P = 0.007, #P = 0.005. (C) Effect of PAI-1 inhibition on clearance of rhFGF23 in WT AKI mice (n = 7) and TM5441 (10 mg/kg per day)–treated WT AKI mice (n = 7). The rhFGF23 was injected into the tail vein, and human cFGF23 (hcFGF23) levels were measured in plasma samples collected at time points 1, 3, 5, 15, 30, 60, and 120 min. (D) The cFGF23 levels at 1 min were considered as 100% to plot percent remaining values of rhFGF23 over the time course of the experiment.

  • Fig. 3 Effect of PAI-1 on circulating levels of phosphate and PTH in AKI and kl/kl mice.

    TM5441 treatment or PAI-1 deficiency did not affect the increased serum phosphate levels (#P < 0.05 versus WT) (A) and elevated plasma levels of PTH (*P < 0.01 versus WT) (B) in kl/kl and FA-treated WT and pai-1−/− mice. mPTH, mouse PTH.

  • Fig. 4 Proteolysis of rhFGF23 by tPA, uPA, and plasmin in vitro.

    (A) Silver-stained SDS-PAGE gel of reactions containing purified recombinant human tPA, uPA, and FGF23. (B) Western blot (WB) using anti–6×His-tag antibody and tPA reactions from (A). (C) Silver-stained SDS-PAGE gel of in vitro time courses of FGF23 proteolysis by tPA and uPA. (D) Silver-stained gel showing degradation of FGF23 by plasmin. (E) Immunoblot of the gel in (D) with anti–6×His-tag antibody shows iFGF23 (lane 2) and a complete degradation of FGF23 by plasmin (lane 3).

  • Fig. 5 Identification of tPA cleavage sites in FGF23 by LC-MS–based top-down proteomics.

    (A) Silver-stained SDS-PAGE gel image of the reaction subjected to proteomics. (B) Example data for proteolytic peptide identification of tPA-induced cleavage of FGF23. Mass spectrum of FGF23 (amino acids 25 to 175) showing the high mass accuracy of the match of MS data to theoretical mass [within 2 parts per million (ppm)]. Adducts spaced by ~98 Da are indicative of incomplete removal of large amounts of phosphate-containing buffer from the reaction mixture. A graphical fragment map of FGF23 (amino acids 25 to 175) is shown after collisionally induced dissociation of the 20+ charge state, demonstrating its confident identification and retention of its disulfide bond (49). PCS, proteoform characterization score. (C) Map of the peptides confidently identified (E ≤ 1 × 10−11) with proteomics from FGF23 in the sample. (D) Table summarizing the amino acid numbers and sequences of identified cut sites denoted by the “^” symbols. R179Q mutation is shown in blue font color.

  • Fig. 6 TM5441 restores tPA-mediated FGF23 proteolysis in the presence of PAI-1.

    (A) Silver-stained SDS-PAGE gel of reactions containing purified recombinant human tPA, PAI-1, and FGF23 with and without the PAI-1 antagonist TM5441. (B) Model depicting that inhibition of PAI-1 leads to restoration of PA- and furin-mediated FGF23 proteolysis.

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