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Supplementary Discussion (Urakawa I. et al)
(2005-05-05504E)
Supplementary Discussion
Controversial aspects on in vitro and in vivo effects of FGF23
OK cells have been used as the most appropriate cell line to address the renal effects of FGF23
in vitro, because the functional regulation of NaPi2a protein in this cell line has been well
documented and supposed to be useful for evaluating the effect of FGF23 in vitro. However,
independent findings about the activity of recombinant FGF23 on OK cells have been controversial
and it remained unclear if FGF23 changes phosphate transport activity on these cells2,15,16,32. Even
among the findings showing some effect of FGF23, the role of heparin has been conflicting, either
that was inhibitory32, stimulative16 or ineffective15. Furthermore, results reporting the binding of
FGF23 to FGFR1(IIIc) was conflicting between studies15,16. These contradictions may be derived
from the diversity of OK cell sublines and the difference in recombinant FGF23 protein preparations
used in these studies. In this study, we employed recombinant FGF23 protein whose activity was
certified in vivo as determined by the reduction of CYP27B1 mRNA abundance within 1 h post
injection6. We also attempted to look the effect of FGF23 using OK cells with high expression
level of NaPi2a associated with a strong sodium-dependent phosphate transport activity. The
sodium-dependent phosphate transport activity was drastically suppressed by parathyroid hormone
within an hour, but did not change by our activity-certified recombinant FGF23. The absence of the
direct action of FGF23 on in vitro phosphate transport activity was not surprising because FGF23
could change NaPi2a in vivo but it requires more than 5h6, whereas PTH can reduce Npt2a protein in
less than half an hour in the same condition. Furthermore, the binding assay in this study clearly
demonstrated that the Klotho-dependent FGF23 binding is extremely stronger than the binding to the
OK cells with or without heparin. Taking into account of the facts that Klotho seems to be involved
in the regulation of NaPi2a (Figure 3h) and that Klotho is expressed on the distal tubules but not on
the proximal tubules in the kidney, now we propose a new hypothesis that the NaPi2a regulation on
the renal proximal tubules by FGF23 is mediated by a distal tubular function.
Interactions between FGF23 and FGFRs and the role of GAGs
A previous study indicated that glucosaminoglycan (GAG) such as heparin can reinforce the
FGF23-FGFR1 interaction and assumed that such types of GAG might be naturally expressed by OK
cells, leading to a hypothesis that such GAG might be a key component to determine the renotropic
effect of FGF2315. However, OK cells did not possess a specific binding property to FGF23 like
other cell types, CHO and HEK293 (Supplementary Figure 2a) assessed by radiolabeled ligand
binding assay. Furthermore, heparin did not potentiate the FGF23 binding to the OK cells
(Supplementary Figure 2c) and other GAGs as well as heparin were not able to substitute for Klotho
in the Egr-1 promoter activation assay (Supplementary Figure 6). These findings clearly indicate that
Supplementary Discussion (Urakawa I. et al)
(2005-05-05504E)
it is possible to detect some heparin-assisted interaction between FGF23 and FGFRs in vitro by
specific assay systems, the presence of Klotho rather than the topical localization of highly sulfated
GAGs is indispensable for generating biological FGF23 response in a tissue-specific manner.
Furthermore, the Klotho-dependent activation of FGFR1(IIIc) required only 0.3 ng/mL of FGF23
(Fig. 2e, Supplementary Figure 4a) whereas GAG-supported activation of FGFR1 in the previous
study15 required more than 1000-fold concentration (25 nM, about 800 ng/mL) of FGF23. Such
complete difference can be explained by the evident difference in Kd values for FGF23 between
Klotho-expressing cells (Kd: 29~79 pM) and OK cells (Kd: 3,320 pM) that have been supposed to
express GAG permissive for FGF23 activity (Fig. 2b and Supplementary Figure 2a-d). Although
heparin could not effectively support the FGF23-FGFR1(IIIc) complex without Klotho, it seemed to
be able to stabilize the FGF23-Klotho-FGFR1(IIIc) complex. Since heparin did not stabilize the
binding between FGF23 and Klotho, heparin probably contributes to the stabilization of the
FGF23-FGFR1(IIIc) interaction in the FGF23-Klotho-FGFR1(IIIc) complex as in a well-known
manner between other FGF-FGFR interactions. In other words, the role of Klotho is likely to
produce such an ordinal FGF-FGFR interaction between FGF23 and FGFR1(IIIc), which barely
happens without Klotho even they have enough chance to contact each other.
References in Supplementary Disscussion
32
Bowe, A. E. et al. FGF-23 inhibits renal tubular phosphate transport and is a PHEX substrate.
Biochem Biophys Res Commun 284, 977-981 (2002).