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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).