The present studies demonstrate that acute intravenous infusions of equimolar doses of full length FGF-23 and FGF-23 176-251 result in similar increases in phosphate excretion. Infusion of FGF-23 fragments 180-251, 180-205 and 184-251 also significantly increase phosphate excretion, although the magnitude of the increase in phosphate excretion is less than the phosphaturic response observed with infusion of full length FGF-23 or FGF-23 fragment 176-251. Of interest, these bioactive fragments also increase sodium excretion when administered over the short-term. This is very likely due to the movement of sodium ions along with phosphate in the proximal tubule. With long term-administration the sodium wasting is no longer seen, perhaps due to increased sodium reabsorption in the distal tubule.
The effects of the acute infusion of full length FGF-23 on phosphate excretion are consistent with the in vivo
studies by Shimada et al
and Schiavi et al
in mice which demonstrate that administration of recombinant full length FGF-23 increases phosphate excretion [11
]. However, in contrast to subsequent studies by Shimada et al
in which neither the N-terminal or C-terminal fragments of FGF-23 affected serum phosphate or phosphate excretion, in the present study, we show that the infusion of the carboxyl fragments of FGF-23 176-251, 180-205, 184-251, and 180-251 all significantly increase phosphate [11
]. Of the peptides tested, only FGF-23 206-251 did not exhibit any biologic activity. The precise reason for the differences in bioactivity of the fragments tested by us compared to those tested by Shimada et al
is not known [11
To validate our in vivo
observations that various C-terminal fragments are bioactive, we also injected the smallest C-terminal fragment (180-205) into Fgf-23−/−
mice, and found a significant decrease in serum phosphate levels when compared to vehicle-injected Fgf-23−/−
]; these results suggest that C-terminal fragments could alter phosphate homeostasis in Fgf-23−/−
mice, and that only a small part of the FGF23 protein has the potential to be used for treatment of diseases with hyperphosphatemia.
The in vitro
studies demonstrate that similar to PTH, FGF23 and FGF23 180-251 inhibit sodium-phosphate transport by reducing the amount of plasma membrane localized NaPi IIa. FGF23 contains a consensus binding domain for FGFRs and several studies have suggested that FGF23 may bind and activate signaling through one or more FGFRs [31
]. Recent in vivo
genetic manipulation studies suggest that both FGF23 and KLOTHO act through a common signaling pathway [30
] and that KLOTHO facilitates FGF23 binding to an FGFR [34
]. Of relevance is the absence of the canonical FGFR binding domain on the bioactive FGF23 180-251 fragment. These results raise the possibility that FGF23 may bind to a novel receptor, in addition to a known FGFR.
The role of FGF-23 in the pathogenesis of ADHR has been shown to be due to mutations in the FGF-23 gene that result in the production of a mutant protein in which the amino acid sequence of a furin proconvertase cleavage site (176RHTR179) is altered (R176Q, R179Q, R179W), rendering the mutant FGF-23 resistant to proteolysis[6
]. The mutant FGF-23 has a prolonged half-life [6
]. Our studies suggest that although furin cleavage between residues 176 and 179 results in a reduction of bioactivity of FGF-23, significant bioactivity persists with fragments that extend from carboxyl terminal amino acid residue 180 up to residue 206. This, in turn, suggests that further cleavage of FGF-23 in the carboxyl terminal domain needs to occur in order render the molecule inactive. Indeed, Campos and others have demonstrated the presence of PHEX cleavage sites at amino acid residue 183 and 215 [35
]. Potential PHEX cleavage sites also exist at residues 186, 188 and 208, all of which may play a role in altering the bioactivity of the FGF-23. Since the 180-205 fragment of FGF-23 is bioactive, it is likely that sites carboxyl-terminal of residue 205, are important in the proteolytic processing and bio-inactivation of FGF-23.
It is interesting to note that patients with tumoral calcinosis have elevated concentrations of FGF-23 measured by an assay that detects carboxyl terminal fragments of FGF-23 [17
]. In contrast, these patients have normal or low-normal concentrations of intact FGF-23. Our data would suggest that the carboxyl terminal fragments circulating in patients with tumoral calcinosis are different than those tested by us. Alternatively, if FGF-23 fragments found in the circulation of patients with tumoral calcinosis are similar to those tested by us, they must be present in concentrations considerably lower than those achieved in our experiments.
In conclusion, we have identified the phosphaturic, bioactive domain of FGF-23 and show that it is present in the 176-205 region of the protein.