FGF23 is processed by proprotein convertases but not by PHEX
Introduction
Autosomal dominant hypophosphatemic rickets (ADHR), tumor-induced osteomalacia (TIO), and X-linked hypophosphatemic rickets (XLH) are characterized by renal phosphate wasting, rickets, and osteomalacia. In addition, patients with these conditions exhibit inappropriately normal 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) serum levels. It has been hypothesized that a common circulating factor with phosphaturic activity is responsible for renal phosphate wasting in these disorders, and fibroblast growth factor 23 (FGF23) has been proposed as a candidate.
FGF23 is produced as a 251-amino acid precursor with the first 24 residues coding for a signal peptide. The secreted protein FGF2325–251 has a weight of approximately 30 kDa. During secretion in HEK293 and COS-7 cells, FGF23 is also processed at the C-terminal end between amino acids 179 and 180 at a possible subtilisin-like proprotein convertase (SPC) cleavage site (RXXR motif) [1]. SPCs are a seven-member family of calcium-dependent serine proteases responsible for the processing of peptide hormones, neuropeptides, adhesion molecules, receptors, growth factors, cell surface glycoprotein, and enzymes. The substrates are cleaved C-terminal of the R-(X)n-R consensus site where n is 0, 2, 4, or 6, and X is any amino acid except Cys [2], [3], [4].
Missense mutations at that cleavage site (R176Q, R179W, and R179Q) prevent proteolytic cleavage and cause ADHR [5], [6], [7], [8], [9], [10]. Furthermore, FGF23 is overexpressed in tumors resected from TIO patients [7], [11], [12], [13], and increased serum levels of FGF23 have been observed in these patients [14], [15]. As serum phosphate levels become normal after resection of the tumors, it is likely that intact FGF2325–251 functions as the phosphaturic factor in these conditions. It is less clear whether cleaved FGF2325–179 causes renal phosphate wasting: the administration of intact FGF23 to rodents and the expression of intact FGF23 in nude mice caused renal phosphate wasting, whereas the administration of FGF2325–179 and FGF23180–251 did not [6].
In addition to FGF23, other proteins such as Frizzled-related protein 4 (FRP-4) [12] and matrix extracellular phosphoglycoprotein (MEPE) [16] are overexpressed in TIO tumors. MEPE overexpression in mice did not cause hypophosphatemia [11]. Recently, however, phosphaturic activity of FRP-4 has been reported [17].
XLH is caused by loss of function mutations in PHEX [18], [19]. Several studies indicate that PHEX, which is mainly expressed in bone and teeth [20], regulates the production or degradation of a phosphaturic hormone [21], [22]. It is a type II membrane protein belonging to the M13 family of zinc endopeptidases that are involved in the activation or degradation of bioactive peptides [23]. Experiments with internally quenched fluorogenic peptide substrates revealed that a secreted recombinant form of PHEX has endopeptidase activity [24], whereas mutated PHEX does not [25]. It was also demonstrated that secreted PHEX (secPHEX) can hydrolyze PTHrP107–139 [26].
It was initially tempting to speculate that FGF23 is a substrate of PHEX [27], [28]. This has been supported by the finding that most, but not all, XLH patients showed elevated FGF23 serum levels [14], [15], [29]. However, although it was initially reported that PHEX degraded FGF23 in an in vitro translation system [30], this has not been confirmed by subsequent experiments [31].
Here, we show that FGF23 is cleaved at the C-terminal end by SPCs during its biosynthesis in HEK293 cells. We analyzed the expression of SPCs in this cell line and in mouse osteoblasts. We further report that co-incubation of untagged PHEX and untagged FGF23 did not provide evidence that FGF2325–179, FGF23180–251, or intact FGF2325–251 are substrates of PHEX.
Section snippets
Construction of expression vectors
For expression of the native FGF23 protein, the full-length FGF23 cDNA was subcloned into the mammalian expression vector pcDNA3.1 (InvitrogenTM). To create a C-terminal FGF23/His and an N-terminal FLAG/FGF23 fusion protein, full-length FGF23 cDNA was subcloned in frame into the expression vectors pcDNA3.1/myc-His (InvitrogenTM) and pFLAG-CMV-3 (Sigma-Aldrich) [7], respectively.
To generate a soluble, secreted form of PHEX (secPHEX), we followed a strategy previously described by Boileau et al.
Inhibition of FGF23 processing
Full-length untagged FGF23 (pcDNA/FGF23) was stably transfected into HEK293 cells. The conditioned medium was harvested and concentrated 1:20. Total protein concentration was between 0.5 and 0.9 μg/μl after concentration, with FGF23 constituting a major protein fraction (Fig. 1A). Western blot analysis was performed using a polyclonal antibody against FGF23173–187 (antiFGF23173–187) that recognizes the C-terminal part of human FGF23. Two products of approximately 30 and 10 kDa were detected,
Discussion
Mutant FGF23 that is resistant to cleavage in ADHR, overproduction of FGF23 by tumors in TIO, and impaired FGF23 degradation due to reduction or loss of PHEX activity in XLH have been proposed as the molecular mechanisms responsible for renal phosphate wasting in these diseases. In the current study, we show that FGF23 is cleaved by SPCs during its biosynthesis in HEK293 cells at the RHTR site and that neither intact FGF23 nor N- or C-terminal peptides are further degraded by PHEX.
Using the SPC
Acknowledgements
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (STR304/2-1) and NIH grants R01AR42228, R01DK063934, and K24-AR02095.
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