The Autosomal Dominant Hypophosphatemic Rickets R176Q Mutation in Fibroblast Growth Factor 23 Resists Proteolytic Cleavage and Enhances in Vivo Biological Potency

Abstract

Missense mutations in fibroblast growth factor 23 (FGF23) are the cause of autosomal dominant hypophosphatemic rickets (ADHR). The mutations (R176Q, R179W, and R179Q) replace Arg residues within a subtilisin-like proprotein convertase (SPC) cleavage site (RXXR motif), leading to protease resistance of FGF23. The goals of this study were to examine in vivo the biological potency of the R176Q mutant FGF23 form and to characterize alterations in homeostatic mechanisms that give rise to the phenotypic presentation of this disorder. For this, wild type and R176Q mutant FGF23 were overexpressed in the intact animals using a tumorbearing nude mouse system. At comparable circulating levels, the mutant form was more potent in inducing hypophosphatemia, in decreasing circulating concentrations of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), and in causing rickets and osteomalacia in these animals compared with wild type FGF23. Parameters of calcium homeostasis were also altered, leading to secondary hyperparathyroidism and parathyroid gland hyperplasia. However, the raised circulating levels of parathyroid hormone were ineffective in normalizing the reduced 1,25(OH) 2 D 3 levels by increasing renal expression of 25(OH)D 3 -1␣-hydroxylase (Cyp40) to promote its synthesis and by decreasing that of 25(OH)D 3 -24-hydroxylase (Cyp24) to prevent its catabolism. The findings provide direct in vivo evidence that missense mutations from ADHR kindreds are gain-of-function mutations that retain and increase the protein's biological potency. Moreover, for the first time, they define a potential role for FGF23 in dissociating parathyroid hormone actions on mineral fluxes and on vitamin D metabolism at the level of the kidney.Renal phosphate wasting is associated with a number of hereditary disorders including X-linked (XLH) 1 and autosomal dominant (ADHR) forms of hypophosphatemic rickets. In addition to hypophosphatemia, patients with these two conditions exhibit decreased or inappropriately normal serum levels of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), as well as rickets and osteomalacia. Interestingly, ADHR encompasses not only the classic presentation of hypophosphatemia and rickets, but also it displays variable penetrance with delayed onset of the disease and an even more perplexing feature, spontaneous resolution of the biochemical defect (1, 2).The genes responsible for XLH and ADHR have now been identified. Through positional cloning, a gene that spans the deleted region Xp22.1 in XLH patients or is mutated in nondeletion patients with the disorder has been identified (designated PEX and subsequently PHEX, for phosphate-regulating gene with homologies to endopeptidases on the X chromosome) (3). The predicted human PHEX gene product exhibits structural similarity to a family of neutral endopeptidases involved in either activation or degradation of peptide hormones. Therefore, PHEX probably functions as a protease, and it may act by processing factor(s) involved in bone mineral metabolism. Extensive mutation analysis of XL...

MEPE has the properties of an osteoblastic phosphatonin and minhibin

“…There is overwhelming evidence implicating the osteoblast as the cell intrinsically defective [13,18,[20][21][22]30,37,50,58,59,67,69,80,101,102] and the HYP osteoblast directly secretes factors (phosphatonins) that impact adversely on renal phosphate uptake and mineralization in vivo and in vitro. FGF23 activating mutations are responsible for the changes in phosphate, vitamin D metabolism and mineralization in ADHR [4,82,97,98] and overexpression of wild-type FGF23 in some but not all OHO tumors is directly or indirectly responsible for the changes in some tumor-induced osteomalacias [15,81,97,99]. Also, a number of groups have reported lack of FGF23 expression in bone/osteoblasts [3,27,29,41,98,104] and others using more sensitive techniques have detected low levels of FGF23 mRNA in bone tissue but not in normal murine osteoblasts [40].…”

“…Recent discoveries have confirmed that autosomal dominant hypophosphatemic rickets (ADHR) is caused by activating mutations in FGF23 [4,82,97,98]. The specific ADHR mutations increase the stability of mutated FGF23 and the uncleaved mutated molecule is phosphaturic and elicits osteomalacia/rickets [4,77,81,98].…”

“…The specific ADHR mutations increase the stability of mutated FGF23 and the uncleaved mutated molecule is phosphaturic and elicits osteomalacia/rickets [4,77,81,98]. Wild-type FGF23 is also expressed in OHO tumors and the tumor over-expression of the wild-type FGF23 molecule is thought to swamp proteolytic mechanisms that normally inactivate full-length FGF23 [11,15,81].…”

Biological activity of FGF-23 fragments

“…The mutant FGF-23 has a prolonged half-life [6,7,9,11,28]. 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.…”

“…In autosomal dominant hypophosphatemic rickets (ADHR), a disease associated with low serum phosphorus and 1 ,25(OH) 2 D concentrations and rickets, mutations in FGF-23 gene result in the expression of a protein that is resistant to proteolysis and with an increased half life [7,9,11,14,28]. Mutations in the gene alter the furin proconvertase recognition site (176RHTR179) in the protein such that the arginine residue at 176 is replaced by glutamine (R176Q) or the arginine residue at amino acid 179 is replaced by a glutamine (R179Q) or tryptophan residue (R179W).…”