Fibroblast growth factor 23 (FGF23) is a phosphaturic factor that suppresses both sodium-dependent phosphate transport and production of 1,25-dihydroxyvitamin D [1,25(OH) 2 D] in the proximal tubule. In vitro studies suggest that FGFR3 is the physiologically relevant receptor for FGF23 in the kidney, but this has not been established in vivo. Here, immunohistochemical analysis of the mouse kidney revealed that the proximal tubule expresses FGF receptor 3 (FGFR3) but not FGFR1, FGFR2, or FGFR4. Compared with wild-type mice, Hyp mice, which have elevated circulating levels of FGF23, exhibited low levels of serum phosphate and 1,25(OH) 2 D, reduced expression of the sodium-dependent phosphate transporter NPT2a in the proximal tubules, and low bone mineral density as a result of osteomalacia. In contrast, neither the serum phosphate nor 1,25(OH) 2 D levels were altered in FGFR3-null mice. For examination of the role of FGFR3 in mediating the effects of FGF23, Hyp mice were crossed with FGFR3-null mice; interestingly, this failed to correct the aforementioned metabolic abnormalities of Hyp mice. Ablation of FGFR4 also failed to correct hypophosphatemia in Hyp mice. Because the ablation of neither FGFR3 nor FGFR4 inhibited the renal effects of excess FGF23, the kidney localization of FGFR1 was investigated. FGFR1 co-localized with Klotho, the co-factor required for FGF23-dependent FGFR activation, in the distal tubule. In summary, neither FGFR3 nor FGFR4 is the principal mediator of FGF23 effects in the proximal tubule, and co-localization of FGFR1 and Klotho suggests that the distal tubule may be an effector site of FGF23. Fibroblast growth factor 23 (FGF23), a phosphaturic factor predominantly produced by osteocytes in bone, 1 causes hypophosphatemia, suppression of 1,25-dihydroxyvitamin D [1,25(OH) 2 D] production, and rickets/osteomalacia. 1-4 Excess FGF23 mediates renal phosphate wasting in hereditary human hypophosphatemic disorders, including autosomal dominant hypophosphatemic rickets, 5,6 Xlinked hypophosphatemia, 7,8 and autosomal recessive hypophosphatemic rickets. 9,10 Elevated FGF23 also mediates hypophosphatemia in several acquired disorders, including tumor-induced osteomalacia, 11 McCune-Albright syndrome, and polyostotic fibrous dysplasia. 12 FGF23 inhibits the sodium-dependent phosphate transporter and CYP27B1 activity in the proximal tubule of the kidney, leading to phosphaturia and reduced production of 1,25(OH) 2 D. 2,11,13,14 FGF23 binds to and activates FGF receptors (FGFR) 1c, 3c, and 4 in cell lines that coexpress Klotho, a transmembrane protein co-factor that determines the tissue specificity of FGF23. 15,16 FGF23 has been shown to inhibit directly phosphate transport and to suppress 1␣-hydroxylase gene expression in isolated proximal tubules and/or proximal tubule-derived cell lines in

Inactivating PHEX (phosphate regulating gene with homologies to endopeptidases on the X chromosome) mutations cause X-linked hypophosphatemia in humans and mice (Hyp) through overproduction of fibroblast growth factor 23 (FGF23) a phosphaturic factor, by osteocytes. Matrix extracellular phosphoglycoprotein (MEPE) is also elevated in Hyp and other hypophosphatemic disorders. In addition, the administration of an ASARM (acidic serineaspartate rich MEPE-associated motif) peptide derived from MEPE causes phosphaturia and inhibits bone mineralization in mice, suggesting that MEPE also plays a role in phosphate homeostasis. Since recent studies found that MEPE binds specifically to PHEX in vitro, we tested the effect of recombinant-MEPE and its ASARM peptide on PHEX enzyme activity in vitro and FGF23 expression in bone marrow stromal cell cultures ex vivo. We found that both recombinant MEPE and synthetic phosphorylated ASARM peptide (ASARM-PO 4 ) inhibit PHEX enzyme activities in an in vitro fluorescent-quenched PHEX enzyme activity assay. The ASARM-PO 4 peptide inhibits PHEX enzyme activity in a dose-dependent manner with a K i of 128 nM and V maxKi of 100%. Recombinant MEPE also inhibits PHEX activity (K i Z2 nM and V maxKi Z26%). Long-term bone marrow stromal cell cultures supplemented with 10 mM ASARM-PO 4 peptide resulted in significant elevation of FGF23 transcripts and inhibition of mineralization. These findings suggest that MEPE inhibits mineralization and PHEX activity and leads to increased FGF23 production. The resulting coordination of mineralization and release of a phosphaturic factor by MEPE may serve a physiological role in regulating systemic phosphate homeostasis to meet the needs for bone mineralization.

Liu S, Tang W, Zhou J, Vierthaler L, Quarles LD. Distinct roles for intrinsic osteocyte abnormalities and systemic factors in regulation of FGF23 and bone mineralization in Hyp mice. Am J Physiol Endocrinol Metab 293: E1636-E1644, 2007. First published September 11, 2007 doi:10.1152/ajpendo.00396.2007.-Xlinked hypophosphatemia (XLH) is characterized by hypophosphatemia and impaired mineralization caused by mutations of the PHEX endopeptidase (phosphate-regulating gene with homologies to endopeptidases on the X chromosome), which leads to the overproduction of the phosphaturic fibroblast growth factor 23 (FGF23) in osteocytes. The mechanism whereby PHEX mutations increase FGF23 expression and impair mineralization is uncertain. Either an intrinsic osteocyte abnormality or unidentified PHEX substrates could stimulate FGF23 in XLH. Similarly, impaired mineralization in XLH could result solely from hypophosphatemia or from a concomitant PHEX-dependent intrinsic osteocyte abnormality. To distinguish between these possibilities, we assessed FGF23 expression and mineralization after reciprocal bone cross-transplantations between wildtype (WT) mice and the Hyp mouse model of XLH. We found that increased FGF23 expression in Hyp bone results from a local effect of PHEX deficiency, since FGF23 was increased in Hyp osteocytes before and after explantation into WT mice but was not increased in WT osteocytes after explantation into Hyp mice. WT bone explanted into Hyp mice developed rickets and osteomalacia, but Hyp bone explanted into WT mice displayed persistent osteomalacia and abnormalities in the primary spongiosa, indicating that both phosphate and PHEX independently regulate extracellular matrix mineralization. Unexpectedly, we observed a paradoxical suppression of FGF23 in juvenile Hyp bone explanted into adult Hyp mice, indicating the presence of an age-dependent systemic inhibitor of FGF23. Thus PHEX functions in bone to coordinate bone mineralization and systemic phosphate homeostasis by directly regulating the mineralization process and producing FGF23. In addition, systemic counterregulatory factors that attenuate the upregulation of FGF23 expression in Hyp mouse osteocytes are present in older mice. PHEX endopeptidase; X-linked hypophosphatemia; fibroblast growth factor 23; rickets; osteomalacia X-LINKED HYPOPHOSPHATEMIA (XLH) is caused by inactivating mutations of the endopeptidase PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) (32), a member of the M13 family of the type II cell-surface zinc-dependent proteases that is predominately expressed in bone (33). The mouse Phex cDNA sequence is highly homologous to that of humans, and a 3Ј deletion of the Phex gene in the Hyp mouse results in an animal model of XLH (4,29). PHEX mutations result in impaired renal tubular reabsorption of phosphate and aberrant regulation of 1,25(OH) 2 D production, leading to hypophosphatemia and defective calcification of cartilage and bone, which result in rickets, osteomalacia, and growth ret...