Inactivating mutations of Phex cause X-linked hypophosphatemia (XLH) by increasing levels of a circulating phosphaturic factor. FGF23 is a candidate for this phosphaturic factor. Elevated serum FGF23 levels correlate with the degree of hypophosphatemia in XLH, suggesting that loss of Phex function in this disorder results in either diminished degradation and/or increased biosynthesis of FGF23. To establish the mechanisms whereby Phex regulates FGF23, we assessed Phex-dependent hydrolysis of recombinant FGF23 in vitro and measured fgf23 message levels in the Hyp mouse homologue of XLH. In COS-7 cells, overexpression of FGF23 resulted in its degradation into N-and C-terminal fragments by an endogenous decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone-sensitive furin-type convertase. Phex-dependent hydrolysis of full-length FGF23 or its N-and C-terminal fragments could not be demonstrated in the presence or absence of decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone in COS-7 cells expressing Phex and FGF23. In a reticulolysate system, apparent cleavage of FGF23 occurred with wild-type Phex, the inactive Phex-3M mutant, and vector controls, indicating nonspecific metabolism of FGF23 by contaminating enzymes. These findings suggest that FGF23 is not a direct Phex substrate. In contrast, by real-time reverse transcriptase PCR, the levels of fgf23 transcripts were highest in bone, the predominant site of Phex expression. In addition, Hyp mice displayed a bonerestricted increase in fgf23 transcripts in association with inactivating Phex mutations. Increased expression of fgf23 was also observed in Hyp-derived osteoblasts in culture. These findings suggest that Phex, possibly through the actions of unidentified Phex substrates or other downstream effectors, regulates fgf23 expression as part of a potential hormonal axis between bone and kidney that controls systemic phosphate homeostasis and mineralization. X-linked hypophosphatemia (XLH)1 is a disorder characterized by defective calcification of cartilage and bone, growth retardation, impaired renal tubular reabsorption of phosphate, aberrant regulation of 1,25(OH) 2 D 3 production, and resistance to phosphorus and vitamin D therapy (1). XLH is caused by inactivating mutations of PHEX (2-5), a member of the M13 family of type II cell surface zinc-dependent proteases that include neprilysin, endothelin-converting enzymes 1 and 2 (6, 7), KELL (8), and DINE/X-converting enzyme (9, 10). The mouse Phex cDNA sequence is highly homologous to that of humans (11,12), and inactivating mutations of Phex are identified in several mouse homologues of XLH, including Hyp, Gy, and Ska1 mice (10,13,14).Current data indicate that Phex regulates the production and/or degradation of a systemic phosphaturic hormone, referred to as phosphatonin (15). The presence of phosphatonin in XLH/Hyp was detected by parabiosis experiments in which Hyp mice transferred the phosphaturic phenotype to normal mice (16). Studies in parathyroidectomized Hyp mice eliminated parathyroid hormone as the responsible phosph...

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