REGULATION OF PHOSPHATE METABOLISM

The circulating concentration of phosphate is dependent on the dietary Pi intake, intestinal absorption, renal filtration and reabsorption, and exchange from intracellular and bone reservoirs. Figure 1 shows the metabolism of Pi in humans. A normal human ingests approximately 1.5 to 2 g of phosphorus per day (20 mg/kg/day). Of this, 1 to 1.2 g (16 mg/kg/day) is absorbed in the proximal intestine and 200 mg (3 mg/kg/day) is secreted into the intestinal lumen giving a net absorption of approximately 1 g (13 mg/kg/day). The absorbed phosphorus enters an extracellular fluid pool and moves in and out of bone as needed (3 mg/kg/day). Approximately 1 g (13 mg/kg/day) is excreted by the kidneys such that equivalent amounts of phosphorus are absorbed and excreted. Thus, under physiologic conditions, the intestine and the kidney function in concert to maintain phosphorus homeostasis and normal plasma Pi concentrations (2.5 to 4.5 mg/dL). Patients with hypophosphatemic disorders such as XLH, ADHR, and TIO may have circulating levels as low as 2 mg/dL, whereas patients with declining renal function can have levels that exceed 6 mg/dL.

Figure 1.

Phosphorus metabolism. 

Full figure and legend (17K)

The major known hormones regulating phosphorus metabolism are 1,25(OH)₂D₃ and PTH^{1,2,4,7,8,9,10,11,12,13}. As described below, PTH inhibits renal phosphate reabsorption and indirectly increases intestinal phosphorus absorption by stimulating the synthesis of 1,25(OH)₂D. Phosphorus has a direct stimulatory effect on the secretion of PTH and elevated serum Pi is believed to be an important contributor to the hyperparathyroidism associated with late stages of chronic renal disease Figure 2^{14,15,16,17,18}.

Figure 2.

Influence of phosphate on parathyroid hormone (PTH) and vitamin D synthesis and activity. Pi is inorganic phosphorus.

Full figure and legend (11K)

1,25(OH)₂D₃ increases intestinal Pi absorption and renal Pi reabsorption^{2,9,10,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35}. The formation of the active form of vitamin D₃, 1,25(OH)₂D₃, is regulated by dietary Pi, circulating serum Pi concentrations and PTH. Extracellular Pi regulates 25(OH)D₃ 1-hydroxylase [1 (OH)ase] synthesis and activity such that low Pi concentrations increase and high Pi concentrations inhibit its synthesis and activity Figure 2^{2,3,4,10,19,20,21,23,24,25,26,27,28,35,36,37,38,39}. This regulation occurs via a mechanism that is independent of PTH as well as the sodium phosphate transporter, NaPi IIa⁴⁰. The adaptation to low Pi diets in the vitamin D endocrine system appears to require the presence of growth hormone and insulin-like growth factors^{41,42,43,44,45,46,47,48}. PTH increases synthesis of 1,25(OH)₂D₃ by increasing the activity of 1 (OH)ase^49,50.

Regulation of phosphate homeostasis in the kidney occurs primarily in the proximal tubule and is controlled by a variety of factors that either increase or decrease Pi reabsorption Table 1^{2,3,4,5,7,8,9,10, 11,12,13,19,20,22,23,51,52,53,54,55, 56,57, 58,
59,60, 61,62,
63,64, 65,66, 67,68, 69,70, 71,72, 73,74, 75,76, 77,78}. Plasma inorganic phosphate is freely filtered at the glomerulus. Approximately 80% of filtered phosphate is reabsorbed along with sodium through specific cotransporters, NaPi IIa, located in the proximal tubule. NaPi IIa is a member of one of three distinct families of sodium phosphate cotransporters Table 2^11,12,79,80. Mice deficient in NaPi IIa have about an 80% reduction in proximal renal phosphate reabsorption, confirming the major role of this specific transporter in phosphate homeostasis⁸¹.

Table 1 - Factors affecting phosphate reabsorption in the proximal tubule.

Full table

Table 2 - The three families of Na⁺/Pi cotransporters^11,12,72.

Full table

Regulation of phosphate reabsorption in the kidney is achieved primarily by controlling the number of the NaPi IIa cotransporters present on the surface of the proximal brush border membranes. Current evidence suggests that acute decreases in dietary Pi lead to recruitment of existing NaPi IIa transporters from an intracellular pool^82,83, whereas chronic changes in dietary Pi regulate NaPi IIa through transcription-dependent methods^83,84. Chronic administration of 1,25(OH)₂D₃ and PTH modulate NaPi IIa mRNA and protein^85,86. Elevated PTH rapidly reduces the number of NaPi IIa transporters by inducing surface retrieval and subsequent lysosomal degradation^{7,8,11,12,57,67,68,69,70,71,72,73,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101}. The mechanism of PTH-induced retrieval involves stimulation of at least two signal transduction pathways that converge on the mitogen-activated protein (MAP) kinase, extracellular regulated kinase (ERK)^102,103. It is currently unclear how stimulation of ERK triggers internalization of NaPi IIa. Targeted disruption of NHERF-I, a cytoplasmic docking protein known to assemble multiple protein signaling complexes, results in decreased brush border NaPi IIa expression and reduced renal phosphate reabsorption¹⁰⁴. The demonstration that NHERF-I interacts with NaPi IIa further supports the possibility that this protein may be an important intermediate in PTH action¹⁰⁵. However, a recent study suggests that NHERF-I, while important for trafficking of NaPi IIa to the membrane, is not involved in PTH-induced membrane retrieval^106,107.

Intestinal absorption of phosphate occurs through both nonregulated passive transport and regulated active mechanisms. The type IIb sodium-phosphate cotransporter located predominantly in the jejunum mediates active intestinal phosphate transport and appears to be regulated at the transcriptional level by 1,25(OH)₂D₃⁹⁰. It, however, does not undergo retrieval from the membrane in response to PTH. The mechanisms controlling the release of phosphate and calcium from intracellular and bone pools are poorly understood and may involve specific regulation of the type III sodium-phosphate transporters, Glvr1 (Pit1) and Ram1 (Pit2)^108,109.

This well-defined paradigm for phosphate regulation may be extended to include the function of one or more members of the emerging class of phosphate regulating proteins, the "phosphatonins"^{74,75,76,77,78,79,110,111,112,113,114,115}. As we will show in subsequent sections, the "phosphatonins"[fibroblast growth factor 23 (FGF23) and frizzled-related protein 4 (FRP4)] also play roles in modulating renal phosphorus reabsorption and 1,25(OH)₂D₃ production. Insufficient information prohibits a clear understanding of the interplay between phosphatonins, PTH, and the vitamin D pathway. However, recent and tantalizing new data have provided enough evidence that focused studies of the phosphatonins are not only relevant but necessary for a complete understanding of phosphate homeostasis.

THE PHOSPHATONINS

A group of pathologic conditions exist in which phosphate levels are altered without significant net changes in serum concentrations of calcium, PTH, and 1,25(OH)₂D₃. These diseases, which are characterized by hypophosphatemia, hyperphosphaturia, and defective bone mineralization include TIO, XLH, and ADHR^{75,76,77,110,111,112,113,114,115,116}. The observation that patients with these diseases generally have normal serum concentrations of PTH and calcium, in association with low or normal serum 1,25(OH)₂D₃ concentrations, is unexpected based on the current understanding of phosphate regulation. Early studies provided compelling evidence that a circulating humoral factor, referred to as "phosphatonin," is likely responsible for the altered phosphate regulation observed in these syndromes^{110,111,112,113,114,115,116,117,118,119,120}. Tumors associated with osteomalacia cause hypophosphatemia when implanted into athymic mice and conditioned media from tumors cultured in vitro specifically reduced phosphate uptake in renal epithelial cells¹¹⁰. These studies suggested that such tumors secrete a factor(s) that can act at a distance to inhibit renal phosphate reabsorption. Evidence that a humoral factor(s) is associated with XLH is derived from studies in hyp mice, the mouse model of XLH. Parabiosis experiments between hyp and normal mice show that a factor present in the circulation of hyp mice causes hypophosphatemia in the normal mouse^117,118. This factor is not PTH, as parathyroidectomy of the hyp mouse (in the parabiotic pair) does not influence the reduction in serum Pi in the normal mouse. In addition, transplantation of a kidney from a normal mouse into a hyp mouse does not correct the hyperphosphaturia and hypophosphatemia present in the recipient; conversely, the reverse procedure is not associated with any alterations in Pi metabolism in the normal mouse¹¹⁹. Additionally, cultures of hyp mouse proximal tubules transport phosphate normally¹²⁰. These data are consistent with the presence of a circulating phosphaturic factor(s) in hyp mice. Although analogous studies cannot be performed for ADHR, the similarity of symptoms associated with ADHR, XLH, and TIO suggest that an increase in expression and/or activity of a phosphatonin is a common feature among these syndromes. Since these early studies, several genomic and proteomic strategies have been utilized in the search for these molecules. Surprisingly, these studies identified three proteins that are associated with one or more of these syndromes, FGF23, FRP4, and matrix extracellular phosphoglycoprotein (MEPE), that may each possess some or all of the properties associated with phosphatonin.

THE SEARCH FOR PHOSPHATONIN

A positional cloning approach was used to discover the affected gene associated with ADHR. Analysis of a large affected family indicated that the gene responsible for this disease was located on chromosome 12p13.3¹²¹. Mutations within a new gene, FGF23, with significant homology to genes within the FGF family, segregated exclusively with ADHR¹²². In four ADHR families, three missense mutations were found clustered within a small region of 2 amino acids. This observation together with the fact that ADHR is an autosomal-dominant disorder suggested that these mutations might result in a gain of function for FGF23.

The impact of these mutations on the structure of FGF23 became clear as several groups expressed the wild-type form of FGF23 in vitro. In addition to the expected 30 kD protein encoded by the full-length FGF23 gene, two smaller fragments were also detected. Expression of FGF23 protein containing carboxy^{123,124,125,126} and amino epitope tags¹²⁷ revealed that a portion of the secreted FGF23 was processed into two discrete fragments of 18 and 12 kD. The sizes of these fragments and their deduced amino terminal sequences are consistent with cleavage at a predicted site for a pro-convertase type enzyme, RXXR^{122,123,124,125}. Importantly, the identified ADHR mutations, R176Q, R179W, and R179Q, replace critical R residues within the consensus cleavage site of FGF23 (176-RHTR-179)^{122,125,126,127}. Processing of the mutated protein is inhibited in vitro^124,126,128 supporting the hypothesis that ADHR mutations interfere with FGF23 processing Figure 3. Although there is no direct evidence that altered activity or levels of a circulating factor(s) results in ADHR, the similarity between this syndrome, XLH, and TIO where there is evidence for a circulating factor, provides a compelling case that FGF23 encodes phosphatonin activity.

Figure 3.

Model depicting fibroblast growth factor 23 (FGF23) processing and potential PHEX cleavage in normal, autosomal-dominant hypophosphatemic rickets (ADHR) or X-linked hypophosphatemic rickets (XLH) individuals. 

Full figure and legend (15K)

Because ADHR shares similar clinical and biochemical symptoms with the tumor-associated disease, TIO, White et al¹²³ examined FGF23 RNA expression in tumors associated with osteomalacia. Northern blot analysis suggested that FGF23 mRNA was expressed at relatively high levels in five tumors isolated from TIO patients and Western blot analysis further revealed detectable levels of FGF23 in a sixth tumor. This observation linking FGF23 to two related diseases, TIO and ADHR, further supported the hypothesis that FGF23 is phosphatonin.

Despite the striking evidence correlating FGF23-associated changes with phosphaturic disorders, the assignment of phosphatonin becomes uncertain when differential expression studies are considered^125,128,129. Although FGF23 was identified in two out of three of these studies, expression profiling also identified at least two additional phosphatonin candidates and other genes potentially involved in the phosphatonin pathway. These approaches have been based on the premise that a phosphaturic protein(s) and/or the gene(s) encoding it, is overexpressed in specific tissues from diseased individuals. Evidence that tumors causing TIO secrete a phosphate-lowering activity¹¹⁰ prompted several investigators to utilize these tumors as a source tissue for the identification of phosphatonin. Rowe et al¹³⁰ were the first to identify a novel TIO-associated gene encoding a potential phosphatonin candidate. A TIO tumor-derived cDNA library was screened with antibodies raised against circulating proteins in sera from a TIO patient. The sera had been extensively preabsorbed against normal human sera to enrich for antibodies that would specifically recognize proteins differentially expressed in sera of TIO patients. The distinct advantage to this strategy was that it detected only those overexpressed genes that encode circulating proteins. Using this approach, MEPE was identified as a novel tumor-associated product. The observations that MEPE is present in the circulation of TIO patients appears to be expressed abundantly in TIO tumors, and is a previously uncharacterized secreted protein, are consistent with the possibility that MEPE is phosphatonin.

Gene expression profiling of TIO tumors using two distinct strategies further extended this work^125,129. In each case, several genes, including FGF23 and MEPE, were identified that were specifically associated with TIO. Shimada et al¹²⁵ generated two cDNA libraries from a single TIO tumor and adjacent normal bone tissue. Probes specific for each library were also generated using subtractive hybridization and used to specifically probe each library. Out of a total of 32,000 clones isolated, 456 clones were unique to the TIO tumor-derived library. Proteins encoded by the selected genes included dentin matrix protein 1 (DMP1), heat shock protein 90 (HSP90), osteopontin (OPN), fibronectin, MEPE, and FGF23. DMP1 and HSP90 were the most abundant candidate proteins identified, whereas significantly fewer clones represented FGF23 and MEPE. Although each of the secreted proteins identified could theoretically function as phosphatonin, the preexisting evidence linking FGF23 to ADHR¹²² and MEPE to TIO¹³⁰ continued to place these two genes high on the list of potential phosphatonin candidates.

A more extensive analysis of TIO-specific genes was provided by serial analysis of gene expression (SAGE)¹²⁹. In this strategy, gene expression profiles were obtained from a variety of tumors resected from TIO patients and histologically matched control tumors that were not associated with TIO. Genes that were overexpressed in the TIO tumors relative to the control tumors were selected and further validated by microarray analysis using RNA isolated from a broad panel of additional control and TIO tumors. This approach ensured that phosphatonin would be selected if it were encoded by a gene induced by a tumor-specific autocrine factor. An additional advantage to this strategy was that a high degree of stringency was imposed on gene selection by identifying genes differentially expressed between multiple control and TIO tumors.

In addition to MEPE, DMP1, and FGF23, SAGE identified other TIO-associated genes¹²⁹. These genes included FRP4, transforming growth factor-1 (TGF-1), secreted serine protease (SP11), a synaptotagmin-like protein, GLVR1, and ANK. Furthermore, the SAGE analysis demonstrated that several genes reported to be linked to TIO by the Japanese group were not specifically associated with the TIO phenotype¹⁰². For example, OPN and fibronectin were elevated in several normal and TIO-associated tumors.

Of interest is the observation that many of the genes identified by SAGE encode proteins associated with osteoblast/chondrocyte differention and/or mineral metabolism. For example, ANK encodes a transporter for pyrophosphate, a mineralization inhibitor^131,132. ANK-deficient mice have a progressive form of arthritis that is accompanied by ectopic mineralization. GLVR1(PIT1) encodes a ubiquitously expressed type III sodium phosphate transporter Table 2 that has been implicated in osteoblast mineralization^{108,109,133,134}. TGF-1 is an important osteoblast differentiation factor and increases expression of GLVR1 mRNA¹³⁵. These and other SAGE-identified genes may represent downstream components of phosphatonin-regulated pathways. The observation that phosphatonin(s) may have induced tumor expression of osteoblast-related genes provides evidence that phosphatonins may have direct actions on bone.

ASSESSING PHOSPHATONIN ACTIVITY

The genomic and proteomic approaches described above identified multiple genes associated with hypophosphatemic disorders. Each of the encoded secreted proteins (or posttranslationally modified variations of the proteins), represent potential phosphatonin candidates. Alternatively, these genes could encode regulators of phosphatonin expression or activity, or represent genes induced by phosphatonin.

The ability to unambiguously identify the gene(s) that encodes phosphatonin is clearly dependent on the definition of its activity. Kumar⁷⁷ proposed that phosphatonin should be identified when the following criteria are met. First, the protein must inhibit phosphate uptake in vitro and in vivo. Second, the factor must inhibit the formation of 1,25(OH)₂D₃ in vivo and in vitro. Third, the protein should be expressed in tumor tissues responsible for the syndrome. Fourth, the concentrations of phosphatonin should be elevated in the serum of patients with each of the disorders. In the case of TIO, surgical resection of the tumor should result in decreased serum concentrations. Finally, urinary cyclic adenosine monophosphate (cAMP) excretion should be normal. It is important to point out that these criteria also assume that a single protein is responsible for the symptoms associated with TIO, XLH, and ADHR. However, the evidence that multiple proteins participate in the pathophysiology of these phosphaturic diseases raises the possibility that phosphatonin activity is due to the combined actions of minimally three proteins.

FGF23

Several approaches have been used to assess the function of FGF23. We have demonstrated that both the native and a mutated form found in ADHR patients inhibits phosphate but not alanine, glucose, or sulfate uptake in renal epithelial cells in vitro¹²⁴. Picomolar concentrations of FGF23 inhibited phosphate transport suggesting that FGF23 may function at concentrations that fall within the normal range for other cytokines Figure 4a^124,136. Differences in clonal cell lines and assay conditions may account for discrepancies in the ability of various groups to detect FGF23 activity in vitro.

Figure 4.

Effect of fibroblast growth factor 23 (FGF23) or frizzled-related protein 4 (FRP4) on phosphate transport. (A) In vitro effects of FGF23 () or FRP4 () on inorganic phosphorus transport (Pi) in opossum kidney cells. (B) FGF23 effects on the percent fractional Pi excretion. Mice were infused for 2 hours with vehicle (), 0.5 g FGF23/2 hour (), or 5 g FGF23/2 hours (). (C) FRP4 effects on the percent fractional Pi excretion. Mice were infused for 2 hours with vehicle (), 0.05 g FRP4/2 hour (), or 0.5 g FRP4/2 hours.

Full figure and legend (24K)

Intraperitoneal administration of recombinant FGF23 in normal mice induced phosphaturia and a small but significant decrease in serum phosphate levels¹²⁵. There was no effect when purified amino or carboxyl FGF23 fragments were injected suggesting that the intact molecule is responsible for biologic activity. In mice injected intravaneously with a dose of 5 g of intact FGF23, a 20% to 25% decrease in serum phosphate and a reduction in renal NaPi IIa protein was observed within 8 hours. 1,25(OH)₂D₃ levels were also reduced but statistically significant changes were observed within 2 hours and reached a maximum decrease within 9 hours after administration of FGF23 [abstract; Shimada T, J Bone Miner Res 17:S425, 2000]. There was no detectable difference in the levels of calcium and PTH. These temporal relationships prompted investigators to suggest that the primary role of FGF23 is to control 1,25(OH)₂D₃ production, and that the effects on phosphate transport are of secondary importance. Our data demonstrate that FGF23 inhibits phosphate transport in vitro within 3 hours Figure 4a and increases phosphate excretion as quickly as 1¹/₂ hours after intravenous infusion of FGF23 Figure 4b. It is possible that FGF23 promotes mobilization of phosphate from bone or intracellular stores in addition to renal excretion, such that a lag period is observed before reductions in serum Pi can be observed.

Long-term exposure to FGF23 by implantation of FGF23-expressing Chinese hamster ovary (CHO-FGF23) cells into athymic nude mice, induced more severe hypophosphatemia, osteomalacia, decreased 1,25(OH)₂D₃ levels, and a corresponding decrease in 25(OH)D₃ 1-hydroxylase mRNA. There was no increase in urinary cAMP concentrations or alterations in the excretion of amino acids¹²⁵. Studies of transgenic mice expressing FGF23 provide further evidence that this factor contributes to many if not all the laboratory abnormalities associated with hypophosphatemic, hyperphosphaturic disorders [abstract; Shimada T, J Bone Miner Res 16:S151, 2001]. FGF23 transgenic mice had significantly reduced serum phosphate and 1,25(OH)₂D₃ concentrations. Furthermore, skeletal abnormalities, including retarded growth, increased osteoid, and curvature of the spine, were also observed.

In vivo experiments also revealed that in addition to inhibiting protein processing, the ADHR mutation results in enhanced phosphaturic activity^137,138. Bai et al¹³⁷ examined the effects of implanted CHO tumors expressing either wild-type FGF23 or the ADHR mutant, FGF23R176Q, on serum and urine phosphate levels. Although approximately equivalent levels of each form of FGF23 protein were produced, FGF23R176Q expression was associated with a greater increase in Pi excretion, decreased serum Pi, and reduced 1,25(OH)₂D₃ concentrations. Interestingly, these experiments also revealed that long-term expression of FGF23 or FGF23R176Q resulted in increased PTH concentrations. Experiments in which naked DNA was injected into rats also demonstrated profound effects of all three ADHR mutants on serum phosphate levels and renal NaPi IIa protein levels [138]. These experiments, which differed in both the method of expressing FGF23 and the animal species, failed to demonstrate the changes in serum Pi that have been observed in mice.

The development of enzyme-linked immunosorbent assays (ELISA) allowing the measurement of FGF23 in the serum of normal and hypophosphatemic patients has provided somewhat unexpected results. As mentioned above, FGF23 is expressed as an intact 30 kD form or processed fragments of 18 and 12 kD. Detectable levels of FGF23 were measured in normal individuals using an assay recognizing an epitope within the carboxy end (recognizes both the 30 and 12 kD forms of the protein)¹³⁹ and by an assay developed to detect only the 30 kD FGF23 protein¹⁴⁰. Increased FGF23 levels were detected in most but not all patients with XLH or TIO^139,140. In TIO patients who exhibited elevated FGF23 concentrations, circulating levels returned to within the normal range upon removal of the tumor^139,140. Singh and Kumar¹⁴¹ demonstrated that patients with humoral hypercalcemia of malignancy had elevated serum FG23 concentrations and patients with hyperparathyroidism had slightly increased FGF23 concentrations. These elevations are not correlated with the decrease in serum phosphate or 1,25(OH)₂D₃ concentrations. The finding that elevated levels of FGF23 are not always linked to hyperphosphaturic hypophosphatemic disorders is surprising and implies that FGF23 is not the sole factor contributing to the pathogenesis of these disorders.

The mechanism by which FGF23 alters phosphate homeostasis is not entirely clear. In vivo studies demonstrated that NaPi IIa protein is reduced after chronic FGF23 administration, raising the possibility that similar to PTH, FGF23 may induce NaPi IIa internalization and degradation^125,138. It is assumed that FGF23 may elicit these and other changes through binding to a membrane-associated receptor such as one of the known FGF receptors (FGFRs). Consistent with the presence of a FGFR binding domain within the amino portion of FGF23 is the finding that FGF23 binds to known FGFRs in vitro [abstract; Pragnell M, J Am Soc Nephrol 12:600A, 2001]¹³⁶. However, in vivo validation will be required to convincingly demonstrate that FGF23 elicits its actions through a known FGFR. Binding and signaling by other FGFs in vivo is dependent not only on the presence of the appropriate receptor but the expression of specific proteoglycans on the cell surface¹⁴². In vitro systems may not adequately reflect the in vivo conditions with regard to proteoglycan and receptor distribution. Thus, differences observed in the ability of heparin to influence the activity of FGF23 within in vitro uptake assays may be due to differential proteoglycan expression of various opossum kidney cell clones used in the phosphate transport assay^{124,125,129,136}. However, the observation that a tyrosine phosphorylation event occurs in response to FGF23 is consistent with the possibility that FGF23 actions are elicited at least in part through a classic FGFR¹³⁶.

FRP4

Additional evidence suggests that at least in the case of TIO, FGF23 may not be the only phosphaturic protein involved in the disease pathogenesis. We have demonstrated that secreted FRP4, a second factor overexpressed in TIO tumors, also has phosphate-lowering activity^56,129. FRP4 is a posttranslationally modified protein that has a predicted molecular weight of 40,000 kD but migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a M_r of 48,000 kD due to glycosylation. It is a member of a protein family consisting of eight members that contain a cysteine-rich domain with high homology to the extracellular domain of membrane-bound Wnt receptors, the frizzled proteins^143,144. The Wnt proteins play an exceptionally important and prominent role in diverse biologic processes ranging from embryo development and cancer formation to neurodegenerative disease¹⁴⁵. There are numerous secreted Wnt ligands of which 19 have been identified in the human genome. These ligands transduce paracrine signals through ten or more membrane-bound receptors, the frizzled proteins. Some but not all FRPs have been shown to bind Wnts and antagonize their actions by serving as a soluble decoy^146,147. Interestingly, there appears to be interconnections between Wnt signaling pathways and other growth factor pathways such as Notch, bone morphogenetic proteins (BMPs), and FGFs^144,148,149. It is appealing to speculate that there may be direct link between the actions of FRP4 and FGF23.

FRP4 inhibits phosphate transport in the classic opossum kidney cell assay and is phosphaturic in vivo Figure 4a and c⁵⁶. Infusion of FRP4 into mice or rats caused increased phosphate excretion within 2 hours and decreased serum Pi within 8 hours of administration. 1,25(OH)₂D₃ serum concentrations were not altered despite FRP4-induced hypophosphatemia. FRP4 did not alter urinary cAMP levels but caused increased phosphorylation of renal -catenin levels, suggesting that this protein may function by opposing the actions of a Wnt. Finally, circulating levels of FRP4 were identified in normal individuals and a patient with TIO. Thus, the severe phosphaturia associated with TIO is likely due to overexpression of at least two proteins, FGF23 and FRP4. It remains to be determined whether FRP4 is also overexpressed in XLH and ADHR patients and contributes to the pathophysiology of these diseases.

MEPE

MEPE encodes a glycosylated protein of approximately 60 kD. Based on gene structure, MEPE appears to be a member of a growing family of extracellular matrix proteins called small integrin-binding ligand N-linked glycoproteins (SIBLINGs). Other family members include OPN, DMP1, and dentin sialoprotein (DSSP)¹⁵⁰. Although MEPE is clearly overexpressed in the tumors of patients with TIO, the data with respect to its role in this syndrome are unclear. Antisera from a patient harboring a TIO tumor blocked an apparent inhibition of phosphate uptake in proximal tubule cells induced by TIO-conditioned media¹²⁸. Although the antisera clearly recognizes MEPE, it is also possible that the sera contains antibodies to proteins such as FGF23 and/or FRP4 that may be present at undetectable but physiologically relevant levels in circulation. Recombinant mammalian-derived MEPE protein failed to inhibit phosphate transport in vitro¹²⁴ or to elicit a phosphaturic response in normal mice¹²⁵. In contrast, Rowe et al reported that MEPE generated in insect cells led to a significant decrease in serum phosphate levels and increased phosphate excretion when injected into mice [abstract; Rowe P, J Bone Miner Res 17:S211, 2002]. Furthermore, implantation of CHO-MEPE cells into nude mice also led to a reduction in serum phosphate and 1,25(OH)₂D₃ levels, and increased phosphate excretion. The reason for the discrepancy among these results is not clear but it is possible that various protein preparations contained different post-translation modifications. Importantly, MEPE knockout animals have increased bone density but do not have altered serum phosphate levels^151,152. This result suggests that MEPE's potential effect on phosphate homeostasis may be overcome by the more general actions of PTH and 1,25(OH)₂D₃ or alternatively may be secondary to the actions of another phosphatonin.

MEPE is most abundantly expressed in bone tissue^130,153. Argiro et al¹⁵³ demonstrated that MEPE expression in an osteoblast line, MC3T3-E1 is significantly induced in mineralizing cultures incubated with -glycerophosphate suggesting MEPE may have a role in mineralization. Furthermore, MEPE knockout animals have increased bone density¹⁵¹. Taken together, these results imply that the primary function of MEPE is to control bone mineralization rather than alter renal phosphate reabsorption.

ONE OR MORE "PHOSPHATONINS"

It is not possible to definitely designate one or all three of the proteins described above as the classic phosphatonin fulfilling all the criteria as originally defined⁷⁷. Each of the proteins has been found to inhibit uptake in vitro, promote phosphaturia in vivo, and are each associated with at least one of the hypophosphatemic, hyperphosphaturic disorders Table 3. FGF23 and FRP4 have been shown to inhibit the normal increase in 1,25(OH)₂D₃ concentrations typically induced by hypophosphatemia. Preliminary evidence also suggests that MEPE may also control 1,25(OH)₂D₃ concentrations. The case that FGF23 is the sole phosphatonin is dramatically weakened by the observation that elevated circulating FGF23 concentrations are not always correlated with the hypophosphatemic rickets/osteomalacia disorders. Furthermore, FGF23 is also elevated under conditions unrelated to these disorders such as humoral hypercalcemia¹⁴¹. Determination of potential circulating levels of MEPE and/or FRP4 in normal and diseased patients may ultimately resolve this paradox.

Table 3 - Comparison of phosphatonin candidate proteins:fibroblast growth factor 23 (FGF23), frizzled-related protein 4 (FRP4), and matrix extracellular phosphoglycoprotein (MEPE).

Full table

For the time being, it is reasonable to assume that all three proteins might play a role in the hypophosphatemic syndromes. However, the term, phosphatonin, may need to be redefined when a more complete understanding of the functional impact of each of these three proteins within temporal and spatial context of the whole organism is available. It is possible that all three proteins work synergistically or in a sequential manner to exert phosphatonin activities. Alternatively, each protein may elicit only a subset of the phosphatonin activities such that all three proteins contribute to the disease symptoms associated with overexpression of the phosphaturic proteins. Currently, various groups are working relatively independently to unravel the functional characteristics of phosphatonin candidates. A complete and accessible set of reagents for each of the three proteins and studies focused on the interactions of these proteins through physical and functional interactions would contribute greatly to the advancement of this field.

THE PHEX CONNECTION

An area of intense interest and debate is the relationship between PHEX and the phosphatonin activity. In 1995, PHEX was identified as the gene defective in XLH patients¹⁵⁴. PHEX was found to have significant homology to a family of endopeptidases that play prominent roles in either activation or inactivation of peptide factors through proteolytic modification. Although there is significant heterogeneity in the types of mutations associated with PHEX, each of the mutations appears to result in loss of functional PHEX at the osteoblast surface^155,156,157. To reconcile preexisting evidence that XLH patients and the murine counterpart, the hyp mouse, contain elevated levels of a circulating phosphaturic factor^{117,118,119,120,158}, the consortium proposed that the primary role of PHEX is to cleave and inactivate phosphatonin¹⁵⁴. The absence of functional PHEX would presumably lead to either inappropriately expressed or elevated levels of circulating phosphatonin. An alternative view is that one or more of the phosphatonins regulates the PHEX substrate(s).

Although degradation by PHEX has never been a requirement for classifying a factor as phosphatonin, it is clear that demonstrating a phosphaturic factor is inactivated when cleaved would make it a stronger candidate. Consistent with the premise that FGF23 is indeed a phosphatonin, crude in vitro proteolysis studies have demonstrated that wild-type FGF23 levels are reduced when coexpressed with PHEX in vitro¹²⁴. Although intact FGF23 is too large to be cleaved by PHEX, it was presumed that smaller FGF23-derived fragments were produced by enzymes present in the lysates. The inability of PHEX to cleave the nonprocessed mutant form of FGF23 suggested that one or both of the processed fragments may be the preferred substrate(s) for PHEX Figure 3. In a recent in vitro study, several small FGF23-derived peptides were cleaved by purified soluble PHEX, thereby identifying several potential PHEX cleavage sites on FGF23^159,160. There was no difference in the specificity of cleavage between the soluble or a membrane-associated PHEX. In a second study, a different FGF23-derived peptide was used as substrate and obvious cleaved fragments were not observed¹⁶¹. The fragment used in the study did include one of the predicted PHEX cleavage sites but the detection method may have been insufficient to differentiate between an intact versus PHEX cleaved product.

Based on these data, we and others have proposed a model that explains the relationship of FGF23 and PHEX in XLH, ADHR, and TIO^75,78,79,112 Figure 3. The model is based on the observations that intact FGF23 or ADHR mutant forms can elicit actions on phosphate transport and 1,25(OH)₂D₃ synthesis but the model also assumes that FGF23 processing is a prerequisite for cleavage and subsequent inactivation by PHEX. Thus in TIO, PHEX cannot efficiently cleave the elevated amounts of FGF23 produced by tumors. In the case of ADHR, mutations lead to increased activity and stability by preventing processing and downstream PHEX cleavage. Finally, in individuals with XLH, FGF23 concentrations remain elevated as PHEX is unavailable for regulation. The apparent role for FGF23 processing is paradoxic since biologic functions for amino and/or carboxy FGF23 fragments, the by-products of FGF23 processing, have not been defined. Identification of specific functions for these fragments coupled to demonstration that they are inactivated by PHEX cleavage will be necessary to definitely demonstrate that FGF23 is a direct target for PHEX.

While the debate remains regarding PHEX and FGF23, several lines of evidence suggest that there may be more than one PHEX substrate and that the pathophysiology of these disorders may not be explained by overexpression and/or increased activity of a single molecule. TIO tumors overproduce multiple proteins, including FGF23, FRP4, and MEPE. Although FGF23 is the only protein to date that has been linked to all three disorders, elevated levels or activity are not always correlated with hypophosphatemia. Hyp-derived osteoblasts transplanted into a normal mouse fail to develop into normal mineralized bone suggesting that the PHEX-deficient osteoblasts can not respond to normal circulating levels of peptide factors such as FGF23^163,164. Hyp-derived osteoblasts fail to mineralize normally in vitro¹⁶⁵, further suggesting that PHEX mutations result in an intrinsic bone defect. Consistent with alterations in mineralization potential is the observation that hyp-derived osteoblasts express reduced levels of multiple matrix-associated proteins important for the mineralization process¹⁶⁶. Conditioned media from hyp bone marrow cells produce a factor(s) that appears to impart mineralization defects in bones in a normal mouse¹³⁹ and can inhibit renal phosphate transport in vitro^167,168. It is not yet known if the same or multiple factors are responsible for the effects on osteoblast mineralization and renal Pi transport.

The data suggesting a role of MEPE in control of mineralization and the observation that MEPE mRNA is elevated in hyp-derived osteoblasts raise the possibility that MEPE may also be a PHEX substrate¹⁵³. Although in vitro cleavage studies indicate that intact MEPE is not a PHEX substrate^124,162, recent data suggest that PHEX may regulate the cleavage of MEPE by a cathepsin D–type processing enzyme¹⁶². In a separate study, purified PHEX cleaved small MEPE-derived peptides in vitro, but like FGF23, biologic functions have not yet been identified for MEPE fragments¹⁶⁰. Interestingly, transplantation of CHO lines coexpressing MEPE and PHEX do not induce hypophosphatemia, whereas CHO lines expressing only MEPE cause phosphaturia and hypophosphatemia in nude mice [abstract; Rowe P, J Bone Miner Res 17:S211, 2002]. These studies suggest that there is indeed a relationship between MEPE and PHEX but it does not address whether PHEX controls MEPE activity or the activity of a protein regulated by MEPE.

PHOSPHATONINS AND NORMAL PHYSIOLOGY

Do phosphatonins contribute to the overall balance of phosphate traditionally considered to be regulated by 1,25(OH)₂D and PTH? The current data provide only hints of the importance of this pathway. One would expect a phosphate-regulating hormone to circulate in detectable amounts. Unlike the majority of FGF family members, detectable levels of FGF23 are present in normal individuals at circulating levels of less than 50 pg/mL. A second line of evidence that FGF23 might play a role in normal physiology is the potential regulation of FGF23 expression by changes in serum phosphate levels. A significant increase in intact FGF23 was observed in rats fed a high phosphate diet whereas a decrease in levels was observed after a low phosphate diet [abstract; Yamashita T, J Am Soc Nephrol 13:577A, 2002]. Small, yet statistically significant, changes in serum FGF23 concentrations were observed using the ELISA against the carboxy fragment in a group of normal humans fed a diet of escalating phosphate concentrations [abstract; Allen HC, J Bone Miner Res 17:S279, 2002]. These results are consistent with the possibility that FGF23 action is controlled by phosphate. The finding that targeted ablation of FGF23 in mice leads to hyperphosphatemia is surprising since the actions of PTH would be expected to normalize phosphate levels [abstract; Shimada T, J Bone Miner Res 17:S1159, 2002]. These results suggest that FGF23 may play a general role in phosphate homeostasis. By extension, additional proteins within the phosphatonin pathway may also be important in normal physiological control of serum Pi.

FRP4 has been shown to also be a circulating protein but Pi changes in its level have not yet been analyzed in patients with dysregulated phosphate. An understanding of how each of the phosphatonin pathway components interact with each other, as well as PTH and 1,25(OH)₂D₃, will provide a clearer understanding of how the body controls phosphate.

THE PHOSPHATONIN PATHWAY IN CHRONIC KIDNEY DISEASE

Finally, it is relevant to consider the contribution of the phosphatonin pathway in chronic kidney disease. It is well recognized that the progression of chronic kidney disease results in decreased circulating levels of 1,25(OH)₂D₃ followed by increased serum concentrations of PTH and ultimately elevated levels of serum phosphate¹⁶⁹. FGF23 is also elevated in individuals with end-stage renal disease¹³⁹. This result is consistent with potential regulation of FGF23 by elevated Pi associated with late stages of chronic kidney disease. It is possible, that like PTH, FGF23 is induced as part of a compensatory mechanism that is induced with declining renal function. Alternatively, FGF23 increases may reflect decreasing clearance by the damaged kidney. Additional investigation will be required to determine whether the pathophysiology of chronic kidney disease results in dysregulation of FGF23 or FGF23 plays a causal role in chronic kidney disease.

CONCLUSION

The growing evidence that several molecules are associated with hypophosphatemia in individuals with XLH, ADHR, and TIO and evidence for distinct phosphate-regulating factors associated with other disorders⁷⁵ suggest that phosphatonins represent a new class of secreted factors that control phosphate homeostasis. These proteins may contribute to global regulation of serum phosphate concentrations by directly inhibiting renal phosphate transport or indirectly through the control of bone mineralization. The need to maintain average levels of phosphate between 2.5 to 4.5 mg/dL is clearly exemplified by the deleterious effects of sustained hypophosphatemia or hyperphosphatemia. Of equal importance is the need to have localized control of phosphate. Pi is known to specifically regulate cell changes such as gene expression and apoptosis^170,171. The existence of multiple phosphatonins, such as FGF23, FRP4, and MEPE, that have distinct and overlapping functions provides the flexibility to maintain a relative balance in serum phosphate while fine-tuning local phosphate concentrations to meet the needs at a specific site.

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Acknowledgments

This work was supported by NIH grants DK25409, DK58546, DK59505 to R.K.