Despite apparent progress in understanding the pathophysiological basis for XLH, few advances have been made in defining an effective treatment for the disease. This failure is related to a unique block to treatments based on genetic complementation in this disorder. Classic complementation therapy has been precluded in this single-gene (PHEX/Phex
) defect disease by the inability to rescue the phenotype by over-expression of PHEX(24–26)
-mice and the failure to define a PHEX substrate.(21)
However, discovery that a major PHEX-dependent abnormality in XLH is increased circulating FGF-23, which is strongly associated with the genesis of the HYP
phenotype, appeared to offer a realistic alternative strategy for treatment via blockade of FGF-23 effects. Unfortunately, this approach has been severely limited by the incomplete response of the disease phenotype to antibody neutralization.(27, 28)
Alternative approaches to modifying FGF-23 effects for a therapeutic purpose requires better understanding the mechanisms underlying the decreased degradation and increased production of this phosphatonin in health and in XLH. In the current studies, we used a variety of traditional biochemical and molecular biological techniques to explore the regulation of FGF-23 degradation and production both in vitro and in vivo. We initially demonstrated that general inhibition of proprotein convertase activity in normal mice increased circulating levels of FGF-23, decreased serum phosphorus, and induced post-transcriptional inhibition of 25(OH)D-1α-hydroxylase activity, changes that recapitulate the cardinal biochemical features of the HYP phenotype. Upon ascertaining that PC2 was a likely candidate covertase contributing to regulating FGF-23, we investigated whether this enzyme can alter FGF-23 degradation and production in vitro.
Proprotein convertases are responsible for the tissue-specific processing of multiple polypeptide precursors in a wide variety of tissues and cell lines.(38, 44, 52, 53)
However, PC2 differs from other proprotein convertases in its specific requirement for a chaperone protein, 7B2. Numerous studies have shown that PC2 maturation and enzyme activity are uniquely regulated by 7B2 and PC2 expressed in the absence of 7B2 is incapable of autoactivation or substrate cleavage.(46, 54–58)
In accord with these observations, we showed that transfection of TMob cells with Sgne1
(7B2) and Pcsk2
(PC2) results in FGF-23 cleavage, while expression of Sgne1
alone has no effect. Moreover, FGF-23 degradation is blocked in TMob cells by Sgne1
RNA-induced knockdown of Sgne1
expression. These findings are consistent with the known presence of a convertase cleavage site in FGF-23 between amino acids 179 and 180 in the Arg-Arg-His-Thr-Arg motif, which in ADHR limits FGF-23 proteolysis and increases serum FGF-23.(8–10)
While many previous studies have shown that furin can mediate cleavage at this site(29, 59, 60)
and PC2 is capable of cleaving either at the Arg-Arg site, or at single arginines in the presence of an upstream arginine,(61)
our investigations indicate that PC2 may play a larger role than furin in this cleavage event in bone.
Subsequent investigations revealed that 7B2•PC2 activity regulates not only FGF-23 degradation, but FGF-23 production as well. Thus, transfection of TMob cells with a Sgne1
(7B2) expression vector significantly decreased Fgf-23
mRNA, whereas an increase in Fgf-23
mRNA was detected upon Sgne1
(7B2) RNAi inhibition of mRNA and protein expression. Further experiments linked the effects of decreased 7B2•PC2 activity on FGF-23 production to reduced proteolysis of proBMP1, which resulted in limited cleavage of DMP1 to its 37-kDa N- and 57-kDa C-terminal fragments. The apparent impact of DMP1 on FGF-23 supports an important role for DMP1 proteolysis in the regulation of Fgf-23
mRNA expression. However, while the unique effects of BMP1 on DMP1 proteolysis were known,(47)
the apparent role of PC2 on proBMP1 cleavage was somewhat unexpected, since Golgi-localized furin represents the dominant convertase responsible for this cleavage event in the only cell type tested thus far, HT1080 cells.(47)
However, the Arg-Ser-Arg-Arg site cleaved within proBMP1 is a PC2 recognition cleavage site,(61)
providing justification for further work to establish the contribution of PC2 to proBMP1 cleavage in bone cells and tissue.
It is important to note that BMP1 is not the only enzyme that cleaves DMP1. Recent studies62
have documented that matrix metalloproteinase-2 (MMP2), a mebrane bound enzyme, also cleaves the intact DMP1. However, the cleavage products include a 42-kDa carboxy-terminal fragment, which appears to have biological effects in dental pulp. The absence of the DMP1 42-kDa carboxy-terminal fragment in the normal and hyp
-mouse bone, or the TMob cells, indicates that the 7B2-PC2 mediated cleavage of DMP1 is dependent on generation of active BMP1.
While these data clearly establish that decreased 7B2•PC2 activity is associated with reduced FGF-23 degradation and increased hormone production in vitro
, the role that such altered enzyme function might play in XLH remained uncertain. Therefore, we examined whether defective expression of either 7B2 or PC2 occurred in hyp
-mice. Bone obtained from the mutant mice exhibited significantly decreased expression of both Sgne1
(7B2) mRNA and 7B2 protein. These decreased levels of 7B2 limit cleavage not only of FGF-23, but of proPC2 as well, as evidenced by the presence of an increased ratio of proPC2 to active PC2. These observations are consistent with previous reports in the Sgne1
and in cell culture,(49, 56)
and support the concept that reduction in 7B2 levels results in decreased bone PC2 activity in XLH.
The possibility that a primary defect in hyp
-mice is reduced 7B2 was confirmed by further studies that revealed expected downstream effects of decreased 7B2•PC2 activity. As previously found,(22, 23)
-mice exhibit significantly increased levels of serum FGF-23, due to both decreased FGF-23 degradation and increased FGF-23 production. Consistent with our in vitro
studies, increased FGF-23 production in hyp
-mouse bone was associated with decreased BMP1-mediated degradation of DMP1 and decreased genesis of the C-terminal DMP1 fragment. While the increase in Fgf-23
mRNA may not result directly from the decreased concentrations of the DMP1 carboxy-terminal fragment, our in vitro
studies and the recent reports that establish transgenic overexpression of the C-terminal DMP1 fragment is essential to rescue the Dmp1
null phenotype and normalize serum FGF-23,(36–38)
support DMP1 proteolysis-mediated regulation of FGF-23 production in the hyp
-mouse. In summary, these results establish that diminished 7B2 production in bone represents a critical step in the pathogenesis of the HYP
phenotype (Figure S3
), although the mechanism(s) by which the Phex
mutation causes downregulation of 7B2 expression in hyp
-mice remains unknown.
While the well-known functional link between 7B2 and PC2 implies that their expression is coordinately regulated, the data obtained in the hyp
-mouse model support the idea of discordant regulation. Indeed, such discordant regulation has been previously reported. Thus, while stimulation of P19 neuronal cells via a protein kinase C-mediated pathway coordinately increases expression of both 7B2 and PC2, protein kinase A-mediated stimulation increases expression of PC2 alone.(61).
Moreover, a growing body of evidence suggests 7B2 expression frequently controls PC2 activity in hormonal peptide production.(63)
The molecular details of the control of 7B2 expression are only now beginning to emerge, with recent studies pointing to promoter polymorphisms which clearly control expression,(58, 64)
i.e., a 5’ regulatory region in Sgne1
(7B2) mRNA, which acts to repress expression(64)
and epigenetic control by differential methylation of the Sgne1
The relative contributions of these various regulatory mechanisms to the control of 7B2 expression in bone is not yet known.
While our in vivo observations support the possibility that 7B2–dependent, PC2-mediated alterations in FGF-23 degradation and production are operative in the hyp mouse osteoblast, the singular role of this abnormality in the genesis of the HYP phenotype was still unclear. To explore this issue further, we sought to pharmacologically normalize PC2 activity in order to increase FGF-23 degradation and decrease production. The effects observed in D6R-treated hyp-mice were indeed remarkable. As anticipated, treatment resulted in a significant increase in the bone Sgne1 (7B2) mRNA and 7B2 protein and no doubt concordant enhancement of 7B2•PC2 enzyme, which resulted in apparent normalization of the HYP phenotype. Indeed, during the five week period of treatment, serum phosphorus levels increased progressively to values maintained from days 21–35 that were no different than those of normal mice. These changes were clearly secondary to a significant increase in the renal Npt2a mRNA and NPT2a protein. Likewise, administration of D6R corrected the abnormal translational regulation of renal 25-hydroxyvitamin D-1α-hydroxylase activity, characteristic of the hyp-mouse.
Further, D6R treatment not only normalized the biochemical abnormalities, but had profound effects on bone architecture, remodeling and mineralization. Thus, bone length was returned to normal in the hyp
mice, as was bone modeling and mineralization. These changes resulted in long bones with normal width and size of the growth plates and diaphyses. Moreover, restoration of mineralization dynamics included establishing a normal mineral apposition rate and eliminating all evidence of excess unmineralized osteoid. Complete resolution of the rickets and osteomalacia in response to D6R treatment is in marked contrast to the effects of alternative therapeutic regimens. Previous treatment strategies for XLH in affected humans and mice, have included vitamin D and phosphorus,(68)
calcitriol and phosphorus,(69)
antibody neutralization of Fgf-23 (28), and PHEX over-expression,(24–26)
all of which invariably remarkably improve the biochemical phenotype, but uniformly fail to heal the bone disease, particularly the osteomalacia. Thus, treatment with D6R influences bone mineralization in an unprecedented fashion.
The effects noted above were those anticipated with the D6R-mediated increase in bone Sgne1 mRNA and protein, and consequent enhanced 7B2•PC2 enzyme activity. Although the molecular mechanism by which D6R increases Sgne1 message levels in bone cells remains unknown, the expected result of such enhanced activity is restoration of normal FGF-23 production and degradation, and accordingly reduction of the stimulus for the abnormal biochemistries and bone morphology and histology present in hyp-mice. Indeed, in response to D6R treatment we observed decreased transcription of Fgf-23 mRNA, along with the predicted enhanced BMP1 activation and elevated proteolytic degradation of DMP1. However, the serum FGF-23 level in the D6R-treated hyp-mice remained elevated when measured using the intact and carboxy-terminal immunoassays. Nevertheless, comparison of the measurements by these techniques revealed a greater decrement when assessed using the intact immunoassay, suggesting that degradation of the intact FGF-23 was enhanced and perhaps normalized.
The explanation for this incongruous elevation of the serum FGF-23 concentration is not immediately apparent. Measurement of serum FGF-23 at multiple dilutions in D6R-treated hyp
-mice produced results that diluted parallel to the standard curve, eliminating co-measurement of an unrelated factor. It is also unlikely that the bioactivity of the circulating FGF-23 is decreased by a limited interaction of the hormone with its receptor, FGF1R, due to either a decrease in the renal klotho protein, a key component of the binary complex that creates FGF1R,(70)
or competitive inhibition of the intact FGF-23 binding to its receptor by the C-terminal FGF-23, consistent with recently reported findings.(71)
It is possible that structural differences in the circulating FGF-23 in treated hyp
-mice may influence measurement of the intact molecule, consistent with the extra-osseous clearance of intact FGF-23, about which little is known. No data are available that indicate such degradation of circulating FGF-23 generates the classic N- and C-terminal fragments. With the absence of any evidence of increased bioactivity at the kidney or bone, it seems plausible that extra-osseous degradation of remarkably elevated serum levels of intact FGF-23 may generate abundant amounts of a bio-inactive FGF-23 fragment, which includes the antigenic epitope(s) recognized by the antibodies used in the ELISA measurement. Since the antigenic epitopes are at the N-terminal and C-terminal cleavage sites, the bioinactive fragment may result from cleavage of the intact FGF-23 at a site(s) distal to the normal N- and C-terminal cleavage site, rendering the molecule bioinactive but preserving the antigenic epitopes. Additional studies, outside the scope of the present investigation, will be necessary to establish with certainty if the elevated serum FGF-23 level, in association with normal bioactivity at kidney and bone, represents interference of hormone binding to its receptor, circulation of a bio-inactive fragment of FGF-23, detected by the ELISA assay, or another event.
Regardless, our studies indicate that the loss-of-function Phex mutation in hyp-mice results in decreased osseous 7B2 production, a protein essential for manifestation of PC2 activity. The loss of 7B2 results in diminished PC2 activity and significantly limits cleavage of FGF-23 and proBMP1 and indirectly, of DMP1. The physiological consequences that ultimately result from the 7B2 defect are increased levels of circulating FGF-23 and consequent renal phosphate wasting, abnormal transcriptional regulation of renal 25-hydroxyvitamin D-1α-hydroxylase activity, and rickets/osteomalacia. Most importantly, we found that the polyarginine D6R apparently restores the activity of PC2, most likely by increasing 7B2 production. D6R thus normalizes the HYP phenotype, correcting phosphate and vitamin D homeostasis, as well as bone modeling and mineralization. While much work remains to establish the cellular and molecular mechanisms for PHEX-7B2 interactions, the findings presented here provide novel insight into the biosynthetic mechanisms of FGF-23 synthesis, and, most importantly, create a biochemical basis for a fully curative drug treatment regimen for XLH.