Anti‐FGF‐23 neutralizing antibodies ameliorate muscle weakness and decreased spontaneous movement of Hyp mice

Fibroblast growth factor 23 (FGF‐23) plays causative roles in the development of several hypophosphatemic rickets/osteomalacia such as X‐linked hypophosphatemic rickets/osteomalacia (XLH) and tumor‐induced rickets/osteomalacia. Patients with hypophosphatemic rickets/osteomalacia often complain of muscle weakness and bone pain that severely affect daily activities of these patients. The purpose of this study was to examine whether anti‐FGF‐23 antibodies, which have been shown to improve hypophosphatemia and rachitic changes of juvenile Hyp mice in a murine model of XLH, also ameliorate hypophosphatemic osteomalacia and affect muscle force and spontaneous motor activity in adult Hyp mice. Repeated injections of anti‐FGF‐23 antibodies increased serum phosphate and 1,25‐dihydroxyvitmain D levels and enhanced mineralization of osteoid in adult Hyp mice, whereas bone length did not change. We found that grip strength was weaker and that spontaneous movement was less in adult Hyp mice than in wild‐type mice. In addition, FGF‐23 antibodies increased grip strength and spontaneous movement. These results suggest that the inhibition of excess FGF‐23 action not only ameliorates hypophosphatemia and impaired mineralization of bone but also improves muscle weakness and daily activities of patients with FGF‐23‐related hypophosphatemic rickets/osteomalacia. © 2011 American Society for Bone and Mineral Research.     

In Vivo Overexpression of Tissue‐Nonspecific Alkaline Phosphatase Increases Skeletal Mineralization and Affects the Phosphorylation Status of Osteopontin

Functional ablation of tissue‐nonspecific alkaline phosphatase (TNAP) (Alpl^?/? mice) leads to hypophosphatasia, characterized by rickets/osteomalacia attributable to elevated levels of extracellular inorganic pyrophosphate, a potent mineralization inhibitor. Osteopontin (OPN) is also elevated in the plasma and skeleton of Alpl^?/? mice. Phosphorylated OPN is known to inhibit mineralization, however, the phosphorylation status of the increased OPN found in Alpl^?/? mice is unknown. Here, we generated a transgenic mouse line expressing human TNAP under control of an osteoblast‐specific Col1a1 promoter (Col1a1‐Tnap). The transgene is expressed in osteoblasts, periosteum, and cortical bones, and plasma levels of TNAP in mice expressing Col1a1‐Tnap are 10 to 20 times higher than those of wild‐type mice. The Col1a1‐Tnap animals are healthy and exhibit increased bone mineralization by micro–computed tomography (µCT) analysis. Crossbreeding of Col1a1‐Tnap transgenic mice to Alpl^?/? mice rescues the lethal hypophosphatasia phenotype characteristic of this disease model. Osteoblasts from [Col1a1‐Tnap] mice mineralize better than nontransgenic controls and osteoblasts from [Col1a1‐Tnap^+/?; Alpl^?/?] mice are able to mineralize to the level of Alpl^+/? heterozygous osteoblasts, whereas Alpl^?/? osteoblasts show no mineralization. We found that the increased levels of OPN in bone tissue of Alpl^?/? mice are comprised of phosphorylated forms of OPN whereas wild‐type (WT) and [Col1a1‐Tnap^+/?; Alpl^?/?] mice had both phosphorylated and dephosphorylated forms of OPN. OPN from [Col1a1‐Tnap] osteoblasts were more dephosphorylated than nontransgenic control cells. Titanium dioxide‐liquid chromatography and tandem mass spectrometry analysis revealed that OPN peptides derived from Alpl^?/? bone and osteoblasts yielded a higher proportion of phosphorylated peptides than samples from WT mice, and at least two phosphopeptides, p(S¹⁷⁴FQVS¹⁷⁸DEQY¹⁸²PDAT¹⁸⁶DEDLT¹⁹¹)SHMK and FRIp(S²⁹⁹HELES³⁰⁴S³⁰⁵S³⁰⁶S³⁰⁷)EVN, with one nonlocalized site each, appear to be preferred sites of TNAP action on OPN. Our data suggest that the promineralization role of TNAP may be related not only to its accepted pyrophosphatase activity but also to its ability to modify the phosphorylation status of OPN.     

Conditional Deletion of Murine Fgf23: Interruption of the Normal Skeletal Responses to Phosphate Challenge and Rescue of Genetic Hypophosphatemia

The transgenic and knockout (KO) animals involving Fgf23 have been highly informative in defining novel aspects of mineral metabolism, but are limited by shortened lifespan, inability of spatial/temporal FGF23 control, and infertility of the global KO. To more finely test the role of systemic and genetic influences in FGF23 production, a mouse was developed that carried a floxed (“f”)‐Fgf23 allele (exon 2 floxed) which demonstrated in vivo recombination when bred to global‐Cre transgenic mice (eIIa‐cre). Mice homozygous for the recombined allele (“Δ”) had undetectable serum intact FGF23, elevated serum phosphate (p < 0.05), and increased kidney Cyp27b1 mRNA (p < 0.05), similar to global Fgf23‐KO mice. To isolate cellular FGF23 responses during phosphate challenge, Fgf23^Δ/f mice were mated with early osteoblast type Iα1 collagen 2.3‐kb promoter‐cre mice (Col2.3‐cre) and the late osteoblast/early osteocyte Dentin matrix protein‐1‐cre (Dmp1‐cre). Fgf23^Δ/f/Col2.3‐cre⁺ and Fgf23^Δ/f/Dmp1‐cre⁺ exhibited reduced baseline serum intact FGF23 versus controls. After challenge with high‐phosphate diet Cre^– mice had 2.1‐fold to 2.5‐fold increased serum FGF23 (p < 0.01), but Col2.3‐cre⁺ mice had no significant increase, and Dmp1‐cre⁺ mice had only a 37% increase (p < 0.01) despite prevailing hyperphosphatemia in both models. The Fgf23^Δ/f/Col2.3‐cre was bred onto the Hyp (murine X‐linked hypophosphatemia [XLH] model) genetic background to test the contribution of osteoblasts and osteocytes to elevated FGF23 and Hyp disease phenotypes. Whereas Hyp mice maintained inappropriately elevated FGF23 considering their marked hypophosphatemia, Hyp/Fgf23^Δ/f/Col2.3‐cre⁺ mice had serum FGF23 <4% of Hyp (p < 0.01), and this targeted restriction normalized serum phosphorus and ricketic bone disease. In summary, deleting FGF23 within early osteoblasts and osteocytes demonstrated that both cell types contribute to baseline circulating FGF23 concentrations, and that targeting osteoblasts/osteocytes for FGF23 production can modify systemic responses to changes in serum phosphate concentrations and rescue the Hyp genetic syndrome. © 2016 American Society for Bone and Mineral Research.     

Genetic Ablation of Osteopontin in Osteomalacic Hyp Mice Partially Rescues the Deficient Mineralization Without Correcting Hypophosphatemia

PHEX is predominantly expressed by bone and tooth-forming cells, and its inactivating mutations in X-linked hypophosphatemia (XLH) lead to renal phosphate wasting and severe hypomineralization of bones and teeth. Also present in XLH are hallmark hypomineralized periosteocytic lesions (POLs, halos) that persist despite stable correction of serum phosphate (P_i) that improves bulk bone mineralization. In XLH, mineralization-inhibiting osteopontin (OPN, a substrate for PHEX) accumulates in the extracellular matrix of bone. To investigate how OPN functions in Hyp mice (a model for XLH), double-null (Hyp;Opn^−/−) mice were generated. Undecalcified histomorphometry performed on lumbar vertebrae revealed that Hyp;Opn^−/− mice had significantly reduced osteoid area/bone area (OV/BV) and osteoid thickness of trabecular bone as compared to Hyp mice, despite being as hypophosphatemic as Hyp littermate controls. However, tibias examined by synchrotron radiation micro-CT showed that mineral lacunar volumes remained abnormally enlarged in these double-null mice. When Hyp;Opn^−/− mice were fed a high-P_i diet, serum P_i concentration increased, and OV/BV and osteoid thickness normalized, yet mineral lacunar area remained abnormally enlarged. Enpp1 and Ankh gene expression were increased in double-null mice fed a high-P_i diet, potentially indicating a role for elevated inhibitory pyrophosphate (PP_i) in the absence of OPN. To further investigate the persistence of POLs in Hyp mice despite stable correction of serum P_i, immunohistochemistry for OPN on Hyp mice fed a high-P_i diet showed elevated OPN in the osteocyte pericellular lacunar matrix as compared to Hyp mice fed a control diet. This suggests that POLs persisting in Hyp mice despite correction of serum P_i may be attributable to the well-known upregulation of mineralization-inhibiting OPN by P_i, and its accumulation in the osteocyte pericellular matrix. This study shows that OPN contributes to osteomalacia in Hyp mice, and that genetic ablation of OPN in Hyp mice improves the mineralization phenotype independent of systemic P_i-regulating factors. © 2020 American Society for Bone and Mineral Research.     

Ablation of Osteopontin Improves the Skeletal Phenotype of Phospho1−/− Mice

PHOSPHO1 and tissue‐nonspecific alkaline phosphatase (TNAP) have nonredundant functions during skeletal mineralization. Although TNAP deficiency (Alpl^?/? mice) leads to hypophosphatasia, caused by accumulation of the mineralization inhibitor inorganic pyrophosphate (PP_i), comparably elevated levels of PP_i in Phospho1^?/? mice do not explain their stunted growth, spontaneous fractures, bowed long bones, osteomalacia, and scoliosis. We have previously shown that elevated PP_i in Alpl^?/? mice is accompanied by elevated osteopontin (OPN), another potent mineralization inhibitor, and that the amount of OPN correlates with the severity of hypophosphatasia in mice. Here we demonstrate that plasma OPN is elevated and OPN expression is upregulated in the skeleton, particularly in the vertebrae, of Phospho1^?/? mice. Liquid chromatography/tandem mass spectrometry showed an increased proportion of phosphorylated OPN (p‐OPN) peptides in Phospho1^?/? mice, suggesting that accumulation of p‐OPN causes the skeletal abnormalities in Phospho1^?/? mice. We also show that ablation of the OPN gene, Spp1, leads to improvements in the skeletal phenotype in Phospho1^?/? as they age. In particular, their scoliosis is ameliorated at 1 month of age and is completely rescued at 3 months of age. There is also improvement in the long bone defects characteristic of Phospho1^?/? mice at 3 months of age. Mineralization assays comparing [Phospho1^?/?; Spp1^?/?], Phospho1^?/?, and Spp1^?/? chondrocytes display corrected mineralization by the double knockout cells. Expression of chondrocyte differentiation markers was also normalized in the [Phospho1^?/?; Spp1^?/?] mice. Thus, although Alpl and Phospho1 deficiencies lead to similar skeletal phenotypes and comparable changes in the expression levels of PP_i and OPN, there is a clear dissociation in the hierarchical roles of these potent inhibitors of mineralization, with elevated PP_i and elevated p‐OPN levels causing the respective skeletal phenotypes in Alpl^?/? and Phospho1^?/? mice. © 2014 American Society for Bone and Mineral Research.     

Tooth root dentin mineralization defects in a mouse model of hypophosphatasia

Tissue‐nonspecific alkaline phosphatase (TNAP) is expressed in mineralizing tissues and functions to reduce pyrophosphate (PP_i), a potent inhibitor of mineralization. Loss of TNAP function causes hypophosphatasia (HPP), a heritable disorder marked by increased PP_i, resulting in rickets and osteomalacia. Tooth root cementum defects are well described in both HPP patients and in Alpl^?/? mice, a model for infantile HPP. In Alpl^?/? mice, dentin mineralization is specifically delayed in the root; however, reports from human HPP patients are variable and inconsistent regarding dentin defects. In the current study, we aimed to define the molecular basis for changes in dentinogenesis observed in Alpl^?/? mice. TNAP was found to be highly expressed by mature odontoblasts, and Alpl^?/? molar and incisor roots featured defective dentin mineralization, ranging from a mild delay to severely disturbed root dentinogenesis. Lack of mantle dentin mineralization was associated with disordered and dysmorphic odontoblasts having disrupted expression of marker genes osteocalcin and dentin sialophosphoprotein. The formation of, initiation of mineralization within, and rupture of matrix vesicles in Alpl^?/? dentin matrix was not affected. Osteopontin (OPN), an inhibitor of mineralization that contributes to the skeletal pathology in Alpl^?/? mice, was present in the generally unmineralized Alpl^?/? mantle dentin at ruptured mineralizing matrix vesicles, as detected by immunohistochemistry and by immunogold labeling. However, ablating the OPN‐encoding Spp1 gene in Alpl^?/? mice was insufficient to rescue the dentin mineralization defect. Administration of bioengineered mineral‐targeting human TNAP (ENB‐0040) to Alpl^?/? mice corrected defective dentin mineralization in the molar roots. These studies reveal that TNAP participates in root dentin formation and confirm that reduction of PP_i during dentinogenesis is necessary for odontoblast differentiation, dentin matrix secretion, and mineralization. Furthermore, these results elucidate developmental mechanisms underlying dentin pathology in HPP patients, and begin to explain the reported variability in the dentin/pulp complex pathology in these patients. © 2013 American Society for Bone and Mineral Research     

Klotho Lacks an FGF23‐Independent Role in Mineral Homeostasis

Fibroblast growth factor‐23 (FGF23) is a bone‐derived hormone regulating vitamin D hormone production and renal handling of minerals by signaling through an FGF receptor/αKlotho (Klotho) receptor complex. Whether Klotho has FGF23‐independent effects on mineral homeostasis is a controversial issue. Here, we aimed to shed more light on this controversy by comparing male and female triple knockout mice with simultaneous deficiency in Fgf23 and Klotho and a nonfunctioning vitamin D receptor (VDR) (Fgf23/Klotho/VDR) with double (Fgf23/VDR, Klotho/VDR, and Fgf23/Klotho) and single Fgf23, Klotho, and VDR mutants. As expected, 4‐week‐old Fgf23, Klotho, and Fgf23/Klotho knockout mice were hypercalcemic and hyperphosphatemic, whereas VDR, Fgf23/VDR, and Klotho/VDR mice on rescue diet were normocalcemic and normophosphatemic. Serum levels of calcium, phosphate, and sodium did not differ between 4‐week‐old triple Fgf23/Klotho/VDR and double Fgf23/VDR or Klotho/VDR knockout mice. Notably, 3‐month‐old Fgf23/Klotho/VDR triple knockout mice were indistinguishable from double Fgf23/VDR and Klotho/VDR compound mutants in terms of serum calcium, serum phosphate, serum sodium, and serum PTH, as well as urinary calcium and sodium excretion. Protein expression analysis revealed increased membrane abundance of sodium‐phosphate co‐transporter 2a (NaPi‐2a), and decreased expression of sodium‐chloride co‐transporter (NCC) and transient receptor potential cation channel subfamily V member 5 (TRPV5) in Fgf23/Klotho/VDR, Fgf23/VDR, and Klotho/VDR mice, relative to wild‐type and VDR mice, but no differences between triple and double knockouts. Further, ex vivo treatment of live kidney slices isolated from wild‐type and Klotho/VDR mice with soluble Klotho did not induce changes in intracellular phosphate, calcium or sodium accumulation assessed by two‐photon microscopy. In conclusion, our data suggest that the main physiological function of Klotho for mineral homeostasis in vivo is its role as co‐receptor mediating Fgf23 action. © 2017 American Society for Bone and Mineral Research.     

Targeted Disruption of NF1 in Osteocytes Increases FGF23 and Osteoid With Osteomalacia‐like Bone Phenotype

Neurofibromatosis type 1 (NF1, OMIM 162200), caused by NF1 gene mutations, exhibits multi‐system abnormalities, including skeletal deformities in humans. Osteocytes play critical roles in controlling bone modeling and remodeling. However, the role of neurofibromin, the protein product of the NF1 gene, in osteocytes is largely unknown. This study investigated the role of neurofibromin in osteocytes by disrupting Nf1 under the Dmp1‐promoter. The conditional knockout (Nf1 cKO) mice displayed serum profile of a metabolic bone disorder with an osteomalacia‐like bone phenotype. Serum FGF23 levels were 4 times increased in cKO mice compared with age‐matched controls. In addition, calcium‐phosphorus metabolism was significantly altered (calcium reduced; phosphorus reduced; parathyroid hormone [PTH] increased; 1,25(OH)₂D decreased). Bone histomorphometry showed dramatically increased osteoid parameters, including osteoid volume, surface, and thickness. Dynamic bone histomorphometry revealed reduced bone formation rate and mineral apposition rate in the cKO mice. TRAP staining showed a reduced osteoclast number. Micro‐CT demonstrated thinner and porous cortical bones in the cKO mice, in which osteocyte dendrites were disorganized as assessed by electron microscopy. Interestingly, the cKO mice exhibited spontaneous fractures in long bones, as found in NF1 patients. Mechanical testing of femora revealed significantly reduced maximum force and stiffness. Immunohistochemistry showed significantly increased FGF23 protein in the cKO bones. Moreover, primary osteocytes from cKO femora showed about eightfold increase in FGF23 mRNA levels compared with control cells. The upregulation of FGF23 was specifically and significantly inhibited by PI3K inhibitor Ly294002, indicating upregulation of FGF23 through PI3K in Nf1‐deficient osteocytes. Taken together, these results indicate that Nf1 deficiency in osteocytes dramatically increases FGF23 production and causes a mineralization defect (ie, hyperosteoidosis) via the alteration of calcium‐phosphorus metabolism. This study demonstrates critical roles of neurofibromin in osteocytes for osteoid mineralization. © 2017 American Society for Bone and Mineral Research.     

Unique roles of phosphorus in endochondral bone formation and osteocyte maturation

The mechanisms by which inorganic phosphate (P_i) homeostasis controls bone biology are poorly understood. Here we used Dmp1 null mice, a hypophosphatemic rickets/osteomalacia model, combined with a metatarsal organ culture and an application of neutralizing fibroblast growth factor 23 (FGF‐23) antibodies to gain insight into the roles of P_i in bone biology. We showed (1) that abnormal bone remodeling in Dmp1 null mice is due to reduced osteoclast number, which is secondary to a reduced ratio of RANKL/OPG expressed by osteoclast supporting cells and (2) that osteoblast extracellular matrix mineralization, growth plate maturation, secondary ossification center formation, and osteoblast differentiation are phosphate‐dependent. Finally, a working hypothesis is proposed to explain how phosphate and DMP1 control osteocyte maturation. © 2011 American Society for Bone and Mineral Research.     

Effects of Extracellular Phosphate on Gene Expression in Murine Osteoblasts

That phosphate homeostasis is tightly linked to skeletal mineralization is probably best underscored by the fact that the phosphaturic hormone FGF23 is primarily expressed by terminally differentiated osteoblasts/osteocytes and that increased circulating FGF23 levels are causative for different types of hypophosphatemic rickets. In contrast, FGF23 inactivation results in hyperphosphatemia, and unexpectedly this phenotype is associated with severe osteomalacia in Fgf23-deficient mice. In this context it is interesting that different cell types have been shown to respond to extracellular phosphate, thereby raising the concept that phosphate can act as a signaling molecule. To identify phosphate-responsive genes in primary murine osteoblasts we performed genome wide expression analysis with cells maintained in medium containing either 1 or 4 mM sodium phosphate for 6 h. As confirmed by qRT-PCR, this analysis revealed that several known osteoblast differentiation markers (Bglap, Ibsp, and Phex) were unaffected by raising extracellular phosphate levels. In contrast, we found that the expression of Enpp1 and Ank, two genes encoding inhibitors of matrix mineralization, was induced by extracellular phosphate, while the expression of Sost and Dkk1, two genes encoding inhibitors of bone formation, was negatively regulated. The ability of osteoblasts to respond to extracellular phosphate was dependent on their differentiation state, and shRNA-dependent repression of the phosphate transporter Slc20a1 in MC3T3-E1 cells partially abolished their molecular response to phosphate. Taken together, our results provide further evidence for a role of extracellular phosphate as a signaling molecule and raise the possibility that severe hyperphosphatemia can negatively affect skeletal mineralization.     

Therapeutic Effects of FGF23 c‐tail Fc in a Murine Preclinical Model of X‐Linked Hypophosphatemia Via the Selective Modulation of Phosphate Reabsorption

Fibroblast growth factor 23 (FGF23) is the causative factor of X‐linked hypophosphatemia (XLH), a genetic disorder effecting 1:20,000 that is characterized by excessive phosphate excretion, elevated FGF23 levels and a rickets/osteomalacia phenotype. FGF23 inhibits phosphate reabsorption and suppresses 1α,25‐dihydroxyvitamin D (1,25D) biosynthesis, analytes that differentially contribute to bone integrity and deleterious soft‐tissue mineralization. As inhibition of ligand broadly modulates downstream targets, balancing efficacy and unwanted toxicity is difficult when targeting the FGF23 pathway. We demonstrate that a FGF23 c‐tail‐Fc fusion molecule selectively modulates the phosphate pathway in vivo by competitive antagonism of FGF23 binding to the FGFR/α klotho receptor complex. Repeated injection of FGF23 c‐tail Fc in Hyp mice, a preclinical model of XLH, increases cell surface abundance of kidney NaPi transporters, normalizes phosphate excretion, and significantly improves bone architecture in the absence of soft‐tissue mineralization. Repeated injection does not modulate either 1,25D or calcium in a physiologically relevant manner in either a wild‐type or disease setting. These data suggest that bone integrity can be improved in models of XLH via the exclusive modulation of phosphate. We posit that the selective modulation of the phosphate pathway will increase the window between efficacy and safety risks, allowing increased efficacy to be achieved in the treatment of this chronic disease. © 2017 American Society for Bone and Mineral Research.     

Loss of skeletal mineralization by the simultaneous ablation of PHOSPHO1 and alkaline phosphatase function: A unified model of the mechanisms of initiation of skeletal calcification

Endochondral ossification is a carefully orchestrated process mediated by promoters and inhibitors of mineralization. Phosphatases are implicated, but their identities and functions remain unclear. Alkaline phosphatase (TNAP) plays a crucial role promoting mineralization of the extracellular matrix by restricting the concentration of the calcification inhibitor inorganic pyrophosphate (PP_i). Mutations in the TNAP gene cause hypophosphatasia, a heritable form of rickets and osteomalacia. Here we show that PHOSPHO1, a phosphatase with specificity for phosphoethanolamine and phosphocholine, plays a functional role in the initiation of calcification and that ablation of PHOSPHO1 and TNAP function prevents skeletal mineralization. Phospho1^?/? mice display growth plate abnormalities, spontaneous fractures, bowed long bones, osteomalacia, and scoliosis in early life. Primary cultures of Phospho1^?/? tibial growth plate chondrocytes and chondrocyte‐derived matrix vesicles (MVs) show reduced mineralizing ability, and plasma samples from Phospho1^?/? mice show reduced levels of TNAP and elevated plasma PP_i concentrations. However, transgenic overexpression of TNAP does not correct the bone phenotype in Phospho1^?/? mice despite normalization of their plasma PP_i levels. In contrast, double ablation of PHOSPHO1 and TNAP function leads to the complete absence of skeletal mineralization and perinatal lethality. We conclude that PHOSPHO1 has a nonredundant functional role during endochondral ossification, and based on these data and a review of the current literature, we propose an inclusive model of skeletal calcification that involves intravesicular PHOSPHO1 function and P_i influx into MVs in the initiation of mineralization and the functions of TNAP, nucleotide pyrophosphatase phosphodiesterase‐1, and collagen in the extravesicular progression of mineralization. © 2011 American Society for Bone and Mineral Research.     

Hexa‐D‐arginine treatment increases 7B2•PC2 activity in hyp‐mouse osteoblasts and rescues the HYP phenotype

Inactivating mutations of the “phosphate regulating gene with homologies to endopeptidases on the X chromosome” (PHEX/Phex) underlie disease in patients with X‐linked hypophosphatemia (XLH) and the hyp‐mouse, a murine homologue of the human disorder. Although increased serum fibroblast growth factor 23 (FGF‐23) underlies the HYP phenotype, the mechanism(s) by which PHEX mutations inhibit FGF‐23 degradation and/or enhance production remains unknown. Here we show that treatment of wild‐type mice with the proprotein convertase (PC) inhibitor, decanoyl‐Arg‐Val‐Lys‐Arg‐chloromethyl ketone (Dec), increases serum FGF‐23 and produces the HYP phenotype. Because PC2 is uniquely colocalized with PHEX in osteoblasts/bone, we examined if PC2 regulates PHEX‐dependent FGF‐23 cleavage and production. Transfection of murine osteoblasts with PC2 and its chaperone protein 7B2 cleaved FGF‐23, whereas Signe1 (7B2) RNA interference (RNAi) transfection, which limited 7B2 protein production, decreased FGF‐23 degradation and increased Fgf‐23 mRNA and protein. The mechanism by which decreased 7B2?PC2 activity influences Fgf‐23 mRNA was linked to reduced conversion of the precursor to bone morphogenetic protein 1 (proBMP1) to active BMP1, which resulted in limited cleavage of dentin matrix acidic phosphoprotein 1 (DMP1), and consequent increased Fgf‐23 mRNA. The significance of decreased 7B2?PC2 activity in XLH was confirmed by studies of hyp‐mouse bone, which revealed significantly decreased Sgne1 (7B2) mRNA and 7B2 protein, and limited cleavage of proPC2 to active PC2. The expected downstream effects of these changes included decreased FGF‐23 cleavage and increased FGF‐23 synthesis, secondary to decreased BMP1‐mediated degradation of DMP1. Subsequent Hexa‐D‐Arginine treatment of hyp‐mice enhanced bone 7B2?PC2 activity, normalized FGF‐23 degradation and production, and rescued the HYP phenotype. These data suggest that decreased PHEX‐dependent 7B2?PC2 activity is central to the pathogenesis of XLH. © 2013 American Society for Bone and Mineral Research     

Proteolytic processing of osteopontin by PHEX and accumulation of osteopontin fragments in Hyp mouse bone,the murine model of X‐linked hypophosphatemia

X‐linked hypophosphatemia (XLH/HYP)—with renal phosphate wasting, hypophosphatemia, osteomalacia, and tooth abscesses—is caused by mutations in the zinc‐metallopeptidase PHEX gene (phosphate‐regulating gene with homologies to endopeptidase on the X chromosome). PHEX is highly expressed by mineralized tissue cells. Inactivating mutations in PHEX lead to distal renal effects (implying accumulation of a secreted, circulating phosphaturic factor) and accumulation in bone and teeth of mineralization‐inhibiting, acidic serine‐ and aspartate‐rich motif (ASARM)‐containing peptides, which are proteolytically derived from the mineral‐binding matrix proteins of the SIBLING family (small, integrin‐binding ligand N‐linked glycoproteins). Although the latter observation suggests a local, direct matrix effect for PHEX, its physiologically relevant substrate protein(s) have not been identified. Here, we investigated two SIBLING proteins containing the ASARM motif—osteopontin (OPN) and bone sialoprotein (BSP)—as potential substrates for PHEX. Using cleavage assays, gel electrophoresis, and mass spectrometry, we report that OPN is a full‐length protein substrate for PHEX. Degradation of OPN was essentially complete, including hydrolysis of the ASARM motif, resulting in only very small residual fragments. Western blotting of Hyp (the murine homolog of human XLH) mouse bone extracts having no PHEX activity clearly showed accumulation of an ～35 kDa OPN fragment that was not present in wild‐type mouse bone. Immunohistochemistry and immunogold labeling (electron microscopy) for OPN in Hyp bone likewise showed an accumulation of OPN and/or its fragments compared with normal wild‐type bone. Incubation of Hyp mouse bone extracts with PHEX resulted in the complete degradation of these fragments. In conclusion, these results identify full‐length OPN and its fragments as novel, physiologically relevant substrates for PHEX, suggesting that accumulation of mineralization‐inhibiting OPN fragments may contribute to the mineralization defect seen in the osteomalacic bone characteristic of XLH/HYP. © 2013 American Society for Bone and Mineral Research.     

Human Heterozygous ENPP1 Deficiency Is Associated With Early Onset Osteoporosis,a Phenotype Recapitulated in a Mouse Model of Enpp1 Deficiency

Biallelic ENPP1 deficiency in humans induces generalized arterial calcification of infancy (GACI) and/or autosomal recessive hypophosphatemic rickets type 2 (ARHR2). The latter is characterized by markedly increased circulating FGF23 levels and renal phosphate wasting, but aberrant skeletal manifestations associated with heterozygous ENPP1 deficiency are unknown. Here, we report three adult men with early onset osteoporosis who presented with fractures in the thoracic spine and/or left radius, mildly elevated circulating FGF23, and hypophosphatemia. Total hip bone mineral density scans demonstrated osteoporosis (Z-score < −2.5) and HRpQCT demonstrated microarchitectural defects in trabecular and cortical bone. Next-generation sequencing revealed heterozygous loss-of-function mutations in ENPP1 previously observed as biallelic mutations in infants with GACI. In addition, we present bone mass and structure data as well as plasma pyrophosphate (PPi) data of two siblings suffering from ARHR2 in comparison to their heterozygous and wild-type family members indicative of an ENPP1 gene dose effect. The skeletal phenotype in murine Enpp1 deficiency yielded nearly identical findings. Ten-week-old male Enpp1 ^asj/asj mice exhibited mild elevations in plasma FGF23 and hypophosphatemia, and micro-CT analysis revealed microarchitectural defects in trabecular and cortical bone of similar magnitude to HRpQCT defects observed in humans. Histomorphometry revealed mild osteomalacia and osteopenia at both 10 and 23 weeks. The biomechanical relevance of these findings was demonstrated by increased bone fragility and ductility in Enpp1 ^asj/asj mice. In summary, ENPP1 exerts a gene dose effect such that humans with heterozygous ENPP1 deficiency exhibit intermediate levels of plasma analytes associated with bone mineralization disturbance resulting in early onset osteoporosis. © 2019 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.     

Pharmacological inhibition of fibroblast growth factor (FGF) receptor signaling ameliorates FGF23‐mediated hypophosphatemic rickets

Fibroblast growth factor 23 (FGF23) is a circulating factor secreted by osteocytes that is essential for phosphate homeostasis. In kidney proximal tubular cells FGF23 inhibits phosphate reabsorption and leads to decreased synthesis and enhanced catabolism of 1,25‐dihydroxyvitamin D₃ (1,25[OH]₂D₃). Excess levels of FGF23 cause renal phosphate wasting and suppression of circulating 1,25(OH)₂D₃ levels and are associated with several hereditary hypophosphatemic disorders with skeletal abnormalities, including X‐linked hypophosphatemic rickets (XLH) and autosomal recessive hypophosphatemic rickets (ARHR). Currently, therapeutic approaches to these diseases are limited to treatment with activated vitamin D analogues and phosphate supplementation, often merely resulting in partial correction of the skeletal aberrations. In this study, we evaluate the use of FGFR inhibitors for the treatment of FGF23‐mediated hypophosphatemic disorders using NVP‐BGJ398, a novel selective, pan‐specific FGFR inhibitor currently in Phase I clinical trials for cancer therapy. In two different hypophosphatemic mouse models, Hyp and Dmp1‐null mice, resembling the human diseases XLH and ARHR, we find that pharmacological inhibition of FGFRs efficiently abrogates aberrant FGF23 signaling and normalizes the hypophosphatemic and hypocalcemic conditions of these mice. Correspondingly, long‐term FGFR inhibition in Hyp mice leads to enhanced bone growth, increased mineralization, and reorganization of the disturbed growth plate structure. We therefore propose NVP‐BGJ398 treatment as a novel approach for the therapy of FGF23‐mediated hypophosphatemic diseases. © 2013 American Society for Bone and Mineral Research.