Pathogenic role of Fgf23 in Dmp1-null mice

Autosomal recessive hypophosphatemic rickets (ARHR), which is characterized by renal phosphate wasting, aberrant regulation of 1alpha-hydroxylase activity, and rickets/osteomalacia, is caused by inactivating mutations of dentin matrix protein 1 (DMP1). ARHR resembles autosomal dominant hypophosphatemic rickets (ADHR) and X-linked hypophosphatemia (XLH), hereditary disorders respectively caused by cleavage-resistant mutations of the phosphaturic factor FGF23 and inactivating mutations of PHEX that lead to increased production of FGF23 by osteocytes in bone. Circulating levels of FGF23 are increased in ARHR and its Dmp1-null mouse homologue. To determine the causal role of FGF23 in ARHR, we transferred Fgf23 deficient/enhanced green fluorescent protein (eGFP) reporter mice onto Dmp1-null mice to create mice lacking both Fgf23 and Dmp1. Dmp1(-/-) mice displayed decreased serum phosphate concentrations, inappropriately normal 1,25(OH)(2)D levels, severe rickets, and a diffuse form of osteomalacia in association with elevated Fgf23 serum levels and expression in osteocytes. In contrast, Fgf23(-/-) mice had undetectable serum Fgf23 and elevated serum phosphate and 1,25(OH)(2)D levels along with severe growth retardation and focal form of osteomalacia. In combined Dmp1(-/-)/Fgf23(-/-), circulating Fgf23 levels were also undetectable, and the serum levels of phosphate and 1,25(OH)(2)D levels were identical to Fgf23(-/-) mice. Rickets and diffuse osteomalacia in Dmp1-null mice were transformed to severe growth retardation and focal osteomalacia characteristic of Fgf23-null mice. These data suggest that the regulation of extracellular matrix mineralization by DMP1 is coupled to renal phosphate handling and vitamin D metabolism through a DMP1-dependent regulation of FGF23 production by osteocytes.     

Dysregulated Gene Expression in the Primary Osteoblasts and Osteocytes Isolated from Hypophosphatemic Hyp Mice

Osteocytes express multiple genes involved in mineral metabolism including PHEX, FGF23, DMP1 and FAM20C. In Hyp mice, a murine model for X-linked hypophosphatemia (XLH), Phex deficiency results in the overproduction of FGF23 in osteocytes, which leads to hypophosphatemia and impaired vitamin D metabolism. In this study, to further clarify the abnormality in osteocytes of Hyp mice, we obtained detailed gene expression profiles in osteoblasts and osteocytes isolated from the long bones of 20-week-old Hyp mice and wild-type (WT) control mice. The expression of Fgf23, Dmp1, and Fam20c was higher in osteocytic cells than in osteoblastic cells in both genotypes, and was up-regulated in Hyp cells. Interestingly, the up-regulation of these genes in Hyp bones began before birth. On the other hand, the expression of Slc20a1 encoding the sodium/phosphate (Na⁺/Pi) co-transporter Pit1 was increased in osteoblasts and osteocytes from adult Hyp mice, but not in Hyp fetal bones. The direct effects of extracellular Pi and 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] on isolated osteoblastic and osteocytic cells were also investigated. Twenty-four-hour treatment with 10⁻⁸ M 1,25(OH)₂D₃ increased the expression of Fgf23 in WT osteoblastic cells but not in osteocytic cells. Dmp1 expression in osteocytic cells was increased due to the 24-hour treatment with 10 mM Pi and was suppressed by 10⁻⁸ M 1,25(OH)₂D₃ in WT osteocytic cells. We also found the up-regulation of the genes for FGF1, FGF2, their receptors, and Egr-1 which is a target of FGF signaling, in Hyp osteocytic cells, suggesting the activation of FGF/FGFR signaling. These results implicate the complex gene dysregulation in osteoblasts and osteocytes of Hyp mice, which might contribute to the pathogenesis.     

Osteocytes but not osteoblasts directly build mineralized bone structures

Bone-forming osteoblasts have been a cornerstone of bone biology for more than a century. Most research toward bone biology and bone diseases center on osteoblasts. Overlooked are the 90% of bone cells, called osteocytes. This study aims to test the hypothesis that osteocytes but not osteoblasts directly build mineralized bone structures, and that defects in osteocytes lead to the onset of hypophosphatemia rickets. The hypothesis was tested by developing and modifying multiple imaging techniques, including both in vivo and in vitro models plus two types of hypophosphatemia rickets models (Dmp1-null and Hyp, Phex mutation mice), and Dmp1-Cre induced high level of β-catenin models. Our key findings were that osteocytes (not osteoblasts) build bone similar to the construction of a high-rise building, with a wire mesh frame (i.e., osteocyte dendrites) and cement (mineral matrices secreted from osteocytes), which is a lengthy and slow process whose mineralization direction is from the inside toward the outside. When osteoblasts fail to differentiate into osteocytes but remain highly active in Dmp-1-null or Hyp mice, aberrant and poor bone mineralization occurs, caused by a sharp increase in Wnt-β-catenin signaling. Further, the constitutive expression of β-catenin in osteocytes recaptures a similar osteomalacia phenotype as shown in Dmp1 null or Hyp mice. Thus, we conclude that osteocytes directly build bone, and osteoblasts with a short life span serve as a precursor to osteocytes, which challenges the existing dogma.     

Skeletal FGFR1 signaling is necessary for regulation of serum phosphate level by FGF23 and normal life span

Fibroblast growth factor (FGF) 23 produced by the bone is the principal hormone to regulate serum phosphate level. Serum FGF23 needs to be tightly regulated to maintain serum phosphate in a narrow range. Thus, we hypothesized that the bone has some phosphate-sensing mechanism to regulate the production of FGF23. Previously we showed that extracellular phosphate induces the phosphorylation of FGF receptor 1 (FGFR1) and FGFR1 signaling regulates the expression of Galnt3, whose product works to increase FGF23 production in vitro. In this study, we show the significance of FGFR1 in the regulated FGF23 production and serum phosphate level in vivo. We generated late-osteoblast/osteocyte-specific Fgfr1-knockout mice (Fgfr1^fl/fl; Ocn^Cre/+) by crossing the Ocn-Cre and the floxed Fgfr1 mouse lines. We evaluated serum phosphate and FGF23 levels, the expression of Galnt3 in the bone, the body weight and life span. A selective ablation of Fgfr1 aborted the increase of serum active full-length FGF23 and the enhanced expression of Galnt3 in the bone by a high phosphate diet. These mice showed more pronounced hyperphosphatemia compared with control mice. In addition, these mice fed with a control diet showed body weight loss after 23 weeks of age and shorter life span. These results reveal a novel significance of FGFR1 signaling in the phosphate metabolism and normal life span.     

Nuclear Isoforms of Fibroblast Growth Factor 2 Are Novel Inducers of Hypophosphatemia via Modulation of FGF23 and KLOTHO

FGF2 transgenic mice were developed in which type I collagen regulatory sequences drive the nuclear high molecular weight FGF2 isoforms in osteoblasts (TgHMW). The phenotype of TgHMW mice included dwarfism, decreased bone mineral density (BMD), osteomalacia, and decreased serum phosphate (P_i). When TgHMW mice were fed a high P_i diet, BMD was increased, and dwarfism was partially reversed. The TgHMW phenotype was similar to mice overexpressing FGF23. Serum FGF23 was increased in TgHMW mice. Fgf23 mRNA in bones and fibroblast growth factor receptors 1c and 3c and Klotho mRNAs in kidneys were increased in TgHMW mice, whereas the renal Na⁺/P_i co-transporter Npt2a mRNA was decreased. Immunohistochemistry and Western blot analyses of TgHMW kidneys showed increased KLOTHO and decreased NPT2a protein. The results suggest that overexpression of HMW FGF2 increases FGF23/FGFR/KLOTHO signaling to down-regulate NPT2a, causing P_i wasting, osteomalacia, and decreased BMD. We assessed whether HMW FGF2 expression was altered in the Hyp mouse, a mouse homolog of the human disease X-linked hypophosphatemic rickets/osteomalacia. Fgf2 mRNA was increased in bones, and Western blots showed increased FGF2 protein in nuclear fractions from osteoblasts of Hyp mice. In addition, immunohistochemistry demonstrated co-localization of FGF23 and HMW FGF2 protein in osteoblasts and osteocytes from Hyp mice. This study reveals a novel mechanism of regulation of the FGF23-P_i homeostatic axis.     

Compound deletion of Fgfr3 and Fgfr4 partially rescues the Hyp mouse phenotype

Uncertainty exists regarding the physiologically relevant fibroblast growth factor (FGF) receptor (FGFR) for FGF23 in the kidney and the precise tubular segments that are targeted by FGF23. Current data suggest that FGF23 targets the FGFR1c-Klotho complex to coordinately regulate phosphate transport and 1,25-dihydroxyvitamin D [1,25(OH)(2)D] production in the proximal tubule. In studies using the Hyp mouse model, which displays FGF23-mediated hypophosphatemia and aberrant vitamin D, deletion of Fgfr3 or Fgfr4 alone failed to correct the Hyp phenotype. To determine whether FGFR1 is sufficient to mediate the renal effects of FGF23, we deleted Fgfr3 and Fgfr4 in Hyp mice, leaving intact the FGFR1 pathway by transferring compound Fgfr3/Fgfr4-null mice on the Hyp background to create wild-type (WT), Hyp, Fgfr3(-/-)/Fgfr4(-/-), and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice. We found that deletion of Fgfr3 and Fgfr4 in Fgfr3(-/-)/Fgfr4(-/-) and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice induced an increase in 1,25(OH)(2)D. In Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice, it partially corrected the hypophosphatemia (P(i) = 9.4 ± 0.9, 6.1 ± 0.2, 9.1 ± 0.4, and 8.0 ± 0.5 mg/dl in WT, Hyp, Fgfr3(-/-)/Fgfr4(-/-), and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice, respectively), increased Na-phosphate cotransporter Napi2a and Napi2c and Klotho mRNA expression in the kidney, and markedly increased serum FGF23 levels (107 ± 20, 3,680 ± 284, 167 ± 22, and 18,492 ± 1,547 pg/ml in WT, Hyp, Fgfr3(-/-)/Fgfr4(-/-), and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice, respectively), consistent with a compensatory response to the induction of end-organ resistance. Fgfr1 expression was unchanged in Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice and was not sufficient to transduce the full effects of FGF23 in Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice. These studies suggest that FGFR1, FGFR3, and FGFR4 act in concert to mediate FGF23 effects on the kidney and that loss of FGFR function leads to feedback stimulation of Fgf23 expression in bone.     

Conditional Deletion of Fgfr1 in the Proximal and Distal Tubule Identifies Distinct Roles in Phosphate and Calcium Transport

A postnatal role of fibroblast growth factor receptor-1 (FGFR1) in the kidney is suggested by its binding to α-Klotho to form an obligate receptor for the hormone fibroblast growth factor-23 (FGF-23). FGFR1 is expressed in both the proximal and distal renal tubular segments, but its tubular specific functions are unclear. In this study, we crossed Fgfr1^flox/flox mice with either gamma-glutamyltransferase-Cre (γGT-Cre) or kidney specific-Cre (Ksp-Cre) mice to selectively create proximal tubule (PT) and distal tubule (DT) Fgfr1 conditional knockout mice (designated Fgfr1^PT-cKO and Fgfr1^DT-cKO, respectively). Fgfr1^PT-cKO mice exhibited an increase in sodium-dependent phosphate co-transporter expression, hyperphosphatemia, and refractoriness to the phosphaturic actions of FGF-23, consistent with a direct role of FGFR1 in mediating the proximal tubular phosphate responses to FGF-23. In contrast, Fgfr1^DT-cKO mice unexpectedly developed hypercalciuria, secondary elevations of parathyroid hormone (PTH), hypophosphatemia and enhanced urinary phosphate excretion. Fgfr1^PT-cKO mice also developed a curly tail/spina bifida-like skeletal phenotype, whereas Fgfr1^DT-cKO mice developed renal tubular micro-calcifications and reductions in cortical bone thickness. Thus, FGFR1 has dual functions to directly regulate proximal and distal tubule phosphate and calcium reabsorption, indicating a physiological role of FGFR1 signaling in both phosphate and calcium homeostasis.     

Protective Roles of DMP1 in High Phosphate Homeostasis

Purpose

Dmp1 (dentin matrix protein1) null mice (Dmp1^−/−) display hypophosphatemic rickets with a sharp increase in fibroblast growth factor 23 (FGF23). Disruption of Klotho (the obligatory co-receptor of FGF23) results in hyperphosphatemia with ectopic calcifications formed in blood vessels and kidneys. To determine the role of DMP1 in both a hyperphosphatemic environment and within the ectopic calcifications, we created Dmp1/Klotho compound deficient (Dmp1^−/−kl/kl) mice.

Procedures

A combination of TUNEL, immunohistochemistry, TRAP, von Kossa, micro CT, bone histomorphometry, serum biochemistry and Scanning Electron Microscopy techniques were used to analyze the changes in blood vessels, kidney and bone for wild type control, Dmp1^−/−, Klotho deficient (kl/kl) and Dmp1^−/−kl/kl animals.

Findings

Interestingly, Dmp1^−/−kl/kl mice show a dramatic improvement of rickets and an identical serum biochemical phenotype to kl/kl mice (extremely high FGF23, hyperphosphatemia and reduced parathyroid hormone (PTH) levels). Unexpectedly, Dmp1^−/−kl/kl mice presented elevated levels of apoptosis in osteocytes, endothelial and vascular smooth muscle cells in small and large blood vessels, and within the kidney as well as dramatic increase in ectopic calcification in all these tissues, as compared to kl/kl.

Conclusion

These findings suggest that DMP1 has an anti-apoptotic role in hyperphosphatemia. Discovering this novel protective role of DMP1 may have clinical relevance in protecting the cells from apoptosis in high-phosphate environments as observed in chronic kidney disease (CKD).     

Antibody-Mediated Activation of FGFR1 Induces FGF23 Production and Hypophosphatemia

The phosphaturic hormone Fibroblast Growth Factor 23 (FGF23) controls phosphate homeostasis by regulating renal expression of sodium-dependent phosphate co-transporters and cytochrome P450 enzymes involved in vitamin D catabolism. Multiple FGF Receptors (FGFRs) can act as receptors for FGF23 when bound by the co-receptor Klotho expressed in the renal tubular epithelium. FGFRs also regulate skeletal FGF23 secretion; ectopic FGFR activation is implicated in genetic conditions associated with FGF23 overproduction and hypophosphatemia. The identity of FGFRs that mediate the activity of FGF23 or that regulate skeletal FGF23 secretion remains ill defined. Here we report that pharmacological activation of FGFR1 with monoclonal anti-FGFR1 antibodies (R1MAb) in adult mice is sufficient to cause an elevation in serum FGF23 and mild hypophosphatemia. In cultured rat calvariae osteoblasts, R1MAb induces FGF23 mRNA expression and FGF23 protein secretion into the culture medium. In a cultured kidney epithelial cell line, R1MAb acts as a functional FGF23 mimetic and activates the FGF23 program. siRNA-mediated Fgfr1 knockdown induced the opposite effects. Taken together, our work reveals the central role of FGFR1 in the regulation of FGF23 production and signal transduction, and has implications in the pathogenesis of FGF23-related hypophosphatemic disorders.     

Distinct roles for intrinsic osteocyte abnormalities and systemic factors in regulation of FGF23 and bone mineralization in Hyp mice

X-linked hypophosphatemia (XLH) is characterized by hypophosphatemia and impaired mineralization caused by mutations of the PHEX endopeptidase (phosphate-regulating gene with homologies to endopeptidases on the X chromosome), which leads to the overproduction of the phosphaturic fibroblast growth factor 23 (FGF23) in osteocytes. The mechanism whereby PHEX mutations increase FGF23 expression and impair mineralization is uncertain. Either an intrinsic osteocyte abnormality or unidentified PHEX substrates could stimulate FGF23 in XLH. Similarly, impaired mineralization in XLH could result solely from hypophosphatemia or from a concomitant PHEX-dependent intrinsic osteocyte abnormality. To distinguish between these possibilities, we assessed FGF23 expression and mineralization after reciprocal bone cross-transplantations between wild-type (WT) mice and the Hyp mouse model of XLH. We found that increased FGF23 expression in Hyp bone results from a local effect of PHEX deficiency, since FGF23 was increased in Hyp osteocytes before and after explantation into WT mice but was not increased in WT osteocytes after explantation into Hyp mice. WT bone explanted into Hyp mice developed rickets and osteomalacia, but Hyp bone explanted into WT mice displayed persistent osteomalacia and abnormalities in the primary spongiosa, indicating that both phosphate and PHEX independently regulate extracellular matrix mineralization. Unexpectedly, we observed a paradoxical suppression of FGF23 in juvenile Hyp bone explanted into adult Hyp mice, indicating the presence of an age-dependent systemic inhibitor of FGF23. Thus PHEX functions in bone to coordinate bone mineralization and systemic phosphate homeostasis by directly regulating the mineralization process and producing FGF23. In addition, systemic counterregulatory factors that attenuate the upregulation of FGF23 expression in Hyp mouse osteocytes are present in older mice.     

FGF23 Suppresses Chondrocyte Proliferation in the Presence of Soluble α-Klotho both in Vitro and in Vivo

Fibroblast growth factor-23 (FGF23) is well established to play crucial roles in the regulation of phosphate homeostasis. X-linked hypophosphatemic rickets (XLH) is characterized by impaired mineralization and growth retardation associated with elevated circulating FGF23 levels. Administration of phosphate and calcitriol is effective in improving growth retardation, but is not sufficient to fully reverse impaired growth, suggesting the existence of a disease-specific mechanism in the development of growth retardation in addition to dysregulated phosphate metabolism. However, the precise mechanisms of growth retardation in XLH remain elusive. Here, we postulated that FGF23 suppressed chondrocyte proliferation in the presence of soluble α-Klotho (sKL). In vitro and ex vivo studies revealed that FGF23 formed a protein complex with sKL through KL1 internal repeat and suppressed the linear growth of metatarsals in the presence of sKL, which was antagonized by co-incubation with neutralizing antibodies against FGF23 or by knocking-down FGFR3 expression. Additionally, FGF23 binding to FGFR3 was enhanced in the presence of sKL. Histologically, the length of the proliferating zone was diminished and was associated with decreased chondrocyte proliferation. FGF23/sKL suppressed Indian hedgehog (Ihh) expression and administration of Ihh protein partially rescued the suppressive effect of FGF23/sKL on metatarsal growth. Intraperitoneal administration of sKL in Hyp mice, a murine model for XLH, caused a decrease in the length of the proliferating zone associated with decreased chondrocyte proliferation without altering circulating phosphate levels. These findings suggest that suppression of chondrocyte proliferation by FGF23 could have a causative role in the development of growth retardation in XLH.     

Transgenic expression of Dspp partially rescued the long bone defects of Dmp1-null mice

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) belong to the Small Integrin-Binding Ligand N-linked Glycoprotein (SIBLING) family. In addition to the features common to all SIBLING members, DMP1 and DSPP share several unique similarities in chemical structure, proteolytic activation and tissue localization. Mutations in, or deletion of DMP1, cause autosomal recessive hypophosphatemic rickets along with dental defects; DSPP mutations or its ablation are associated with dentinogenesis imperfecta. While the roles and functional mechanisms of DMP1 in osteogenesis have been extensively studied, those of DSPP in long bones have been studied only to a limited extent. Previous studies by our group revealed that transgenic expression of Dspp completely rescued the dentin defects of Dmp1-null (Dmp1^−/−) mice. In this investigation, we assessed the effects of transgenic Dspp on osteogenesis by analyzing the formation and mineralization of the long bones in Dmp1^−/− mice that expresses a transgene encoding full-length DSPP driven by a 3.6-kb rat Col1a1 promoter (referred as “Dmp1^−/−;Dspp-Tg mice”). We characterized the long bones of the Dmp1^−/−;Dspp-Tg mice at different ages and compared them with those from Dmp1^−/− and Dmp1^+/− (normal control) mice. Our analyses showed that the long bones of Dmp1^−/−;Dspp-Tg mice had a significant increase in cortical bone thickness, bone volume and mineral density along with a remarkable restoration of trabecular thickness compared to those of the Dmp1^−/− mice. The long bones of Dmp1^−/−;Dspp-Tg mice underwent a dramatic reduction in the amount of osteoid, significant improvement of the collagen fibrillar network, and better organization of the lacunocanalicular system, compared to the Dmp1^−/− mice. The elevated levels of biglycan, bone sialoprotein and osteopontin in Dmp1^−/− mice were also noticeably corrected by the transgenic expression of Dspp. These findings suggest that DSPP and DMP1 may function synergistically within the complex milieus of bone matrices.     

Deletion of Fgfr1 in Osteoblasts Enhances Mobilization of EPCs into Peripheral Blood in a Mouse Endotoxemia Model

Endothelial progenitor cells (EPCs) contribute to neovascularization and vascular repair, and may exert a beneficial effect on the clinical outcome of sepsis. Osteoblasts act as a component of “niche” in bone marrow, which provides a nest for stem/progenitor cells and are involved in the formation and maintenance of stem/progenitor cells. Fibroblast growth factor receptor 1 (FGFR1) can regulate osteoblast activity and influence bone mass. So we explored the role of FGFR1 in EPC mobilization. Male mice with osteoblast-specific knockout of Fgfr1 (Fgfr1^fl/fl;OC-Cre) and its wild-type littermates (Fgfr1^fl/fl) were used in this study. Mice intraperitoneally injected with lipopolysaccharide (LPS) were used to measure the number of circulating EPCs in peripheral blood and serum stromal cell-derived factor 1α (SDF-1α). The circulating EPC number and the serum level of SDF-1α were significantly higher in Fgfr1^fl/fl;OC-Cre mice than those in Fgfr1^fl/fl mice after LPS injection. In cell culture system, SDF-1α level was also significantly higher in Fgfr1^fl/fl;OC-Cre osteoblasts compared with that in Fgfr1^fl/fl osteoblasts after LPS treatment. TRAP staining showed that there was no significant difference between the osteoclast activity of septic Fgfr1^fl/fland Fgfr1^fl/fl;OC-Cre mice. This study suggests that targeted deletion of Fgfr1 in osteoblasts enhances mobilization of EPCs into peripheral blood through up-regulating SDF-1α secretion from osteoblasts.     

Fibroblast growth factor receptor 1 regulates the differentiation and activation of osteoclasts through Erk1/2 pathway

To elucidate the direct role and mechanism of FGFR1 signaling in the differentiation and activation of osteoclasts, we conditionally inactivated FGFR1 in bone marrow monocytes and mature osteoclasts of mice. Mice deficient in FGFR1 (Fgfr1^−/−) exhibited misregulated bone remodeling with reduced osteoclast number and impaired osteoclast function. In vitro assay demonstrated that the number of tartrate-resistant acid phosphatase (TRAP) positive osteoclasts derived from bone marrow monocytes of Fgfr1^−/− mice was significantly diminished. The bone resorption activity of mature osteoclasts derived from Fgfr1^−/− mice was also suppressed. Further analysis showed that the osteoclasts with FGFR1 deficiency exhibited downregulated expression of genes related to osteoclastic activity including TRAP and MMP-9. The phosphorylation of Erk1/2 mitogen-activated protein (MAP) kinase was also decreased. Our results suggest that FGFR1 is indispensable for complete differentiation and activation of osteoclasts in mice.     

Phosphatonins: From Discovery to Therapeutics

ObjectivePhosphate is crucial for cell signaling, energy metabolism, nucleotide synthesis, and bone mineralization. The gut-bone-parathyroid-kidney axis is influenced by parathyroid hormone, 1,25-dihydroxyvitamin D, and phosphatonins, especially fibroblast growth factor 23 (FGF23). These hormones facilitate maintenance of phosphate homeostasis. This review summarizes current knowledge regarding the phosphate homeostasis, phosphatonin pathophysiology, and clinical implications of FGF23-related hypophosphatemic disorders, with specific focus on burosumab treatment.MethodA focused literature search of PubMed was conducted.ResultsPhosphatonins including FGF23, secreted frizzled-related protein 4, matrix extracellular phosphoglycoprotein, and fibroblast growth factor 7 play a pathogenic role in several hypophosphatemic disorders. Excess FGF23 inhibits sodium-dependent phosphate cotransporters (NaPi-2a and NaPi-2c), resulting in hyperphosphaturia and hypophosphatemia. Additionally, FGF23 suppresses 1,25-dihydroxyvitamin D synthesis in the proximal renal tubule, and thus, it indirectly inhibits intestinal phosphate absorption. Disorders of FGF23-related hypophosphatemia include X-linked hypophosphatemia (XLH), autosomal dominant hypophosphatemic rickets, autosomal recessive hypophosphatemic rickets, fibrous dysplasia/McCune-Albright syndrome, and tumor-induced osteomalacia (TIO). Complications of conventional therapy with oral phosphate and vitamin D analogs comprise gastrointestinal distress, hypercalcemia, nephrocalcinosis, and secondary/tertiary hyperparathyroidism. In both children and adults with XLH and TIO, the anti-FGF23 antibody burosumab exhibits a favorable safety profile and is associated with healing of rickets in affected children and improvement of osteomalacia in both children and adults.ConclusionThe treatment paradigm for XLH and TIO is changing based on data from recent clinical trials. Research suggest that burosumab is effective and safe for pediatric and adult patients with XLH or TIO.     

Notch Signaling in Osteocytes Differentially Regulates Cancellous and Cortical Bone Remodeling

Notch receptors play a role in skeletal development and homeostasis, and Notch activation in undifferentiated and mature osteoblasts causes osteopenia. In contrast, Notch activation in osteocytes increases bone mass, but the mechanisms involved and exact functions of Notch are not known. In this study, Notch1 and -2 were inactivated preferentially in osteocytes by mating Notch1/2 conditional mice, where Notch alleles are flanked by loxP sequences, with transgenics expressing Cre directed by the Dmp1 (dentin matrix protein 1) promoter. Notch1/2 conditional null male and female mice exhibited an increase in trabecular bone volume due to an increase in osteoblasts and decrease in osteoclasts. In male null mice, this was followed by an increase in osteoclast number and normalization of bone volume. To activate Notch preferentially in osteocytes, Dmp1-Cre transgenics were crossed with Rosa^Notch mice, where a loxP-flanked STOP cassette is placed between the Rosa26 promoter and Notch1 intracellular domain sequences. Dmp1-Cre^+/−;Rosa^Notch mice exhibited an increase in trabecular bone volume due to decreased bone resorption and an increase in cortical bone due to increased bone formation. Biomechanical and chemical properties were not affected. Osteoprotegerin mRNA was increased, sclerostin and dickkopf1 mRNA were decreased, and Wnt signaling was enhanced in Dmp1-Cre^+/−;Rosa^Notch femurs. Botulinum toxin A-induced muscle paralysis caused pronounced osteopenia in control mice, but bone mass was preserved in mice harboring the Notch activation in osteocytes. In conclusion, Notch plays a unique role in osteocytes, up-regulates osteoprotegerin and Wnt signaling, and differentially regulates trabecular and cortical bone homeostasis.     

Knockout of Nuclear High Molecular Weight FGF2 Isoforms in Mice Modulates Bone and Phosphate Homeostasis

We previously reported that targeted overexpression of the fibroblast growth factor 2 (FGF2) high molecular weight (HMW) isoforms in osteoblastic lineage cells in mice resulted in phenotypic changes, including dwarfism, rickets, osteomalacia, hypophosphatemia, increased serum parathyroid hormone, and increased levels of the phosphatonin FGF23 in serum and bone. This study examined the effects of genetically knocking out the FGF2HMW isoforms (HMWKO) on bone and phosphate homeostasis. HMWKO mice were not dwarfed and had significantly increased bone mineral density and bone mineral content in femurs and lumbar vertebrae when compared with the wild-type (WT) littermates. Micro-computed tomography analysis of femurs revealed increased trabecular bone volume, thickness, number, and connective tissue density with decreased trabecular spacing compared with WT. In addition, there was significantly decreased cortical porosity and increased cortical thickness and sub-periosteal area in femurs of HMWKO. Histomorphometric analysis demonstrated increased osteoblast activity and diminished osteoclast activity in the HMWKO. In vitro bone marrow stromal cell cultures showed there was a significant increase in alkaline phosphatase-positive colony number at 1 week in HMWKO. At 3 weeks of culture, the mineralized area was also significantly increased. There was increased expression of osteoblast differentiation marker genes and reduced expression of genes associated with impaired mineralization, including a significant reduction in Fgf23 and Sost mRNA. Normal serum phosphate and parathyroid hormone were observed in HMWKO mice. This study demonstrates a significant negative impact of HMWFGF2 on biological functions in bone and phosphate homeostasis in mice.     

Adamts1 is highly induced in rachitic bones of FGF23 transgenic mice and participates in degradation of non-mineralized bone matrix collagen

Transgenic mice overexpressing fibroblast growth factor 23 (FGF23) in osteoblasts have a rachitic bone phenotype. These mice display hypomineralized bones, increased expression of osteoblast markers, but osteoclast numbers are unaltered or slightly reduced. Paradoxically, they show increased serum levels of the bone resorption marker CTX, a type I collagen degradation fragment. Here we analyzed a matrix metalloproteinase- (MMP-) like secreted protease, Adamts1, that has previously been associated with osteoblastic type I collagen breakdown in vitro. Bones from FGF23 transgenic (tg) mice displayed increased Adamts1 protein upon both immunohistological staining and Western blotting. We further found Adamts1 protein together with excessively degraded type I collagen in the non-mineralized bone fraction of FGF23 tg mice. A similar degradation pattern of type I collagen was noticed upon forced expression of Adamts1 in osteoblastic cells in vitro. Importantly, these Adamts1-expressing osteoblastic cells exhibited increased release of CTX fragments when cultured on demineralized bone discs. Together, these results demonstrate for the first time that Adamts1 can be highly induced in bone tissue and that this MMP-like protease can increase osteoblastic release of CTX fragments from non-mineralized bone. Thus, Adamts1 potentially contributes to the increased serum levels of CTX in rickets/osteomalacia.