Eva S. Liu

Calcitonin Administration in X-Linked Hypophosphatemia

To the Editor: In X-linked hypophosphatemia, phosphate wasting results from increased circulating levels of fibroblast growth factor 23 (FGF-23).1 Administration of calcitonin to a patient with onc...

1,25-Dihydroxyvitamin D Alone Improves Skeletal Growth, Microarchitecture, and Strength in a Murine Model of XLH, Despite Enhanced FGF23 Expression

X‐linked hypophosphatemia (XLH) is characterized by impaired renal tubular reabsorption of phosphate owing to increased circulating FGF23 levels, resulting in rickets in growing children and impaired bone mineralization. Increased FGF23 decreases renal brush border membrane sodium‐dependent phosphate transporter IIa (Npt2a) causing renal phosphate wasting, impairs 1‐α hydroxylation of 25‐hydroxyvitamin D, and induces the vitamin D 24‐hydroxylase, leading to inappropriately low circulating levels of 1,25‐dihydroxyvitamin D (1,25D). The goal of therapy is prevention of rickets and improvement of growth in children by phosphate and 1,25D supplementation. However, this therapy is often complicated by hypercalcemia and nephrocalcinosis and does not always prevent hyperparathyroidism. To determine if 1,25D or blocking FGF23 action can improve the skeletal phenotype without phosphate supplementation, mice with XLH (Hyp) were treated with daily 1,25D repletion, FGF23 antibodies (FGF23Ab), or biweekly high‐dose 1,25D from d2 to d75 without supplemental phosphate. All treatments maintained normocalcemia, increased serum phosphate, and normalized parathyroid hormone levels. They also prevented the loss of Npt2a, α‐Klotho, and pERK1/2 immunoreactivity observed in the kidneys of untreated Hyp mice. Daily treatment with 1,25D decreased urine phosphate losses despite a marked increase in bone FGF23 mRNA and in circulating FGF23 levels. Daily 1,25D was more effective than other treatments in normalizing the growth plate and metaphyseal organization. In addition to being the only therapy that normalized lumbar vertebral height and body weight, daily 1,25D therapy normalized bone geometry and was more effective than FGF23Ab in improving trabecular bone structure. Daily 1,25D and FGF23Ab improved cortical microarchitecture and whole‐bone biomechanical properties more so than biweekly 1,25D. Thus, monotherapy with 1,25D improves growth, skeletal microarchitecture, and bone strength in the absence of phosphate supplementation despite enhancing FGF23 expression, demonstrating that 1,25D has direct beneficial effects on the skeleton in XLH, independent of its role in phosphate homeostasis.

Phosphate Interacts With PTHrP to Regulate Endochondral Bone Formation

Phosphate and parathyroid hormone related peptide (PTHrP) are required for normal growth plate maturation. Hypophosphatemia impairs hypertrophic chondrocyte apoptosis leading to rachitic expansion of the growth plate; however, the effect of phosphate restriction on chondrocyte differentiation during endochondral bone formation has not been examined. Investigations were, therefore, undertaken to address whether phosphate restriction alters the maturation of embryonic d15.5 murine metatarsal elements. Metatarsals cultured in low phosphate media exhibited impaired chondrocyte differentiation, analogous to that seen with PTHrP-treatment of metatarsals cultured in control media. Because phosphate restriction acutely increases PTHrP expression in cultured metatarsals, studies were undertaken to determine if this increase in PTHrP plays a pathogenic role in the impaired chondrocyte differentiation observed under low phosphate conditions. In contrast to what was observed with wild-type metatarsal elements, phosphate restriction did not impair the differentiation of metatarsals isolated from PTHrP heterozygous or PTHrP knockout mice. In vivo studies in postnatal mice demonstrated that PTHrP haploinsufficiency also prevents the impaired hypertrophic chondrocyte apoptosis observed with phosphate restriction. To determine how signaling through the PTH/PTHrP receptor antagonizes the pro-apoptotic effects of phosphate, investigations were performed in primary murine hypertrophic chondrocytes. Receptor activation impaired phosphate-induced Erk1/2 phosphorylation specifically in the mitochondrial fraction and decreased levels of mitochondrial Bad, while increasing cytosolic phospho-Bad. Thus, these data demonstrate that phosphate restriction attenuates chondrocyte differentiation as well as impairing hypertrophic chondrocyte apoptosis and implicate a functional role for the PTH/PTHrP signaling pathway in the abnormalities in chondrocyte differentiation and hypertrophic chondrocyte apoptosis observed under phosphate restricted conditions.

Increased Circulating FGF23 Does Not Lead to Cardiac Hypertrophy in the Male Hyp Mouse Model of XLH

Serum levels of fibroblast growth factor 23 (FGF23) markedly increase with renal impairment, with FGF23 levels correlating with the presence of left ventricular hypertrophy (LVH) and mortality in patients with chronic kidney disease (CKD). FGF23 activates calcineurin/nuclear factor of activated T cell (NFAT) signaling and induces hypertrophy in murine cardiomyocytes. X-linked hypophosphatemia (XLH) is characterized by high circulating levels of FGF23 but, in contrast to CKD, is associated with hypophosphatemia. The cardiac effects of high circulating levels of FGF23 in XLH are not well defined. Thus, studies were undertaken to define the cardiac phenotype in the mouse model of XLH (Hyp mice). Echocardiographic and histological analyses demonstrated that Hyp left ventricles (LVs) are smaller than those of wild-type mice. Messenger RNA expression of cardiac hypertrophy markers was not altered in the LV or right ventricle of Hyp mice. However, the Hyp LVs had increased expression of the NFAT target genes NFATc1 and RCAN1. To determine whether phosphate alone can induce markers of hypertrophy, differentiated C2C12 myocytes were treated with phosphate. Phosphate treatment increased expression of cardiac hypertrophy markers, supporting a primary role for phosphate in inducing LVH. Although previous studies showed that increased circulating FGF23 and phosphate levels are associated with LVH, our results demonstrated that in XLH, high circulating levels of FGF23 in the setting of hypophosphatemia do not induce cardiac hypertrophy.

c-Raf promotes angiogenesis during normal growth plate maturation

Extracellular phosphate plays a key role in growth plate maturation by inducing Erk1/2 (Mapk3/1) phosphorylation, leading to hypertrophic chondrocyte apoptosis. The Raf kinases induce Mek1/2 (Map2k1/2) and Erk1/2 phosphorylation; however, a role for Raf kinases in endochondral bone formation has not been identified. Ablation of both A-Raf (Araf) and B-Raf (Braf) in chondrocytes does not alter growth plate maturation. Because c-Raf (Raf1) phosphorylation is increased by extracellular phosphate and c-Raf is the predominant isoform expressed in hypertrophic chondrocytes, chondrocyte-specific c-Raf knockout mice (c-Raff/f;ColII-Cre+) were generated to define a role for c-Raf in growth plate maturation. In vivo studies demonstrated that loss of c-Raf in chondrocytes leads to expansion of the hypertrophic layer of the growth plate, with decreased phospho-Erk1/2 immunoreactivity and impaired hypertrophic chondrocyte apoptosis. However, cultured hypertrophic chondrocytes from these mice did not exhibit impairment of phosphate-induced Erk1/2 phosphorylation. Studies performed to reconcile the discrepancy between the in vitro and in vivo hypertrophic chondrocyte phenotypes revealed normal chondrocyte differentiation in c-Raff/f;ColII-Cre+ mice and lack of compensatory increase in the expression of A-Raf and B-Raf. However, VEGF (Vegfa) immunoreactivity in the hypertrophic chondrocytes of c-Raff/f;ColII-Cre+ mice was significantly reduced, associated with increased ubiquitylation of VEGF protein. Thus, c-Raf plays an important role in growth plate maturation by regulating vascular invasion, which is crucial for replacement of terminally differentiated hypertrophic chondrocytes by bone. Summary: The chondrocyte-specific ablation of c-Raf in mice results in a delay in vascular invasion and growth plate maturation due to increased ubiquitin-dependent degradation of VEGF.

Raf Kinases are Essential for Phosphate Induction of Erk1/2 Phosphorylation in Hypertrophic Chondrocytes and Normal Endochondral Bone Development.

Hypophosphatemia causes rickets by impairing hypertrophic chondrocyte apoptosis. Phosphate induction of MEK1/2-ERK1/2 phosphorylation in hypertrophic chondrocytes is required for phosphate-mediated apoptosis and growth plate maturation. MEK1/2 can be activated by numerous molecules including Raf isoforms. A- and B-Raf ablation in chondrocytes does not alter skeletal development, whereas ablation of C-Raf decreases hypertrophic chondrocyte apoptosis and impairs vascularization of the growth plate. However, ablation of C-Raf does not impair phosphate-induced ERK1/2 phosphorylation in vitro, but leads to rickets by decreasing VEGF protein stability. To determine whether Raf isoforms are required for phosphate-induced hypertrophic chondrocyte apoptosis, mice lacking all three Raf isoforms in chondrocytes were generated. Raf deletion caused neonatal death and a significant expansion of the hypertrophic chondrocyte layer of the growth plate, accompanied by decreased cleaved caspase-9. This was associated with decreased phospho-ERK1/2 immunoreactivity in the hypertrophic chondrocyte layer and impaired vascular invasion. These data further demonstrated that Raf kinases are required for phosphate-induced ERK1/2 phosphorylation in cultured hypertrophic chondrocytes and perform essential, but partially redundant roles in growth plate maturation.

Hormonal Regulation of Osteocyte Perilacunar and Canalicular Remodeling in the Hyp Mouse Model of X-Linked Hypophosphatemia

Osteocytes remodel their surrounding perilacunar matrix and canalicular network to maintain skeletal homeostasis. Perilacunar/canalicular remodeling is also thought to play a role in determining bone quality. X‐linked hypophosphatemia (XLH) is characterized by elevated serum fibroblast growth factor 23 (FGF23) levels, resulting in hypophosphatemia and decreased production of 1,25 dihydroxyvitamin D (1,25D). In addition to rickets and osteomalacia, long bones from mice with XLH (Hyp) have impaired whole‐bone biomechanical integrity accompanied by increased osteocyte apoptosis. To address whether perilacunar/canalicular remodeling is altered in Hyp mice, histomorphometric analyses of tibia and 3D intravital microscopic analyses of calvaria were performed. These studies demonstrate that Hyp mice have larger osteocyte lacunae in both the tibia and calvaria, accompanied by enhanced osteocyte mRNA and protein expression of matrix metalloproteinase 13 (MMP13) and genes classically used by osteoclasts to resorb bone, such as cathepsin K (CTSK). Hyp mice also exhibit impaired canalicular organization, with a decrease in number and branching of canaliculi extending from tibial and calvarial lacunae. To determine whether improving mineral ion and hormone homeostasis attenuates the lacunocanalicular phenotype, Hyp mice were treated with 1,25D or FGF23 blocking antibody (FGF23Ab). Both therapies were shown to decrease osteocyte lacunar size and to improve canalicular organization in tibia and calvaria. 1,25D treatment of Hyp mice normalizes osteocyte expression of MMP13 and classic osteoclast markers, while FGF23Ab decreases expression of MMP13 and selected osteoclast markers. Taken together, these studies point to regulation of perilacunar/canalicular remodeling by physiologic stimuli including hypophosphatemia and 1,25D.

Clinical Disorders of Phosphate Homeostasis

Significant advances have been made over the past few decades in the understanding of the regulation of phosphate homeostasis. The discovery of fibroblast growth factor 23 and its interactions with other mineral hormones such as parathyroid hormone and 1,25 dihydroxyvitamin D have helped elucidate the pathophysiology underlying disorders of phosphate balance. The coordination of three organs, including the intestine, kidney, and bone, is necessary to maintain phosphate homeostasis. Identification of the genetic defects in skeletal disorders of hypophosphatemia and hyperphosphatemia, such as different forms of hypophosphatemic rickets or tumoral calcinosis, has provided insight into the molecular regulation of phosphate-modulating hormones. The studies on the mouse models of these diseases have been invaluable in the understanding of mineral metabolism. The combination of basic, translational, and clinical investigations into the regulation of phosphate balance has provided the foundation for the design of novel therapeutics for phosphate disorders.

Hormonal Regulation of Osteocyte Perilacunar and Canalicular Remodeling in the Hyp Mouse Model of X-Linked Hypophosphatemia: OSTEOCYTE PERILACUAR AND CANALICULAR REMODELING IN HYP MICE

Osteocytes remodel their surrounding perilacunar matrix and canalicular network to maintain skeletal homeostasis. Perilacunar/canalicular remodeling is also thought to play a role in determining bone quality. X‐linked hypophosphatemia (XLH) is characterized by elevated serum fibroblast growth factor 23 (FGF23) levels, resulting in hypophosphatemia and decreased production of 1,25 dihydroxyvitamin D (1,25D). In addition to rickets and osteomalacia, long bones from mice with XLH (Hyp) have impaired whole‐bone biomechanical integrity accompanied by increased osteocyte apoptosis. To address whether perilacunar/canalicular remodeling is altered in Hyp mice, histomorphometric analyses of tibia and 3D intravital microscopic analyses of calvaria were performed. These studies demonstrate that Hyp mice have larger osteocyte lacunae in both the tibia and calvaria, accompanied by enhanced osteocyte mRNA and protein expression of matrix metalloproteinase 13 (MMP13) and genes classically used by osteoclasts to resorb bone, such as cathepsin K (CTSK). Hyp mice also exhibit impaired canalicular organization, with a decrease in number and branching of canaliculi extending from tibial and calvarial lacunae. To determine whether improving mineral ion and hormone homeostasis attenuates the lacunocanalicular phenotype, Hyp mice were treated with 1,25D or FGF23 blocking antibody (FGF23Ab). Both therapies were shown to decrease osteocyte lacunar size and to improve canalicular organization in tibia and calvaria. 1,25D treatment of Hyp mice normalizes osteocyte expression of MMP13 and classic osteoclast markers, while FGF23Ab decreases expression of MMP13 and selected osteoclast markers. Taken together, these studies point to regulation of perilacunar/canalicular remodeling by physiologic stimuli including hypophosphatemia and 1,25D.

Molecular analysis of enthesopathy in a mouse model of hypophosphatemic rickets

ABSTRACT The bone tendon attachment site known as the enthesis comprises a transitional zone between bone and tendon, and plays an important role in enabling movement at this site. X-linked hypophosphatemia (XLH) is characterized by impaired activation of vitamin D, elevated serum FGF23 levels and low serum phosphate levels, which impair bone mineralization. Paradoxically, an important complication of XLH is mineralization of the enthesis (enthesopathy). Studies were undertaken to identify the cellular and molecular pathways important for normal post-natal enthesis maturation and to examine their role during the development of enthesopathy in mice with XLH (Hyp). The Achilles tendon entheses of Hyp mice demonstrate an expansion of hypertrophic-appearing chondrogenic cells by P14. Post-natally, cells in wild-type and Hyp entheses similarly descend from scleraxis- and Sox9-expressing progenitors; however, Hyp entheses exhibit an expansion of Sox9-expressing cells, and enhanced BMP and IHH signaling. These results support a role for enhanced BMP and IHH signaling in the development of enthesopathy in XLH. Summary: The BMP and IHH signaling pathways play a role in normal postnatal enthesis maturation. Misregulation of these pathways contributes to the development of enthesopathy in X-linked hypophosphatemia.