X-linked hypophosphatemia (XLH) is characterized by impaired renal tubular reabsorption of phosphate due 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 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.

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-Raf(f/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-Raf(f/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-Raf(f/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.